<%BANNER%>

Zinc Transporter Expression in Mature Red Blood Cells and Differentiating Erythroid Progenitor Cells

University of Florida Institutional Repository
Permanent Link: http://ufdc.ufl.edu/UFE0021446/00001

Material Information

Title: Zinc Transporter Expression in Mature Red Blood Cells and Differentiating Erythroid Progenitor Cells
Physical Description: 1 online resource (50 p.)
Language: english
Creator: Ryu, Moon-Suhn
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: erythrocytes, erythropoiesis, erythropoietin, metallothionein, mtf1, slc30a1, slc39a10, zinc, zip10, znt1
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Animal and human studies have shown that the in vitro uptake rate of 65Zn by red blood cells (RBCs) is inversely related to the subject's zinc status. The capability of RBCs to take up zinc may be a remnant of an earlier developmental stage of erythrocytes as zinc is essential for the activities of several proteins formed during the erythroid differentiation, e.g., carbonic anhydrase, Cu2+/Zn2+-superoxide dismutase, and zinc finger transcription factors such as erythroid Kruppel-like factor (EKLF) and GATA-1. Conversely, excessive intracellular free zinc ions can interfere with incorporation of ferrous ions into heme at the final stage of erythroid differentiation. Therefore, intracellular zinc homeostasis during erythroid differentiation must be tightly regulated. To examine the hypothesis that the expression of zinc transporters would be involved in the strategic mechanism of erythroid zinc homeostasis, transporters in the membrane fraction of RBCs were screened by western analyses, and only Zip10 and ZnT1 were detectable among the zinc transporters tested. Thereafter, erythroid progenitor cells were prepared from spleens of phenylhydrazine (PHZ)-treated anemic mice for the characterization of transporter gene expression during the terminal stage of erythropoiesis. Differentiation of cells into reticulocytes was induced by erythropoietin (EPO)-treatment in vitro. Hemoglobin (Hb) synthesis and expression of erythroid delta-aminolevulinic acid synthase (ALAS-2) mRNA were measured for the confirmation of the ex vivo erythroid differentiation. Transcript levels of each transporter gene and other genes associated with zinc metabolism were quantified by quantitative real-time PCR (qRT-PCR). Temporal trends in expression of each gene were observed. Briefly, following addition of EPO, Zip10 mRNA levels peaked prior to the time-point when metal-responsive transcription factor-1 (MTF-1) transcripts reached its first peak level, and decreased dramatically afterwards. For ZnT1 mRNA, EPO-dependent expression was initiated later than Zip10 and was sustained until the experimental time-course was over. Metallothionein-1 (MT-1) transcript abundance decreased rapidly after addition of EPO and stayed lower than the 0 h basal levels until 48 h. Expression trends of Zip10 and ZnT1 were further confirmed by western blots utilizing total cell lysates and membrane fractions of these cells. This is the first study conducted to determine which zinc transporters are expressed in the erythroid system. The results presented here suggest that Zip10 and ZnT1 expression is induced in response to EPO. Furthermore, they could be the zinc transporters most directly involved in the regulation of intracellular zinc homeostasis in differentiating erythroid progenitor cells and circulating RBCs. The results may also be a route whereby RBCs accumulate excessive amounts of zinc during malaria.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Moon-Suhn Ryu.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Cousins, Robert J.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021446:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021446/00001

Material Information

Title: Zinc Transporter Expression in Mature Red Blood Cells and Differentiating Erythroid Progenitor Cells
Physical Description: 1 online resource (50 p.)
Language: english
Creator: Ryu, Moon-Suhn
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: erythrocytes, erythropoiesis, erythropoietin, metallothionein, mtf1, slc30a1, slc39a10, zinc, zip10, znt1
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Animal and human studies have shown that the in vitro uptake rate of 65Zn by red blood cells (RBCs) is inversely related to the subject's zinc status. The capability of RBCs to take up zinc may be a remnant of an earlier developmental stage of erythrocytes as zinc is essential for the activities of several proteins formed during the erythroid differentiation, e.g., carbonic anhydrase, Cu2+/Zn2+-superoxide dismutase, and zinc finger transcription factors such as erythroid Kruppel-like factor (EKLF) and GATA-1. Conversely, excessive intracellular free zinc ions can interfere with incorporation of ferrous ions into heme at the final stage of erythroid differentiation. Therefore, intracellular zinc homeostasis during erythroid differentiation must be tightly regulated. To examine the hypothesis that the expression of zinc transporters would be involved in the strategic mechanism of erythroid zinc homeostasis, transporters in the membrane fraction of RBCs were screened by western analyses, and only Zip10 and ZnT1 were detectable among the zinc transporters tested. Thereafter, erythroid progenitor cells were prepared from spleens of phenylhydrazine (PHZ)-treated anemic mice for the characterization of transporter gene expression during the terminal stage of erythropoiesis. Differentiation of cells into reticulocytes was induced by erythropoietin (EPO)-treatment in vitro. Hemoglobin (Hb) synthesis and expression of erythroid delta-aminolevulinic acid synthase (ALAS-2) mRNA were measured for the confirmation of the ex vivo erythroid differentiation. Transcript levels of each transporter gene and other genes associated with zinc metabolism were quantified by quantitative real-time PCR (qRT-PCR). Temporal trends in expression of each gene were observed. Briefly, following addition of EPO, Zip10 mRNA levels peaked prior to the time-point when metal-responsive transcription factor-1 (MTF-1) transcripts reached its first peak level, and decreased dramatically afterwards. For ZnT1 mRNA, EPO-dependent expression was initiated later than Zip10 and was sustained until the experimental time-course was over. Metallothionein-1 (MT-1) transcript abundance decreased rapidly after addition of EPO and stayed lower than the 0 h basal levels until 48 h. Expression trends of Zip10 and ZnT1 were further confirmed by western blots utilizing total cell lysates and membrane fractions of these cells. This is the first study conducted to determine which zinc transporters are expressed in the erythroid system. The results presented here suggest that Zip10 and ZnT1 expression is induced in response to EPO. Furthermore, they could be the zinc transporters most directly involved in the regulation of intracellular zinc homeostasis in differentiating erythroid progenitor cells and circulating RBCs. The results may also be a route whereby RBCs accumulate excessive amounts of zinc during malaria.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Moon-Suhn Ryu.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Cousins, Robert J.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021446:00001


This item has the following downloads:


Full Text
xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID E20101113_AAAAHY INGEST_TIME 2010-11-13T10:03:12Z PACKAGE UFE0021446_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES
FILE SIZE 119232 DFID F20101113_AABFEV ORIGIN DEPOSITOR PATH ryu_m_Page_15.jp2 GLOBAL false PRESERVATION BIT MESSAGE_DIGEST ALGORITHM MD5
ee40517726a8727343d42a117d1e5cfe
SHA-1
17112de768b62734765e492270fda09a9f64466a
4666 F20101113_AABEXY ryu_m_Page_04thm.jpg
62df9abd4e884ecb124ed028ece8f2b3
19cab65caf563667cc6136e87ef4e7646bf7bf20
12964 F20101113_AABFJS ryu_m_Page_35.QC.jpg
5d97eae05ee810d54922026407557745
224d90fd799b2c43f596fe3787f8221499d1b4c1
116255 F20101113_AABFEW ryu_m_Page_16.jp2
d818a22d31c6a45b2113e16e21d84082
028039148822254b6298417834bfbb997ecabaff
2127 F20101113_AABEXZ ryu_m_Page_43.txt
da9780cbc858fe035ce2e3d1a0d4e752
2c9d9b95dbb657f9f62bb8b1239acf710f793975
1320 F20101113_AABFJT ryu_m_Page_02thm.jpg
8800d4d23bf9d3819117f9963b0a2d34
020181cba2444d59d5f883a5d8b19c44c434fcdf
120628 F20101113_AABFEX ryu_m_Page_18.jp2
3cdbf40b58e9198d86caddab10af67ae
fc248b45a429abecf0211c88be89c66fc7f5852d
26125 F20101113_AABFJU ryu_m_Page_28.QC.jpg
dcad49a82de39c6fb226c35851720fd8
f40cad2818c153c70c6144b520c12a607a87baad
44291 F20101113_AABFCA ryu_m_Page_35.jpg
1d57cd1f5b61bbe7defacaf25012820d
d08a938a5c13ef8c4e7fc6cef119b874a1f81560
113721 F20101113_AABFEY ryu_m_Page_20.jp2
e28439fb98364381a41b0d8ecf32d544
4a373f46a0398f832f15bc099991fc75100106c1
4221 F20101113_AABFJV ryu_m_Page_03.QC.jpg
6d3943868defa3c2d3e6ed9fe8f1c9b5
e08405aafae88f4fde9bfd8b774efd8ffd67806d
54046 F20101113_AABFCB ryu_m_Page_41.pro
35f170a69a5c535b917719b01af2dcd8
143002082c481dfb43397144b36d3c25bc823511
108967 F20101113_AABFEZ ryu_m_Page_21.jp2
cece4cdc3e6615a9ac4463bf6593be8e
f89cf70bdbbf752c1ccb34dccfce56b7e4447f0b
26013 F20101113_AABFJW ryu_m_Page_18.QC.jpg
c0dd633d95fed195437715e934cda7b1
30ff3e89660491552748784d0c1b415b0523f274
6972 F20101113_AABFCC ryu_m_Page_24thm.jpg
65aff267b1d13a22be7ae2de7bcc5db2
01c5641b6da55f73ef1b184a60d2c0e798e5c4ff
5131 F20101113_AABFJX ryu_m_Page_44thm.jpg
b9e3f6545e05738adb4bc79f3e3c2d14
000872b6a41e6c96d693268c972a9e837153c5ae
1053954 F20101113_AABFHA ryu_m_Page_48.tif
9c163d2c884cb1cbc5833c947534025f
22b2e418a2c5305e0c0caebbe6630921ee03b627
10671 F20101113_AABFJY ryu_m_Page_36.QC.jpg
e483cb529396899b0ca1acfac834024c
597ccf01a8a09cdfa0265ee1562571b18f00879f
25271604 F20101113_AABFCD ryu_m_Page_31.tif
d88cefeaf7e8214a30fa7d6a4f0749b3
8edc9f2600758005e32b821fcd7e7f6b728113ac
F20101113_AABFHB ryu_m_Page_50.tif
cc810df379c5dfc7bd8ffc1ad625f2ef
3e81cbe2ddebcb824a8b53e32ac78e6857c67b91
21572 F20101113_AABFJZ ryu_m_Page_30.QC.jpg
36bdbcbe23f4739059c86f5aada2a4d9
9d8e0a4958c51e6ba9af8de8b73b7bb7ac703ede
8434 F20101113_AABFHC ryu_m_Page_01.pro
4e7cc9d047a0ed36e4b79e33b3aac1b5
8b523b6b0c9d0520f2678fa8b98ec5a0c7bd9f20
6948 F20101113_AABFCE ryu_m_Page_43thm.jpg
736871539e8515be33fe5fbd2950e9eb
80caaa095d80109076e208ed058e4f61231865d3
4143 F20101113_AABFHD ryu_m_Page_03.pro
2572221bd7568777598fe779a0fb46f0
05eb93ecd49fa1da3ccdfc669d760f7c270cdb18
20145 F20101113_AABFCF ryu_m_Page_35.pro
703e661188f565a80c4b9514c8c7ac10
5b97794fec4a3ee1dd8d295308f69409c065a713
66835 F20101113_AABFHE ryu_m_Page_05.pro
36f6094694d692bd169ff4106bc4d214
d52b5adede275d06f028892b3f4951d844a9e778
6640 F20101113_AABFCG ryu_m_Page_38thm.jpg
4639e46dbebe5b2736e32aa1862c18f7
3711a7442e69da5efa7ab5191c218923ee18b17a
6080 F20101113_AABFHF ryu_m_Page_07.pro
749e96db5c3fc094b5d01751d8be7c06
87fc71235f0a7d4ba1be4c6c59a5c3300087f21f
252 F20101113_AABFCH ryu_m_Page_07.txt
cf534041a2ea3a38d700da3d9baee37e
4f79a5bc53d76bdb66a61417c50f2e3d93a8ea32
30289 F20101113_AABFHG ryu_m_Page_08.pro
24f62f7362c1955ad155b564591a4d85
518b88217a2b1ed4e8125dc26977ccc68cd1ecae
F20101113_AABFCI ryu_m_Page_49.tif
8c199a66f7c9c679100fc0cfe74aded8
5ba88196d7af39a7248b0bdd5e8d0b9df43aadde
53789 F20101113_AABFHH ryu_m_Page_12.pro
3a9d334c90a8f205e86c06057824d7a6
ea543d23aa946183c1721e5dbce83f2423eb8857
26390 F20101113_AABFCJ ryu_m_Page_45.pro
ad6e9466a6bcf2f357a64975aa68aff0
073b3c6008755e60ddf6b918e030c8d32b928801
29149 F20101113_AABFHI ryu_m_Page_13.pro
51f57284c27218072abce3a70ffd4da9
34417cd942328c0ff090072ed0048d12d048a784
14468 F20101113_AABFCK ryu_m_Page_13.QC.jpg
eba77b6d3ff0d680933fdced2fe066fe
41141cdd574456658bc66acc2632b1c505d863a6
F20101113_AABFCL ryu_m_Page_47.tif
a67113094e1ece23ae652cb1819e35ac
f252c63cc84c5dfeebcfdebd9e0136fe621e3c99
55745 F20101113_AABFHJ ryu_m_Page_18.pro
aeae87079fcf43a2832f67fa78a0183e
bcf8752af1e7c29c97563b1759e92d5a75f48f0a
7041 F20101113_AABFCM ryu_m_Page_23thm.jpg
f6a409b161e93f669d2bc290068716dd
4e67d4fc60ddde0d9a0937563c3b874191b35afe
48701 F20101113_AABFHK ryu_m_Page_19.pro
5212601a25a6ab1f3e294875992ba793
386fb3c65724c78e12da9de6f82373c8f1f9e888
6840 F20101113_AABFCN ryu_m_Page_22thm.jpg
4d63291e214fc8267b7c2f1139cef599
76d31d1fbdfc62f08d4eee77c2bb96afcd18797b
49995 F20101113_AABFHL ryu_m_Page_21.pro
1358243a3869af0e364f10d655256577
eaf0dfacceb1fdb9ad886457bb729d043aee21f3
F20101113_AABFCO ryu_m_Page_41.tif
0bdd5d8725d365332ed2342adb7da591
320e318dfc4e2eeb1dcbdeb3f6c398e1943bfdab
50906 F20101113_AABFHM ryu_m_Page_22.pro
315573467e1e7c9f5ff479c84ce5addf
7f144f0e91964b5b757f16053779ec027f01f17e
2154 F20101113_AABFCP ryu_m_Page_16.txt
c913045b567ab47624fb0402d4ca8ef5
25963e08a2c0a08c491c172ab3d65cbf30d21a82
52743 F20101113_AABFHN ryu_m_Page_24.pro
4ca7d083f6b50cef813a73461b685431
8c926a4601ad1e5a89ce59bd1e9ca00bfbf37dd0
348 F20101113_AABFCQ ryu_m_Page_06.txt
15080204bb06e48ba5091f4f277acfd4
59941b5421082d3f0dacacb75ff3fc2af6778838
40806 F20101113_AABFHO ryu_m_Page_25.pro
b4e8b994294b87414c65dba790131195
5254b199fa0cc291348e92b5ab47a72837a1d957
2864 F20101113_AABFCR ryu_m_Page_05.txt
035afb0758521c2ecfc83ba9726d18d8
776c3976a884dc30f3877d719f6ab23ed89393a1
13231 F20101113_AABFHP ryu_m_Page_26.pro
2314e5c2fa5279a9a36cd0f7a4815c6c
a8215d40ba23a614c4cc1b1c1071414ee09802b1
54193 F20101113_AABFCS ryu_m_Page_43.pro
96d272a8410c057fc641c52345741292
21856299510ec080120085b0c1e006199a13ff12
54317 F20101113_AABFHQ ryu_m_Page_29.pro
982f64f59b579935bb0d1e841bca9793
be4a6c1f34dcb024ad655fbdfdfc899efbcd0fbf
5861 F20101113_AABFCT ryu_m_Page_30thm.jpg
326cdee8cfcd1dc9c6231efcbc0515d2
9035eced0688a07a28d9dbca2c37221b6d225f35
42874 F20101113_AABFHR ryu_m_Page_30.pro
5059c5d7607c61af334d0d1ead456859
d0e72f148f745dde6505546d4d250f51157302b0
7035 F20101113_AABFCU ryu_m_Page_15thm.jpg
d79f5cac56f1fdb774917dee8c2e7245
4e8d329690342f4e970af6270de9eedfcea9749d
19786 F20101113_AABFHS ryu_m_Page_31.pro
4ffc20da952f13efb4211e56d554b8ec
a0862afecbfa1a79c1f533a64f4e2dd2c08f1442
2229 F20101113_AABFCV ryu_m_Page_39.txt
05c1541aa3221228c3d98d25b05bf5d7
6f90730dc7c4587974798c4518e8177da95de354
31973 F20101113_AABFHT ryu_m_Page_34.pro
a540d0f98ddecf684fa45fb7db82efb7
5f74bd0c382e417548006aa0666329f638080d78
F20101113_AABFCW ryu_m_Page_35.tif
af393caa647888ff1108129fb3322c54
d0cbb563c7961e75e4f8066edb83335f3b42deea
15013 F20101113_AABFHU ryu_m_Page_36.pro
6c711cc2a2e5865ede9f083f7f9c0782
04cad652d78b36ec8997829fcc33d5ebf368e3e1
1759 F20101113_AABFCX ryu_m_Page_03thm.jpg
1520593561da93ccf40febc4365f80ea
53ae125caa58a548d3d4e0eb3bef9b871ae3315f
53211 F20101113_AABFHV ryu_m_Page_38.pro
5f075d52b0696948416e62690c204c80
3866f8f0dacdd1f97d4e877687f10b325c0e3f54
F20101113_AABFAA ryu_m_Page_46.tif
dbe24872456db1b01c795a22a812c959
1426e9045638e2b2a6cd92e223c7e9f07c6323e8
108159 F20101113_AABFCY ryu_m_Page_14.jp2
6b5401a9e4d2cee069bdb380317e2f48
2ecd0972ed0b01de3f8b66eb2d9327ed165a81b5
56425 F20101113_AABFHW ryu_m_Page_40.pro
3164bc838758be455c2be66ba0ed7860
d8228ae4c6001de115570791ac4aed18d3c4ee1d
25528 F20101113_AABEYA ryu_m_Page_22.QC.jpg
06804e4c7df2cb597b9b7c1c209c79f2
d69530f0876c8dadb6b820be4aa38a5350457947
F20101113_AABFAB ryu_m_Page_23.tif
616c7d98e514539f5a7ad9061caeceab
0833cb3f9cc2701252b519c371bc4ad4fd9676ca
3838 F20101113_AABFCZ ryu_m_Page_08thm.jpg
98491e8f6ff46a41baf9e6cb9a103752
44c62f2d42fbb924be0cf681c10d0287e38f9242
54771 F20101113_AABFHX ryu_m_Page_42.pro
9829a053f338edd1fa9fc99f5238d29e
5851ae39c215d49fc5757d10d9f9125d41b19c40
6846 F20101113_AABEYB ryu_m_Page_16thm.jpg
08b37b2faba16386be5f575f28628f19
67785459d4c6d1dd0144d5f596b627ef13974fa7
36469 F20101113_AABFHY ryu_m_Page_44.pro
d1c0f25a3180e26c072e85ef93401d8b
97db90da8267f01c2ff7b700c27bd10def8bb141
26078 F20101113_AABEYC ryu_m_Page_29.QC.jpg
02e9de29d412a09bc50c6fe2e9998f58
babcc8bfb27964e3714a00d565b509d0ac769f7c
4993 F20101113_AABFAC ryu_m_Page_02.jp2
3d7306fd6879746b7d9f58e8b20041d4
0f1e3880869abbb27a903aa89d2e91baf76b0cab
118775 F20101113_AABFFA ryu_m_Page_23.jp2
6db1e472b82a4192a2343ce568820cd1
c768afba5b62fea9294dd3adf46db2a95b1db0a9
60277 F20101113_AABFHZ ryu_m_Page_46.pro
691f70e0272f0d97b38cbaefb476a2bf
d295d89d7264843a0d6f7040f03e4b0ebb65f5a1
38854 F20101113_AABEYD ryu_m_Page_37.pro
a7a5be2bbcd1b752161a11f83d3d9b74
ce3fa7e602c6609a8a8b70d5a160c6badd854bb5
6230 F20101113_AABFAD ryu_m_Page_09thm.jpg
d44605c6410a5cd2c82c639fbc30e173
200a58a2b8f57c4243b6895b4da141432f160968
105179 F20101113_AABFFB ryu_m_Page_27.jp2
649994d7988bef9a2ce0afc08647a567
1b3a8b163fea5486c5bf29960b09a25c03320c08
7922 F20101113_AABEYE ryu_m_Page_32.pro
a4ce5e934fb964b354324b3831324495
981eb3405032fc2a09b90e2061cdd2cc0276ce9a
800 F20101113_AABFAE ryu_m_Page_02.pro
2497f27dcc5b1c4fbd6092a671a0bc54
4283428dd1ca8dd7b163286219768e2157565ec9
119403 F20101113_AABFFC ryu_m_Page_28.jp2
857a5fc49e454430eae86db681170828
a0360487642755decbc180d31ac2b8fbd58da081
F20101113_AABEYF ryu_m_Page_39.tif
dffc327b287a8d6ddbc8fa36a91eaa5a
1117f83a3cf76ea16ec35bac0877f2638d5989e3
235 F20101113_AABFAF ryu_m_Page_03.txt
24917e590c48af7989a4c54210211138
5a9a095fb16e2e5fdbfa91dcce19053c7208488e
11480 F20101113_AABFKA ryu_m_Page_50.QC.jpg
e1bdcfa6d56eb4250dc1040152745e9a
c4918e1e07722a5a03230576c2539e70ba54c628
117003 F20101113_AABFFD ryu_m_Page_29.jp2
53e0b3568473e5fd0b21c31791c030a2
1927bada7acbb160560bc55aaa0b8588faa01ab2
57987 F20101113_AABEYG ryu_m_Page_10.jpg
1e64bcd109405c4aea9988ca9c620e5b
4c6e79eb427fa5171378367840d2a3753491c7b8
969 F20101113_AABFAG ryu_m_Page_31.txt
b3b89746a180ce9573515484ed32a1f0
71487a8decfe8fbe0f60590bb19cb058ac47224e
3352 F20101113_AABFKB ryu_m_Page_36thm.jpg
696a28f3bdb89eaac2568d82736d4749
df53c69d87d67e1f0ab3cf74042e2d418ca93241
93132 F20101113_AABFFE ryu_m_Page_30.jp2
35fbe2fa270a83d6825ec0015171c2ff
acf3d102644c849775e1d4e75abdb265628d84e8
F20101113_AABEYH ryu_m_Page_24.tif
b2332b8beaf6f65c31795fe87341de2b
ab09f84e4c5d052f6c55f25b3ea22ab45343dc61
789 F20101113_AABFAH ryu_m_Page_36.txt
408ec40f015ac333c92d064da167c3ef
3c9e78a08c94fa97a15c161ccd407b3273eaa69f
76713 F20101113_AABFKC UFE0021446_00001.xml FULL
3a4fe6ac357c853b9f2200e80b4ec6a0
54de7a1d023ba26eaa7f73b8fdfa1bdab4dd20b4
220880 F20101113_AABFFF ryu_m_Page_32.jp2
ca703b6bd55b5dc274c1fadd2ce7689f
4385bfea7e60322ca6c3b96629627b54068da82f
38940 F20101113_AABEYI ryu_m_Page_10.pro
366ed59bbf8db610cc9345cb0eb1a232
bb403f1c47941580104d107a826c227fb18dc142
3903 F20101113_AABFAI ryu_m_Page_31thm.jpg
3b3c5a4ef58165c0f3e40b3219b797b1
7e3697375c93080d9014784b3c78a27809589a1a
1599 F20101113_AABFKD ryu_m_Page_06thm.jpg
e27e56805a3928959f1bee0a01409e94
34dfa8143db9d2f662eeeb1e31a7818d8b73827b
722418 F20101113_AABFFG ryu_m_Page_33.jp2
4bc1beb0c5173a12d508f920f09e0fd9
65c663880c7f8856e2f104ee26f9af54c1c27bef
2319 F20101113_AABEYJ ryu_m_Page_49thm.jpg
e8a15722c5ece9d6aacd546b0277c3fc
653d4a5b0dbdcbb1c5a17ad881a8f3cc9d476f8f
47875 F20101113_AABFAJ ryu_m_Page_27.pro
6a302e414d73e976ffb7c2fae17af621
93c208eda32e7555764a8d53a8f9ec4a8869fa85
5022 F20101113_AABFKE ryu_m_Page_07.QC.jpg
5e358f0cf3f7328cb56308b502ec211c
b648d429234fc66237f6c96916aa144981138f83
26243 F20101113_AABEYK ryu_m_Page_23.QC.jpg
9ad6ed882ae2fff45f8cee223161c608
f37620621088d228ed5d34ff4a7413040eeba96c
598551 F20101113_AABFAK ryu_m_Page_31.jp2
481bd6aafb8391f9660f8f1160a4a70a
4e11db164ee5562f84596af5cea56b6bbca1171c
14382 F20101113_AABFKF ryu_m_Page_08.QC.jpg
94d0db95b510f7d4303ea4ee0969c8fd
8862bc45f52eb7957f7b40f06a9e897eebe9a752
797679 F20101113_AABFFH ryu_m_Page_34.jp2
f30c95e66dd82377a2335d8cfc47655d
11c26f18203e102fc155b83160e47ef38a0aaabb
23402 F20101113_AABEYL ryu_m_Page_19.QC.jpg
12b7d912867424da2fda07d02c9f69c0
d33bfb14859c83b27506910fe861f20e6a9712a5
77391 F20101113_AABFAL ryu_m_Page_41.jpg
d9d08177ed6f6669a19b7098e45a1cef
0e4de399c37a3bff88dda46fb562bfe92743d71a
19466 F20101113_AABFKG ryu_m_Page_10.QC.jpg
ae1a19a21ebf179a5573a273ad7eace4
72c2a454d122969c73647c94c1fcfd148732bc13
458619 F20101113_AABFFI ryu_m_Page_36.jp2
73ab43ec57b552f27976d56346459fd1
2ef0657dfdfe6eafd53d62318f59398909fbfa33
2012 F20101113_AABEYM ryu_m_Page_27.txt
070f704d073910ba20b6304d9b24bf25
a12fba76f662c9d3097aa7a1c6baa7c2eee4ce0c
49072 F20101113_AABFAM ryu_m_Page_33.jpg
de23635b9256c4149861d2037d096822
975f50f6c52d8811363167c2252f9a1070c49a30
5551 F20101113_AABFKH ryu_m_Page_10thm.jpg
3f9875d45b50c9c2dae3ff8028c04104
ffd14b8279a9844f99a1c1d88da40925aa6616ce
884024 F20101113_AABFFJ ryu_m_Page_37.jp2
312abd9fcf92b463b3d8dbe2625db72a
e22f9be78d58883cf1b807a0c490f466037f0bcc
159749 F20101113_AABEYN ryu_m_Page_06.jp2
db644f7a931e244271ea42ef633bf8f8
fca2a58f53cd51962aeda5c88c43701e50e982e6
2432 F20101113_AABFAN ryu_m_Page_47.txt
d972ec99c1b04eb318784c379d72b9cf
5c578b69d3e0ffbb1405a670757285fd387128e0
25246 F20101113_AABFKI ryu_m_Page_11.QC.jpg
62646e940afb4043c02d739444100036
c704706b220d1a24626c5466ffd021ac08fc3932
113625 F20101113_AABFFK ryu_m_Page_38.jp2
5d01bfd843332190b76de1be1b9213c8
6ec231aabbf9e68e36cc2f77569ee209d0100f99
31960 F20101113_AABEYO ryu_m_Page_04.pro
9a1445c48671924e0a9eae0da8e17efb
53516d3089f99b8333cee91a905a0d42c40f7617
47103 F20101113_AABFAO ryu_m_Page_09.pro
8aa66d08a971dcc9de71d784ab9287c1
341224dc2c1b6508d3ec9acab478c2267375f33e
6923 F20101113_AABFKJ ryu_m_Page_12thm.jpg
2e0f61509364f7312e90ffc6c51dc10c
6b256e0c5124b866779c6022ab375d8d4ad0d6f0
121122 F20101113_AABFFL ryu_m_Page_40.jp2
ff1f3d76c839e0a8e409d0857f759207
9d76627c9d85b42f1c14489b23d0cf56c7009557
103063 F20101113_AABEYP ryu_m_Page_09.jp2
f3af5ac3758d8d1088fd4ac6cb2df098
a89f89933d5ca8b825dba0285df1b529acece014
5257 F20101113_AABFAP ryu_m_Page_05thm.jpg
e3f5e1080cd79f63aec9f030e9ce2595
9042b21d24a9343292c846a599601f9daa1cab9d
3953 F20101113_AABFKK ryu_m_Page_13thm.jpg
fca4e82471390a6381b3bbf20f390930
f2eeaee7f956dd9c38c895ff4044dc83331f3907
115477 F20101113_AABFFM ryu_m_Page_41.jp2
6fa5760236c98e66d3e4a01702532934
adc19122a5062ac885c58fc1f3cb5577735dbc4a
13189 F20101113_AABEYQ ryu_m_Page_06.jpg
026118610a443bfc9f42a49aae66745d
d5aab229e96cca665ab804ce4e4a47c25ae4fff1
56309 F20101113_AABFAQ ryu_m_Page_28.pro
4f7fccaafef5634b6218852a4ea78545
f13740f37ecaeab56b65f5a46cd74470c16398b5
25818 F20101113_AABFKL ryu_m_Page_15.QC.jpg
98195d00adde638fe703e4b679e3fe46
e6f6ce47c89d8197fdf3f59dd45f17319e968bef
116926 F20101113_AABFFN ryu_m_Page_42.jp2
5681d31677db7ce783313e6d58fd6917
59069b33b982253efd22b340ee6c4c1d9020f2b0
6451 F20101113_AABEYR ryu_m_Page_14thm.jpg
d6091bc0868a563bc375f6a482a08c66
99ce81cd5dfe342a6e29e5be3cfceca43e84a598
F20101113_AABFAR ryu_m_Page_27.tif
bb13aac6f465a89de29a87f774c0e5a2
67c78a5f625906450a99c33b6fe0cd881f3da9a3
115310 F20101113_AABFFO ryu_m_Page_43.jp2
436cfd614053081ff69cb69fd1831322
60f33c700185c2ceb54ef80bc0e42c5992a859bb
F20101113_AABEYS ryu_m_Page_16.tif
4b9f9c6bc07dd24f45ade96fe85b079d
1467ddcaf2baebe918892c3ef8839e100cf4c4c6
18381 F20101113_AABFAS ryu_m_Page_44.QC.jpg
20b7d0c62f97bdf1f91ca72f11543989
415773a59a2ea98c0a22f7b64e51554cccd47e7e
25924 F20101113_AABFKM ryu_m_Page_16.QC.jpg
23667503ed6adcaf25e13cbd651f3b32
c7742227dbd2e164d91b6c3c5ad88f09c1b2af68
80727 F20101113_AABFFP ryu_m_Page_44.jp2
ea5813c41b2f15780caeb85aee3cddb5
b1957f0e317dc4171ccb23fd764c598daa9b83e0
F20101113_AABEYT ryu_m_Page_18.tif
a60c0d1a2f62bd33399a553e5307f4eb
e62585c017c2e16a2972556a143b349c5dac7480
16705 F20101113_AABFAT ryu_m_Page_34.QC.jpg
91f50ddb5f276b35f789259be3f43754
b3f171ea4d57f0e5c40562529feb1b5dc43948fe
25472 F20101113_AABFKN ryu_m_Page_17.QC.jpg
076132e7c034cfa00bdb0280791de08c
2dbccb331faefb2c1da31e21cbf23f23c0474068
844292 F20101113_AABFFQ ryu_m_Page_45.jp2
64252eecdad92f65f558d865e5d81874
ff805e7090df9107444b109e428bf4423bf772a7
21900 F20101113_AABFAU ryu_m_Page_32.jpg
7f958873aac9502322e3417e555fb68a
80ba2fbe1f42450371337f61e8592d2cd62656e9
6955 F20101113_AABFKO ryu_m_Page_17thm.jpg
d4bca29917b7651e23a51550e07f3788
444e6afa69380b6a76ca70a306d9ce97ecca1b82
125881 F20101113_AABFFR ryu_m_Page_46.jp2
7f0d0ca65d7e029bcb819d8e8fcb177c
e2e0e2b4ee8be84409cb114cf1b158eb0fa41ea7
75623 F20101113_AABEYU ryu_m_Page_12.jpg
306551b77366d6133237a92f4852bfa9
436f99881d7fb214359bab265199c1be12f7f3b2
2069 F20101113_AABFAV ryu_m_Page_22.txt
70fe72379c8ac897d6e3085fa57e5957
ebf9b56170980cbe1bc0df15a057df473de71f88
6982 F20101113_AABFKP ryu_m_Page_18thm.jpg
75e47c6f5239b1de86039704a6b1d3cc
9efbd182018523336805f88c7d78d1aa384cec69
127388 F20101113_AABFFS ryu_m_Page_47.jp2
cef60d69ab3d93db44d2728cb1dfe77b
b37d8646da0051dff004c9d11744fcd1d9c6417f
488 F20101113_AABEYV ryu_m_Page_01.txt
9c046c4f23edd1d8db49e1c1fcedf5ac
74ea85367efcfb149a93ae61e9e2c1d99bade7cc
5351 F20101113_AABFAW ryu_m_Page_06.pro
98f479d313b368a4b7cfa6450cac1a53
03e56211efcaeb5fad68834e6f91702e832fc1e6
6279 F20101113_AABFKQ ryu_m_Page_19thm.jpg
b3db25d0fa4079a53fc44854aabd8864
4bbe19f5f3c0aa7a03f91986bb75b6590e4b5b96
134490 F20101113_AABFFT ryu_m_Page_48.jp2
bfaa52219e6f62f3555ffa616028c002
3b29289596fef51b16064b1a6491665e5b251a4b
104600 F20101113_AABEYW ryu_m_Page_19.jp2
a83440a64580c25c808c5ba045e428b9
0877bcbaaba05f03cbb093ff9dfe88a67a5cd224
24924 F20101113_AABFAX ryu_m_Page_12.QC.jpg
e07ea2c72fcf84dfe7379e35726aa243
19ab80c0287879845198755ead5914aad01fd361
25459 F20101113_AABFKR ryu_m_Page_20.QC.jpg
a057d8e281df2d65fb588a9e9289f923
b3023878b08dc03e1f04903e15cc89528ceea639
28448 F20101113_AABFFU ryu_m_Page_49.jp2
af5452e30864f28140d25a5e704d35fc
1544fafe0e31a1680c6fbbe809dd1c7097d88ea6
25279 F20101113_AABEYX ryu_m_Page_01.jp2
73da72a1bd1bfca0f6dfd14028ff9da0
073addef4c167d83ea3a37b2b72bc9d82079dbdf
24130 F20101113_AABFAY ryu_m_Page_14.QC.jpg
c624079716e2e25b865c687f9bec39aa
f6e5ace79f3753a692de14068882fb9864b0844e
6754 F20101113_AABFKS ryu_m_Page_21thm.jpg
f81105f54e2b438fba1f750141d34e4c
7ff8b9095a84760f097782c19c68af626dbc6045
48142 F20101113_AABFFV ryu_m_Page_50.jp2
a011242d4258f1ede7d7bbc9fff5a811
94e99b825418f4d3800be12ae6dabd9080ff3390
1549 F20101113_AABEYY ryu_m_Page_10.txt
b953f4b2e8aafa24f4771294d264fe7f
ac1e22df862459e9fa96b43ccf73facc568bb8d4
6829 F20101113_AABFAZ ryu_m_Page_41thm.jpg
97ab9b63233033889242eb1dc733f671
de0b129223ea99ac0541833f5382e91f0a333be3
21024 F20101113_AABFKT ryu_m_Page_25.QC.jpg
04b443c12db91577537841c02d69abe2
ca16120276e8a7769e1422688b49c29aa90800a2
F20101113_AABFFW ryu_m_Page_01.tif
ba37746f10cdbc1bae63b8c4eecc083c
a1bc1af41b0c3150c0d707335296421751f7fc70
16505 F20101113_AABEYZ ryu_m_Page_04.QC.jpg
e767c8155c12862222a329b4700b1ab5
00ca60566bd312e638f7aa537bbd8718fc3265b5
5629 F20101113_AABFKU ryu_m_Page_25thm.jpg
7b7e04ea7b840dfe5fc393c32a3f5902
c50e99db67067dd504f0d1d2684186f0964a43bf
F20101113_AABFFX ryu_m_Page_02.tif
af0526971b2e89e0f64ffad549523542
fbc11266083a77f331ba747370db53d1c2454ebd
6470 F20101113_AABFKV ryu_m_Page_27thm.jpg
98f4daa03a9858e8653e35f5b05d5c6e
7e69109bddab95cd90ee6814e0487a1163e1a568
2121 F20101113_AABFDA ryu_m_Page_41.txt
4230e5d04524aa4328335b34dbc2570f
0b9bc0541bc598c1ac66835f74ef865963a7acb2
F20101113_AABFFY ryu_m_Page_04.tif
b0bcdde1d5f90491a9c31d69f8998576
9715915c697f01a145e70055752d3bdbef57c190
7203 F20101113_AABFKW ryu_m_Page_28thm.jpg
74ff153e1c52e948cc46b3ed489a8988
d5655257161e4d76f0614be893f349b380cf753e
23212 F20101113_AABFDB ryu_m_Page_27.QC.jpg
e9206c2a7196f3b6fd28ac7629cc1ce3
47f8ab507224ab4167c1b01c508ca801cb837f7e
F20101113_AABFFZ ryu_m_Page_05.tif
124f2d7148bf98359cc6b49ca3183dc0
61626ea728af915442d0aedb16f53e7a65df6a7d
6938 F20101113_AABFKX ryu_m_Page_29thm.jpg
1d7e9a7976efc96b406a5b2a90511779
cdae8dab441ddee8946c02ec3a3b6c27e4f73e4a
93114 F20101113_AABFDC ryu_m_Page_25.jp2
00b3293aefa9e1364e0f5ef771e23670
3a2950722c2066e6576c395d02e2e983efa53554
6507 F20101113_AABFKY ryu_m_Page_32.QC.jpg
c9a6410190922584431a425edfb325f8
d90064f96514a93a5fc863307bf30bde300d2fbc
59735 F20101113_AABFDD UFE0021446_00001.mets
738731233652713cd6c416336763fa23
d78b613cf62631f238923ebe5891a79321794401
60206 F20101113_AABFIA ryu_m_Page_47.pro
1bff6103f03b7c947e7c84f49fbeda39
c3cc7b43427f0dac8033700565d0ebcda8e8fbe8
4369 F20101113_AABFKZ ryu_m_Page_33thm.jpg
d6982e5ca98db78d5557f72d27853fec
0fa20f915f17fd5372b0b72a890aeb11120f29a5
63582 F20101113_AABFIB ryu_m_Page_48.pro
28c3347d0e09cf4b4094d6b6d88b3d39
4040b841dd1680ad3c51f1539ea74d2b56ea5c48
85 F20101113_AABFIC ryu_m_Page_02.txt
9c7a67aa79a9ad45408c78f0c22ee03e
2e8f6472f13f75a1dfceabca6a9b0f51d423fca8
1304 F20101113_AABFID ryu_m_Page_04.txt
122368e8e5a4232b97a7080572d7ad19
ab96b2f7298a7e7cd771a1a3c3a1ec609f8d1442
9870 F20101113_AABFDG ryu_m_Page_02.jpg
b55de9de8c1c4d94d7eb6e78f1408e6e
ba32226648214c3c2effae0d1af083e4d5731e36
1213 F20101113_AABFIE ryu_m_Page_08.txt
f26bddb8acd1ad29e86af66a11437c4c
ae457e00f1c442f59f2bad1a5d2a4003076d76ff
14532 F20101113_AABFDH ryu_m_Page_03.jpg
e95b19d9b10d2cc877c8b5cfc08ee8ab
98e44a42e456bbef3883e069474cb4a46761bf93
2053 F20101113_AABFIF ryu_m_Page_09.txt
398d8558df5face64640e7e934cda637
283dbf00a32b8477520dad6d20427a166d561e67
77329 F20101113_AABFDI ryu_m_Page_05.jpg
3875cce4eeff4457158cb620ff221aac
c1198b0475bc210b251ec0dc497da242d5236038
2188 F20101113_AABFIG ryu_m_Page_11.txt
d342979d5c20aafc090b29ed8859517c
44d271521a467eb294310d02d0c3843c1748f07e
17914 F20101113_AABFDJ ryu_m_Page_07.jpg
e599c356cbb6eee603f1f84cb511af0c
ae2660f1c8e6f21c1f5c9af3d36583349a157408
2117 F20101113_AABFIH ryu_m_Page_12.txt
be21884a31536f8ea0fcaeb2a6bcbf0f
422e837af1e0b587a5280ea36b18af6b2ae2e203
76929 F20101113_AABFDK ryu_m_Page_11.jpg
b410c351248add38e1d9a50cc5e94a89
0ca877965d37b2740b897b45bb5e6413b6a75c8e
1175 F20101113_AABFII ryu_m_Page_13.txt
5a226864ccf84f72582e38473125cdb1
e2f2cfc5bf991672c8a392181c5fb23f0fc37d10
45017 F20101113_AABFDL ryu_m_Page_13.jpg
e832570884f4268b9d39facea52d25f0
eea5d1d298fcc2311137006fcc02cf157ae9ca26
2095 F20101113_AABFIJ ryu_m_Page_14.txt
84858ee909fcddf651d5eb934293331b
802570efa94508754e0bd7876a177fef81c1ca0a
72736 F20101113_AABFDM ryu_m_Page_14.jpg
cf39944dc2fb22f12d02fe3c6272792f
c2da6b05a59dcbe22e1c55efa7130ea6d6e3aea5
78611 F20101113_AABFDN ryu_m_Page_15.jpg
3ca5871332ac62a4f612356b9104d414
3284a5f95753323a46f14ab8ec3b93de1ba5360e
2164 F20101113_AABFIK ryu_m_Page_15.txt
d3b4af64ea24f35392316724dac8bdd6
731fb259b995972501e6c60103a7d0b717756683
77475 F20101113_AABFDO ryu_m_Page_16.jpg
75e7b612b94b4c50edadab9d43986c09
eb932e819843e4f8623e171a2cac498e54d9d5ea
2214 F20101113_AABFIL ryu_m_Page_18.txt
ae0ecc37cd0b5daf5628ea132d0b8a06
759ad29b105687fe944104703e3d1a328134efd6
78046 F20101113_AABFDP ryu_m_Page_17.jpg
9d86a87c8a621cd2d5eb95cb1c0378e6
6a13b7863d8bf39361f74e119eb4d703edf4a3f8
1927 F20101113_AABFIM ryu_m_Page_19.txt
713ef5a44118307ad0eba13abad68c36
7427457989c89e5c8cca0dc85259edd8f1510d74
78591 F20101113_AABFDQ ryu_m_Page_18.jpg
e4c0eb3f52a689c6d338d66ef5c25049
ecae606b4df4b2750ada58d305cceb5827e44283
2159 F20101113_AABFIN ryu_m_Page_23.txt
a0cfd7e92a7928935bc04404bbb6ad88
8b4b6cd44f58250e7c780eb604896bbea71f4104
71029 F20101113_AABFDR ryu_m_Page_19.jpg
9e3d94ae395a1bb42ff195c5682a580b
9c890a1570dde82db53ed7be3aa42439e57e4e09
2133 F20101113_AABFIO ryu_m_Page_24.txt
ea88a5a2d1c0d5e5df9478defba010d3
388fb20dd14e9083ff59d511017ae460ccba379c
76460 F20101113_AABFDS ryu_m_Page_20.jpg
1934b8dae5d18470e79a309eb6d6575e
7e9c32f9e8a0a85c523dacef8b185f7d26c0d3a4
1657 F20101113_AABFIP ryu_m_Page_25.txt
f8128b2d567cf818ffd4ec0458437f3a
115bd38a7dd008152ad71277cbeb2cae909560b9
72567 F20101113_AABFDT ryu_m_Page_21.jpg
4580f1af53582b8f72fb34d1e1414c1f
b6b668d3655d7c9cd8900873d3c54a59a7095866
542 F20101113_AABFIQ ryu_m_Page_26.txt
453ccdd15daa5c1ce255c42943b01b83
2451943f970ad4606beea8dc64d638e433bef2a8
75957 F20101113_AABFDU ryu_m_Page_22.jpg
4d01834c5ef5766492b9ebcd0e02c447
756b16e479dca8309ea94964011fd94b4f0313fa
2225 F20101113_AABFIR ryu_m_Page_28.txt
7eb68c5a335ce07788e8fd3051d5da82
329215dc02ad739e66065666770d806647c37297
77104 F20101113_AABFDV ryu_m_Page_24.jpg
03638f195556f42c285877a67b9c7066
06e5117b43455b8c46501fb117a616c95ed5e240
2146 F20101113_AABFIS ryu_m_Page_29.txt
88f061c7bbfc5496a63153ed9fcac468
546c4e75e7647ff22ea0fa7c32a80429ed3ef846
63547 F20101113_AABFDW ryu_m_Page_25.jpg
9759574c7802adbf94ce7333154728dc
08385928dcc4910f950fe1b197e88af98d7fd0ab
1729 F20101113_AABFIT ryu_m_Page_30.txt
98996c604175e18f4de273fb7571850c
d5196a6160e889bcc1ca09d1d641cbe7cd1daaab
385 F20101113_AABFIU ryu_m_Page_32.txt
4865cc933c4d4947fc4fc95b30f8dfe3
355c03d13fb1abee25af40ede96ac930aef8f9c9
29148 F20101113_AABFDX ryu_m_Page_26.jpg
d347a0767f20a718fa392fc7cae5541d
b1c81edaf02f87e3689246176636647a821ed828
1117 F20101113_AABFIV ryu_m_Page_33.txt
0671d8ae46455d5ee57b14002773c6f1
772b6ff22df43535e3c7a1dc4da9576c7ddc6b8d
18328 F20101113_AABFBA ryu_m_Page_45.QC.jpg
f5b76eeb1f559ea47469f520ac7bbbdc
231bc7008e44f7a7ca1cb27fdc93b82ef6703031
71300 F20101113_AABFDY ryu_m_Page_27.jpg
eab770a50956cb5abd4e89b6720393a4
4a122d4bb374acf2c1861d35ca2ecae61f43337d
1415 F20101113_AABFIW ryu_m_Page_34.txt
890eb4fc7a3104ea50ec901da4f91bc3
ce3782091db3714ac07f894a48aa5edfdaf26720
614426 F20101113_AABEZA ryu_m_Page_35.jp2
b499a44926d7d460bb4aedeb948622d4
7603852dcb76ae370f67fea3a40340421b3e8fd3
114723 F20101113_AABFBB ryu_m_Page_12.jp2
a4d921a4b4e77b227bd9e7f86aea6a4b
4d90ed0d454d45a63fb12fb5035c7ac216d77afe
80797 F20101113_AABFDZ ryu_m_Page_28.jpg
5242d9e130e23c657854801faba73d5a
70bc4a488cfe033e409970fd8fdbd5967cecefab
953 F20101113_AABFIX ryu_m_Page_35.txt
770b9b5268e4dcd8586c6ff6013b1794
4b17fca48920b751d7dc1dbe2e8f3a558c735dc4
20514 F20101113_AABEZB ryu_m_Page_50.pro
cf22b16fba4de955d770ea5269e856e3
95f3eb481b177c57e9a6bde7d7eee42a07d7e4e3
7559 F20101113_AABFBC ryu_m_Page_01.QC.jpg
9fbf2ed988589b3a21393d7c39b5239e
faf35ad358d037d7a47293b66e11c655a7c14ee1
F20101113_AABFGA ryu_m_Page_06.tif
f6865e3866b4d2bb80087ea68e3d471f
b1c988e3f3c52c96eacec906eba4c5a8ef6de4ac
1793 F20101113_AABFIY ryu_m_Page_37.txt
d334d95045832828157caaf0222d7ee3
abf79f31966b34bd7546dbe7bbd75bb1a562d58c
26213 F20101113_AABEZC ryu_m_Page_47.QC.jpg
1c40705d783ad4d4d06ca441d2f45165
a824b9615a55ab777e3a8eb98a5c0e58169589a4
F20101113_AABFGB ryu_m_Page_08.tif
42107956ff84fe9b7bbceb0f70652c8d
3b8f8b6fa608b133528e67e22fd7cd51d4cab5df
2173 F20101113_AABFIZ ryu_m_Page_38.txt
17cda4d9b8712cbeb8cbddfb59f8526c
e167fe6c79bccc2819a2c5e8eb9d79f675198516
24849 F20101113_AABEZD ryu_m_Page_01.jpg
0ac041dfaf9078b2dbff9cc067447a9f
b26488719045b84608f5398c17c8f79b382afd66
2043 F20101113_AABFBD ryu_m_Page_21.txt
1789b55bc2190cffea09c696b5de92fb
4b869d15d4b20a4ca34cc86cdc14f381149fe36e
F20101113_AABFGC ryu_m_Page_09.tif
3d560fb2375cd9a44d18b366eb1245e8
53dce4e4284f97dfc730226e760247c72d55d715
79674 F20101113_AABEZE ryu_m_Page_23.jpg
003fe140f8a47597a06face762cb784c
c5f444f8f3641790657a419ba554b025cfc574f6
116474 F20101113_AABFBE ryu_m_Page_24.jp2
6eec8826a36d32a1c54872762921e045
7107cd7a04fd53df3cf3c8648d4202f9d6f45c90
F20101113_AABFGD ryu_m_Page_13.tif
103a1019b666a6115d542fec5c678d11
8b519294c0c591cf6fd24be96cea53a4b6b9be05
2199 F20101113_AABEZF ryu_m_Page_17.txt
4cbeb35aeb43e6185d4dab20dfab57dc
4c6b02aef8c2ab1c04c3c950886dfac6bb8c6298
53762 F20101113_AABFBF ryu_m_Page_11.pro
ebc7ac145d0a9db2d3f63e9591ed5c10
1de675b8d64ac1b5ce8c5ea382f6c0f849f455fe
4372 F20101113_AABFLA ryu_m_Page_35thm.jpg
27181b4531498768d36392d51313410a
badb1a9e60946664800cf1d745579955c38f7292
F20101113_AABFGE ryu_m_Page_14.tif
7ce34c48ed8d3ab381c552ad1088e19d
e14b356d4e45437c0c4a254be2c262beaf1fa384
19234 F20101113_AABEZG ryu_m_Page_33.pro
2b929dfa279005953143862b6726615c
542406a7492a4a5742e7c655db4774bb9b734ec0
55200 F20101113_AABFBG ryu_m_Page_17.pro
f5d7e745d78cea09ae49b01169fd9738
dd89ca6e4c92328f9aaf737a32a0a7686ab5a42d
17977 F20101113_AABFLB ryu_m_Page_37.QC.jpg
816de9299982e920064e4ea72dee7388
c2e0411f78de8e59d21aa4332c59e744f99bf31e
F20101113_AABFGF ryu_m_Page_15.tif
9b1dfc954e57295c3604f294cf468c9e
da0fecbb0ebf7ea01180bee02a3c34f9979fe51f
77371 F20101113_AABEZH ryu_m_Page_29.jpg
bab3259a1134961380e29368de97afb8
0d0ff6a3304128192d60a5ffed78a3ebe99b7a94
50387 F20101113_AABFBH ryu_m_Page_14.pro
fc5f4ddb4347bd2666f9b6996a0c786d
cb53049990842e776220130b6f3cfac707e25d2f
26388 F20101113_AABFLC ryu_m_Page_39.QC.jpg
8b12e66a1d5443d085f7110959944bb6
50b2fa6b8b0220376d99c15db0f27d9de67fb655
F20101113_AABFGG ryu_m_Page_17.tif
cfb8e376ccbe1abb88453e1cc18abc9b
fc2d236a20561a5a781c66fbf257fbb80ba6c28d
54432 F20101113_AABEZI ryu_m_Page_16.pro
b2887125e1721c28c908ac280bfbf85d
a5e87c0c9727e7d6ad7e1ca1ac3d08c1af1b77ae
49285 F20101113_AABFBI ryu_m_Page_08.jpg
6333deeea83a35a3c373c3ccb8840d12
311b100d786678b84f3cc1f3257c0e9874d0bb74
7038 F20101113_AABFLD ryu_m_Page_40thm.jpg
5a763291f1bd9daeefbb63bf9d8b6072
1bf69429c4cf4548a32c7fb72b17f2584be97ca1
F20101113_AABFGH ryu_m_Page_19.tif
0df7ebc958ca7f93d8ffd89e632efab4
8b9c53029a983beeea5bda7d2529e9d2b91abd86
2361 F20101113_AABEZJ ryu_m_Page_32thm.jpg
c9ae2f9c7132b172b9d6b7332dfc9a09
1f6cb221a331a04d79f9278e567c861f36565ddf
2801 F20101113_AABFBJ ryu_m_Page_26thm.jpg
68a69bc8813d71fa6acd63b48c90a436
7513d830ef34018e233d1fcdd6cd241982f4a6f4
24986 F20101113_AABFLE ryu_m_Page_41.QC.jpg
d35b74e16ca0a4e8e1b76f71dde21a7c
94ffbfb135a875d229b2e8fe7bafacab54c0ce29
6968 F20101113_AABEZK ryu_m_Page_39thm.jpg
9e548b0134064f20910d300def3d140a
e303ecc7389ae28d82174798fd2f7477b42d3a80
F20101113_AABFBK ryu_m_Page_12.tif
8ac5a5ae7cb212599c781969b0639572
070b2724015abd78695fad71bf28d84602027224
6895 F20101113_AABFLF ryu_m_Page_42thm.jpg
2a7fc83513d37c22b11ccb1df2ec7212
bad305ed5fd10041da3a0ae23e081b860ab08788
F20101113_AABFGI ryu_m_Page_20.tif
5e3a5b94ba0bcbd55e197b16ea77e8c7
b589299b3c3b0655ee796c052db22b952448dc54
56797 F20101113_AABEZL ryu_m_Page_39.pro
598538d96e2143a8c3a5e49fca6029ec
ae518fd4f41effa9fa476cc17d15c68862a98690
1051985 F20101113_AABFBL ryu_m_Page_08.jp2
ca84eaf0d261d1165f237d735e01a20e
9123355daf01c63c6d347fd821941740345bd215
25621 F20101113_AABFLG ryu_m_Page_43.QC.jpg
9c5051c3b74933265110d88f02fee9e2
40166efed1bcee9b216f42d1c3054dceca775690
F20101113_AABFGJ ryu_m_Page_21.tif
6af8a1193d4aec888cb38680d1675180
11721aaf7303506ef8ddc4f3f417aa363071fd43
12482 F20101113_AABEZM ryu_m_Page_31.QC.jpg
e9c7e798533b5869db9b0f390bf82fcf
4f59e0ed3b9afe24e3f361fee924d206c9c42d71
51966 F20101113_AABFBM ryu_m_Page_20.pro
5da1fdb28290a2e2d4e045c2ab228cfc
a8bafa3c7d827f51ba87c44ddd81198557fd661d
26189 F20101113_AABFLH ryu_m_Page_46.QC.jpg
0cae3ed52324ad267a0f04955d165ffe
0304a07b214c3ada8bea29ae2050363b2f8b16bc
F20101113_AABFGK ryu_m_Page_22.tif
ae07eada14d5c42629ce658030db3726
42e07e33b850628942c0e47b3e18fdb83bd9ba71
54256 F20101113_AABEZN ryu_m_Page_23.pro
5252d06e213af748cd0a3d2a7ecb7b98
4516c78d857a26be033b42d66ebce9b5d749e7e6
F20101113_AABFBN ryu_m_Page_10.tif
861e7ede9a94aeb08331136ab2e44b03
59a83f9a5c62b76216fddea7aa953e93325addf9
7030 F20101113_AABFLI ryu_m_Page_46thm.jpg
9cd9f2bcc84561d02c3f1348f9a715ee
ae9267b7ff8a721ed6f4c237d232f9e3e37306b5
F20101113_AABFGL ryu_m_Page_25.tif
2390c0ff90074f23307e55fea4e186cb
2a18fef0575644f721c0f636ae87507e98721840
82941 F20101113_AABEZO ryu_m_Page_46.jpg
78922b515eff9cb4e8c7d63f4df0e3d2
bd5c7a1e8f9786350beefb8b14c44c1ce4b62aa6
113014 F20101113_AABFBO ryu_m_Page_22.jp2
5a07c79d9394b5a558096c8c8e5cc460
83c36c38b3185f21ea3f1190acb25bc1d5003a05
6873 F20101113_AABFLJ ryu_m_Page_47thm.jpg
fb7f76235624cd357e2d49de25ddc6df
a8db81edb3e1d22dd5656e1842dade9ae2b01a8a
F20101113_AABFGM ryu_m_Page_26.tif
8396447941cd2783fe0bc2833d01be32
fed19e6a5b499f5cacbbfb713b557d09fc4f8a63
120779 F20101113_AABEZP ryu_m_Page_39.jp2
9153aea40799957a375327a4105fa44d
949980480ad1ca55c3442961778a21d5cf73e2b3
2153 F20101113_AABFBP ryu_m_Page_42.txt
31247a72898a1f616df8380a9cff7b5f
3f7af42132a1e99d2634762260e8f85e0da2ff81
26555 F20101113_AABFLK ryu_m_Page_48.QC.jpg
39a190edb4b54b1b658f0d260653f1dc
37695e2a765ac944af6d01ca1b30ccb80e31dabb
F20101113_AABFGN ryu_m_Page_28.tif
5c2fe179744b7cc3e25666d945ee236d
ec5652974ef5aa300cfd10d366a1fc0ba828ca82
F20101113_AABEZQ ryu_m_Page_07.tif
be607ad2c31d98b0afb7dcd3e8ca24e4
b33bbb1ff13b3cdb3c6805f871d857b9b8c3033b
1447 F20101113_AABFBQ ryu_m_Page_44.txt
ea1117a969764a10195791dcc692350f
9969374423633efc252a4eb59390432288da6113
6989 F20101113_AABFLL ryu_m_Page_48thm.jpg
6515cfdec20d839fab5655d17e94c86a
d768b00a552bfb06a76cd1d27d9a287647f043db
F20101113_AABFGO ryu_m_Page_29.tif
0edbfd4af0c542a7b3cd7ed7b19aee67
cd841661eb1754ab9ac7cf4c8e92be631e66a40f
78454 F20101113_AABEZR ryu_m_Page_42.jpg
3a90bc1774f1a7cab24cb62bcd18f403
9650760e1daea02c3a4c54b725b8bfdd29431f73
25272 F20101113_AABFBR ryu_m_Page_24.QC.jpg
42ece91aeeaed33e7da62a015107f2ed
2ab7a60c607eb128f5a1a1f2518a48c5ecfeaa2e
3506 F20101113_AABFLM ryu_m_Page_50thm.jpg
8297be2af3f80b78f345e559dccd8f72
fe08d1c73f20faa374f8357018468d917dd9dabf
F20101113_AABFGP ryu_m_Page_30.tif
d9481de46e96af1072be3e6d68066244
019b955c3170965fa5ea252b0aa7b092e387a9a5
F20101113_AABEZS ryu_m_Page_40.tif
335269e3e74f7d00665ded57f2339ad3
1194db395e711b435cc63483191a34af58a33a8d
F20101113_AABFBS ryu_m_Page_03.tif
95a0f84630740a022d0a76929c334a88
ec746ea950a77b94129d84ed897dfba6e77573d3
F20101113_AABFGQ ryu_m_Page_32.tif
f18a085a96fdf9053a84e0c46ca19a06
c3b64e51c2a15b31492935e70248013cc22c7362
F20101113_AABEZT ryu_m_Page_11.tif
c73baa9e1b22c12062a637713428e0db
117ad475239a3e610829a5a8f770821186de1b55
2157 F20101113_AABFBT ryu_m_Page_20.txt
fac28848858700f0fd6508122256baf0
02c9141c08f8e18c394e0797770d3c9fbea816df
F20101113_AABFGR ryu_m_Page_33.tif
effc67fb36bde8dc28be478afa0c0bdd
54737d4b2f9eeb9d0c901e713028bacef11e758f
36243 F20101113_AABEZU ryu_m_Page_26.jp2
52e58be64d6fb36cfefb7a0c480991ad
d942718f6cc960d5c8ce48c6222d4237a637bdde
6980 F20101113_AABFBU ryu_m_Page_49.QC.jpg
c23c2b2d0404aa9b99e1c7cd1758284d
94d846420a022d1921e103f358a66c3ddd4e29be
8423998 F20101113_AABFGS ryu_m_Page_34.tif
d0c051203a40f641e87b90f78dec6adf
9f724499fc3908e34fab2c5c8f2efa489c8079b5
50749 F20101113_AABEZV ryu_m_Page_04.jpg
19f02cc36c2510666b5779be11562140
4ef5567e544c2a0a95be4889231f2fdf0a284921
6954 F20101113_AABFBV ryu_m_Page_11thm.jpg
afc68b8a9581f46e0a5cd9017d9bc863
aed01fad462b1f048d44b59408799b9f60957a85
21614 F20101113_AABEZW ryu_m_Page_09.QC.jpg
83634a2d55f1beb073fc27ff12327980
fa130d0d92bbd58deabe3b14eb18e0df14f221f4
19813 F20101113_AABFBW ryu_m_Page_05.QC.jpg
8245ab8940557858461484cc44057c45
bac03707e8589bd38c6187f1f289f44f5097ebc3
F20101113_AABFGT ryu_m_Page_36.tif
deb535c5cef63d29f1e97f008d3d863b
cb42a93904384949b1181f05ac58ae3ccfb66fdb
11664 F20101113_AABEZX ryu_m_Page_49.pro
211d330983490359469377092d2eee27
a9ec2b7ec84f1bce6a8c0433ef41c5cadb1e7a99
853 F20101113_AABFBX ryu_m_Page_50.txt
ca0a78f3963d27989ffc9364eb8f6f91
a03d3d29c51600ea8ac2f99f361bbf7a2be0eb63
F20101113_AABFGU ryu_m_Page_37.tif
3fbf1fd08dc1a518be0cdc412fccca49
2df9ec19e031525ba2bce5948bceb528827d2796
70910 F20101113_AABEZY ryu_m_Page_09.jpg
573be8ffb7db68ea1ed1469c6f684528
e9849ab81b85dcedf3129a500bcab61451768aa2
5413 F20101113_AABFBY ryu_m_Page_45thm.jpg
fa03e10867f20649b5d8442d265e9d8e
2d850f4beba313eca52aa7fa362963c3105994fc
F20101113_AABFGV ryu_m_Page_38.tif
5dd56c5c81e77cf1e931d04425b5c41b
9c1b2a1617c24cf0e8033fcf078c888a09e37ad8
54689 F20101113_AABEZZ ryu_m_Page_15.pro
d142f8d29ad03c5ff5ebea1b14fef713
e8feaffd40f18892403a8ed80e143a8e9a0e27c1
24366 F20101113_AABFBZ ryu_m_Page_38.QC.jpg
109aea2f9e3316e80e94e2b70339cc3e
c598a842c2c158d78160028370187f5c24d2c88a
F20101113_AABFGW ryu_m_Page_42.tif
94661512fd0cea9fa017f3ab6137ab09
887244249c3d95fff393fa8069002f5ad2904c14
F20101113_AABFGX ryu_m_Page_43.tif
14e05d51d1c293fb08f893cb97fe9d83
248a7228776f35bdb8ec9aa6556c31b3b28e8489
65469 F20101113_AABFEA ryu_m_Page_30.jpg
df4a2cabe77d81fc30e664faa29d316a
5b471724af43bb6d868f2501130c58aea6370a22
F20101113_AABFGY ryu_m_Page_44.tif
c0f341b620fa84eed6fda2b90f404d2e
35bf35772abd13dae1ae99ccc8a7ca4f65e9db1d
41469 F20101113_AABFEB ryu_m_Page_31.jpg
7278961d0a8acad2dc1c30532620d3bb
3e2797d57c7013a963843efd63c80c6c309fdbd9
F20101113_AABFGZ ryu_m_Page_45.tif
664f86446a2b9ac39213fb83d364e473
14fa948c822a249f9a0c91a0eb9b437de7aa26fd
58431 F20101113_AABFEC ryu_m_Page_34.jpg
4818e7a81713f372bc1bab96bca9a65b
6e2c3f06eaa36b6580625a3e4088ae48c5b0c763
2208 F20101113_AABFJA ryu_m_Page_40.txt
bc91993a6cdd2b7c5b397c5d2faf2be7
896bbbe262b6dbdd24d96bb958618441f6c6e9e2
35737 F20101113_AABFED ryu_m_Page_36.jpg
860ccc3501e66bed319111cadd43e3f9
d56eae70390cd173149e43cbc4994add57fa7056
1441 F20101113_AABFJB ryu_m_Page_45.txt
2ab0fd21fdef3f17f2704148125876f9
52d799b97e436ee4075ab203a631eac930f58df8
63565 F20101113_AABFEE ryu_m_Page_37.jpg
946b1007e7fcbd4abd3c318310dfd478
0e288237277feedf5ad08798738d0757d914989a
2424 F20101113_AABFJC ryu_m_Page_46.txt
12ff01bfb6c6295ea48515b95b4d433a
b63d82d54ea08bff62a65ca5a7f010cd75b9dc49
76027 F20101113_AABFEF ryu_m_Page_38.jpg
efacb40940fa7819040323933cb7bde6
6781d231783e7479cdfe802233e009dd089bfad9
2567 F20101113_AABFJD ryu_m_Page_48.txt
fa3d406e987efa7cd6458ab2d296259c
c3432516d143898ee512c40f650c6efe705e5bd8
481 F20101113_AABFJE ryu_m_Page_49.txt
5cdf9c0135729e8f9e674b05ed6678cf
028ddbd58e2a82e56004f3395cfc82c64d442cb0
79884 F20101113_AABFEG ryu_m_Page_39.jpg
976685ad4ab1edff20522ff5a5c52485
6aee86ab8fb7ad0a3a234aba53c689180ad2e470
2221 F20101113_AABFJF ryu_m_Page_01thm.jpg
ffcbb82c701c364959f2899fca137779
a043fd368c5bf065035acefd89a65be3fdf7adaa
80695 F20101113_AABFEH ryu_m_Page_40.jpg
20b06a92eba55555a013965bf77483af
a1eb1a989c3b261734ba14cc2116e263c36b3ca1
868976 F20101113_AABFJG ryu_m.pdf
c9c007603bd5eb1e45371b7cf0505e5d
af03a7bbd7e8880cd20f7fba0eb8d8c7c8876336
77680 F20101113_AABFEI ryu_m_Page_43.jpg
44d2333a9ac5040d6e99c2e538324326
ee68c534e65e3343f441ac8d9c6cccaefab1986d
4616 F20101113_AABFJH ryu_m_Page_34thm.jpg
6396f090cb105cb77a285b17c898572b
793254f00295848e541d9cfe559afed9147045b1
54896 F20101113_AABFEJ ryu_m_Page_44.jpg
3113a0eb2a5f00f4b49645e1482eb4d8
842a62adf7335c136f15130efb92260aed8e1dc3
14661 F20101113_AABFJI ryu_m_Page_33.QC.jpg
02f588ec841ae3fac8ce5b6daaefb2df
4c6f9f3712ba756287814abf751deb7aac4b3ccd
61504 F20101113_AABFEK ryu_m_Page_45.jpg
dd8d8da2626f5aea06b6079465de851c
e7c005fcc72de38e1affb88d5a126d93a2b3da2b
26489 F20101113_AABFJJ ryu_m_Page_40.QC.jpg
d7adc9898126a2c38b87352ffe0432e1
79697f4972f2c7e1e68beb138e7274b090ba680a
90262 F20101113_AABFEL ryu_m_Page_47.jpg
a6d4c4c084521c2b7daaf90d64b96ced
d8defa4ceb8cb21c676a981ddf1b782ff795c5e2
6943 F20101113_AABFJK ryu_m_Page_20thm.jpg
a284721e1a77fe19dbdd08f954df0ca4
63e8e989c07155a1edd71b42fca48d60e4644115
90673 F20101113_AABFEM ryu_m_Page_48.jpg
4aec2ac406ca9557fda61ec1ba60b195
61fd8c6dadae105293c22210ac1e2b10bd2f5e5e
24798 F20101113_AABFEN ryu_m_Page_49.jpg
84bd25ae73d9e95cad44e08742e153c7
5b9b3a6ecfb2db604cf2ca7dbc80aece9f04f9e5
5051 F20101113_AABFJL ryu_m_Page_37thm.jpg
0dad9a7aa684e0eb66b57ff68bac38ff
15f55e276208ff1acf6f7e0461e28bae43c5badf
35532 F20101113_AABFEO ryu_m_Page_50.jpg
8601c4b1c88732100269d5c55b4e2b91
e96db1fb4b9d77bf895370f1f9585fd9217a3b7a
24263 F20101113_AABFJM ryu_m_Page_21.QC.jpg
e7c2adec8c6511173955d1a6c3efa7db
43b5efbf052f6c820a9b848f9df8ce1c2bdc1f51
12193 F20101113_AABFEP ryu_m_Page_03.jp2
c070300e9448536a20dc110f2a6def7b
fa2474ae70b77b4f71d3f99964ec37ce6b410a86
8445 F20101113_AABFJN ryu_m_Page_26.QC.jpg
75b78133ead79e65b460f7b5583f1f46
08b55a48de251f25ef71b47e9e1e4405e43b6c6c
71151 F20101113_AABFEQ ryu_m_Page_04.jp2
6ca457698a181ed085bb3f579d776a56
82cc844a4f8584a730d65c9121dff11d04957979
25755 F20101113_AABFJO ryu_m_Page_42.QC.jpg
dd6a96f7abe9662cfeb915fbf7c6777c
2ace394d50acd48dfa95e690bf4aee259e65e7e3
1051980 F20101113_AABFER ryu_m_Page_05.jp2
14a119b9d4cbef7c83961eb566d3fe98
4010120c3de57ce3b1623ce3d622bc7f487739b3
86085 F20101113_AABFES ryu_m_Page_10.jp2
22f2cd5484f486d60c7b68bf60fcedb1
73f71a37d3a289157358229ba121c9a34b4fddf5
3998 F20101113_AABFJP ryu_m_Page_06.QC.jpg
22c9f9b0ee025137e7246eb97e46aabe
a10a3f21f349a87e244031e2c56bf8bf46820588
115933 F20101113_AABFET ryu_m_Page_11.jp2
9bee24531f166488e47a8a8420fd41ca
09bc2402d816ae3581f81dca3a40f62fdd15184d
259713 F20101113_AABEXW ryu_m_Page_07.jp2
213c1d85cf850ad1e896cdafca94eda1
76b90ab375acd0835747deee8291a84a373521f5
1805 F20101113_AABFJQ ryu_m_Page_07thm.jpg
d3aa7be3f8c419b5536ff8c5ed918be6
1fb3878da01beb57acd409d3233c4c2a3619dd71
64416 F20101113_AABFEU ryu_m_Page_13.jp2
6916e7e853a4fc52c6e8f899cad62ca6
e1c6c72bee234d230444c846511aa684bfe0ee03
119448 F20101113_AABEXX ryu_m_Page_17.jp2
b9e459bd6a2c01c968df4a1088ebf4a3
2410eadd3baa1dd3b21407ae8481a799dc6e5341
3074 F20101113_AABFJR ryu_m_Page_02.QC.jpg
6b9a0f5b889367b3c75e1f8a71d0a005
168d134c9ba4d2f08703e4923abf7331e77b4bf5







ZINC TRANSPORTER EXPRESSION IN MATURE RED BLOOD CELLS AND
DIFFERENTIATING ERYTHROID PROGENITOR CELLS























By

MOON-SUHN RYU


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2007

































O 2007 Moon-Suhn Ryu


































To my grandfather, Hong-Ryol Ryu (1911-1995). The lessons from him have always inspired me
to pursue the delight to be wise with the right insights.









ACKNOWLEDGMENTS

First of all, I thank my beloved parents for their endless love and trust on me throughout

my life. Their guidance and support have been my greatest source of wisdom and confidence. I

also thank my younger sister, Jin-Suhn Ryu, for always sharing her happiness with me through

the sweetest and brightest stories from far away in South Korea. Additionally, I express my

gratitude to my wonderful lab mates, Juan P. Liuzzi, Louis A. Lichten, Liang Guo, Shou-mei

Chang, and Tolunay Beker Aydemir, for their encouragement and advices from the beginning

and the end of this study. The suggestions from their own experiences were always reliable and

helpful for me, especially, when encountering any factors that could have led me into frustration.

Finally, I render my deep appreciation to my supervisory committee members, Dr. Robert J.

Cousins, Dr. Mitchell D. Knutson, and Dr. David R. Allred, who shared their precious time to

give me the insights to move forward into the appropriate direction. Especially, I thank Dr.

Robert J. Cousins for being a great model for my future plans as a successful scientist and for

giving me the opportunity to continue my doctoral studies under his supervision.












TABLE OF CONTENTS


page

ACKNOWLEDGMENT S .............. ...............4.....


LI ST OF T ABLE S ................. ...............7................


LI ST OF FIGURE S .............. ...............8.....


AB S TRAC T ......_ ................. ............_........9


CHAPTER


1 INTRODUCTION ................. ...............11.......... ......


2 LITERATURE REVIEW ................. ...............14................


Erythrop oi esi s............... .. ... .. ..... ..... ....... .......... ...........1
In Vitro Models of Terminal Erythroid Differentiation ................. ................ ......... .15
Zinc Metabolism during Terminal Erythroid Differentiation ................. .......................16
Zinc Status and Anemia............... ...............17.

Erythroid Zinc Trafficking System ............._. ...._... ...............18...

3 MATERIALS AND METHODS .............. ...............20....


Preparation of RBC Membranes............... ........ ..........2
Production of Primary Erythroid Progenitor Cells ................. ...............20...............
Cell Culture............... ...............21
o-Dianisidine Staining .................... ...............2
RNA Isolation ............... ......... ...............__ .............2
cDNA Synthesis and Quantitative RT-PCR ...._ ......_____ .......___ ............2
Protein Isolation......................... ..........2
Affinity Purification of Antibodies ............_ ..... ..__ ...............24..
Western Analysis............... ...............24
Statistical Analysis............... ...............25

4 RE SULT S .............. ...............27....


Expression of Zinc Transporters in RBC Membranes ......____ ..... ... ._ ..........._....27
Confirmation of Erythroid Differentiation ................. ...............27................
Effects of EPO on Zipl10 and ZnT 1 Transcript Levels ...._._._._ ............. ......._.._.....2
Protein Expression of Zipl10 and ZnT 1 during Differentiation ................ ........_.. ........29
Effects of EPO on MT-1 and MTF-1 Transcript Levels .............. ...............30....

5 DI SCUS SSION ............ ............ ............... 8....











LIST OF REFRENCES .............. ...............46....

BIOGRAPHICAL SKETCH .............. ...............50....


































































6









LIST OF TABLES


Table page

3-1 Zinc transporters screened in erythrocyte ghosts and peptides used for the production
and affinity purification of antibodies against each respective protein. ............................26










LIST OF FIGURES


Figure page

4-1 Zinc transporter expression in mature red blood cells ................. ......... ................31

4-2 Induction of splenomegaly by phenylhydrazine-inj section ................. .......__. ..........32

4-3 Indicators of EPO-mediated terminal erythroid differentiation in vitro. ...........................33

4-4 Relative Zipl0 mRNA abundance during terminal erythroid differentiation. ................... 34

4-5 Relative ZnT1 mRNA abundance during terminal erythroid differentiation. ...................34

4-6 Zipl10 protein expression during terminal erythroid differentiation. ................ ...............35

4-7 ZnT 1 protein expression during terminal erythroid differentiation ................. ...............36

4-8 Relative MT-1 mRNA abundance during terminal erythroid differentiation.. ..................37

4-9 Relative MTF-1 mRNA abundance during terminal erythroid differentiation. .................37

5-1 Putative model for the contribution of erythroid zinc transporters to the homeostatic
regulation of zinc during terminal erythroid differentiation. .............. ....................4









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

ZINC TRANSPORTER EXPRESSION IN MATURE RED BLOOD CELLS AND
DIFFERENTIATING ERYTHROID PROGENITOR CELLS

By

MOON-SUHN RYU

August 2007

Chair: Robert J. Cousins
Maj or: Food Science and Human Nutrition

Animal and human studies have shown that the in vitro uptake rate of 65Zn by red blood

cells (RBCs) is inversely related to the subj ect' s zinc status. The capability of RBCs to take up

zinc may be a remnant of an earlier developmental stage of erythrocytes as zinc is essential for

the activities of several proteins formed during the erythroid differentiation, e.g., carbonic

anhydrase, Cu2+/Zn2+-Superoxide dismutase, and zinc finger transcription factors such as

erythroid Kriippel-like factor (EKLF) and GATA-1. Conversely, excessive intracellular free zinc

ions can interfere with incorporation of ferrous ions into heme at the final stage of erythroid

differentiation. Thus, intracellular zinc homeostasis during erythroid differentiation must be

tightly regulated.

To examine the hypothesis that the expression of zinc transporters would be involved in

the strategic mechanism of erythroid zinc homeostasis, transporters in the membrane fraction of

RBCs were screened by western analyses, and only Zipl0 and ZnT1 were detectable among the

zinc transporters tested. Erythroid progenitor cells were prepared from spleens of

phenylhydrazine (PHZ)-treated anemic mice for the characterization of transporter gene

expression during the terminal stage of erythropoiesis. Differentiation of cells into reticulocytes

was induced by erythropoietin (EPO)-treatment in vitro. Hemoglobin (Hb) synthesis and









expression of erythroid 6-aminolevulinic acid synthase (ALAS-2) mRNA were measured for the

confirmation of the ex vivo erythroid differentiation. Transcript levels of each transporter gene

and other genes associated with zinc metabolism were quantified by quantitative real-time PCR

(qRT-PCR). Temporal trends in expression of each gene were observed. Briefly, following

addition of EPO, Zipl0 mRNA levels peaked prior to the time-point when metal-responsive

transcription factor-1 (MTF-1) transcripts reached its first peak level, and decreased dramatically

afterwards. For ZnT1 mRNA, EPO-dependent expression was initiated later than Zipl0 and was

sustained until the experimental time-course was over. Metallothionein-1 (MT-1) transcript

abundance decreased rapidly after addition of EPO and stayed lower than the 0 h basal levels

until 48 h. Expression trends of Zipl0 and ZnT 1 were further confirmed by western blots

utilizing total cell lysates and membrane fractions of these cells.

This is the first study to determine which zinc transporters are expressed in the erythroid

system. The results presented here suggest that Zipl10 and ZnT1 expression is induced in

response to EPO. Furthermore, they could be the zinc transporters most directly involved in the

regulation of intracellular zinc homeostasis in differentiating erythroid progenitor cells and

circulating RBCs. The results may also be a route whereby RBCs accumulate excessive amounts

of zinc during malaria.









CHAPTER 1
INTTRODUCTION

Zinc is an essential element which is ubiquitously distributed in the human body, and its

deficiency is known to cause reduced growth, hypogonadism, visceromegaly, and hematological

abnormalities associated with iron deficiency anemia (1). The functional properties of zinc can

be categorized by its catalytic, structural and regulatory roles in numerous metabolic processes of

the biological system (2). Differential expression of zinc transporters is known to be involved in

the regulatory mechanism of the intracellular zinc homeostasis in various tissues and cell types

(3). There are two distinct gene families of zinc transporters composed of ten ZnT and fourteen

Zip transporter genes, respectively. ZnT proteins facilitate the removal of cytosolic free zinc

either by exporting through the plasma membrane or by sequestering zinc in vesicles, while the

Zip transporters function in an opposite manner as a pathway for zinc influx from plasma or

vesicles.

In mature red blood cells (RBCs), the zinc concentrations is about fifteen times larger than

that in the plasma (4), and more than 90% of that is known to function as a component essential

for the activity of zinc metalloenzymes, carbonic anhydrase and Cu2+/Zn2+-Superoxide dismutase

(5). Various routes of circulating RBCs whereby zinc influx occurs have been reported by

classical studies, of which suggested mechanisms involve the Cl-/HCO3- anion exchanger activity,

a neutral complex formation with thiocyanate, salicylate ions, and the chelation by amino acids

(6,7). The calcium-dependent zinc efflux by a Ca2+/Zn2+ exchanger has been considered as the

mechanism for the cellular zinc export from circulating RBCs (8). Additionally, there have been

animal and human studies with zinc defieient subj ects implying the expression of zinc-

responsive intrinsic factors involved in the RBC zinc transport system (9-11). In these studies,

RBCs from zinc defieient groups consistently revealed higher 65Zn uptake rates than those









collected from normal subj ects when cultured in conditions with identical zinc contents in vitro.

Even though the modulation of zinc uptake rate is likely to be influenced by the zinc transporter

expression, there have been no reports related to the determination of erythroid zinc transporters

so far.

Proteins involved in the zinc metabolism of mature RBCs are likely to be remnants from

earlier developmental stages as the capability for additional gene expression or protein

production is deprived by enucleation at the final step of erythropoiesis. A study showing

increased zinc uptake by the bone marrow during induced erythropoiesis in zinc deficiency

supports the necessity of minimal amount of zinc during erythroid differentiation (12). One of

the most well-studied features of zinc in erythroid differentiation is its incorporation into zinc

finger transcription factors which are responsible for the expression of essential proteins

involved in events of terminal erythroid maturation (13,14). Additionally, zinc metalloenzymes,

such as carbonic anhydrase and Cu2+/Zn2+-Superoxide dismutase, are produced during

erythropoiesis (15,16). Some clinical studies of zinc treatment for anemia have shown that it can

reverse anemic symptoms by increasing the production of hemoglobin (Hb) and, consequently,

facilitate the formation of normal RBCs (17, 18). However, in converse, there have been reports

of sideroblastic anemia caused by zinc intoxication as well (19,20). This may be due to the

interference by excessive free zinc ions with incorporation of ferrous ions into protoporphyrin

during the heme biosynthetic pathway (21). Based on these findings, it seems critical for the

erythroid intracellular zinc level to be tightly regulated during late stage erythroid differentiation

so that any adverse effects introduced by inadequate or excessive zinc supply can be avoided.

With consideration of these aspects related to the erythroid zinc metabolism, this study was

designed upon the following hypotheses:










* Hypothesis 1: As in other tissues and cell types, certain ZnT and Zip proteins would be
expressed in mature red blood cells for the maintenance of zinc homeostasis during
circulation.

* Hypothesis 2: Zinc transporters detected in mature RBCs would be remnants from
preceding developmental stages. Thus, the expression of respective transporters would occur
during the EPO-mediated differentiation of late stage erythroid progenitor cells.

Consequently, the maj or aim of this study was to determine which zinc transporters may be

involved in the erythroid zinc trafficking system, and characterize the transporter expression

during RBC maturation. Initially, the zinc transporters expressed in circulating erythrocytes were

identified at the protein level. Thereafter, temporal trends of each respective transporter

expression in differentiating erythroid progenitor cells were determined. For a more

comprehensive understanding of the zinc metabolism during terminal erythroid differentiation,

mRNA levels of other zinc metabolism genes, M~T-1 and M~TF-1, were also measured in

differentiating cells.









CHAPTER 2
LITERATURE REVIEW

Erythropoiesis

Red blood cell (RBC) production initiates from a pluripotential hematopoietic stem cell

and sequential differentiation of each intermediate cell type in the hematopoietic hierarchy

depends on the activation by lineage-specific growth factors (22). Normally, the site of

erythropoiesis is the bone marrow and in specific conditions, such as anemic subjects and

embryos, the spleen and the liver becomes the major site of erythropoiesis, respectively (23).

Once pluripotential hematopoietic stem cells become erythroid progenitor cells after carrying out

several steps of erythropoiesis, they exclusively differentiate into RBCs as a response to a

specific glycoprotein termed erythropoietin (EPO) (24). Erythroid progenitor cells have been

classified into two types, the burst-forming unit-erythroid (BFU-E) and the colony-forming unit-

erythroid (CFU-E). Both types require EPO for further differentiation; however, the BFU-E

requires other growth factors such as interleukin-3 or granulocyte-macrophage colony

stimulating factor (GM-CSF) in addition to EPO, while the CFU-E does not (24).

During the terminal stage of erythropoiesis, i.e., CFU-E differentiation, critical events for

normal RBC formation occur. For instance, hemoglobin (Hb) biosynthesis and enucleation

happen as a response to EPO-induction, and characterize the unique properties of erythrocytes

(24). Most adverse effects of nutrient deficiencies or toxicities-related to anemia occur during

this stage of RBC production. For example, in iron deficiency, an inadequate supply of ferrous

ions yields hypochromic anemia by introducing an increased zinc/iron ratio in erythroblasts

whi ch l ead s the ferrochel atas e-faci litate d reacti on to pro duce zinc protop orphyri n rather than the

essential component ofHb, heme (25). Vitamin B6 deficiency results in sideroblastic anemia









because pyridoxal 5-phosphate is a cofactor for erythoid 6-aminolevulinic acid synthase (ALAS-

2) activity (26).

In Vitro Models of Terminal Erythroid Differentiation

As normal RBC formation depends on the sequential events during the terminal stage of

erythropoiesis, several ex vivo models representing this step have been developed for

hematological studies. For instance, splenocytes and bone marrow cells from phenylhydrazine

(PHZ)-treated or Friend virus (FVA)-infected anemic animal models, and erythroleukemia

(1VEL) cell lines have been commonly used (23,27). Because of the absence of nucleus in mature

RBCs, the use of these systems is necessary especially in approaches for the characterization of

erythroid gene expression and the understanding functions of the respective proteins. Hodges et

al. suggested that erythroblasts from PHZ-treated anemic mice show the highest homology to the

in vivo erythroid system by evaluating the responsiveness of several erythroid specific genes in

three different types of in vitro models after EPO-induction (27). In this cell model, sequential

trends in expression of each gene were detected and the time-points of maximum mRNA

abundance were shown to be dependent on the metabolic needs of the relevant protein activity

during erythroid maturation.

Not only reliable ex vivo systems but also protocols for the confirmation of terminal

erythroid differentiation are well-developed. Staining Hb, of which peroxidase activity yields a

brownish-red product in staining solutions with 0-dianisidine or benzidine, has been used as a

classical method for erythroid differentiation assessment (27,28). Evaluating the expression of

genes only induced in differentiating erythroids, such as ALAS-2 (27,29), is another reliable and

also safer way to determine the differentiation status. This is especially relevant when the

carcinogenic risks of the above reagents are concerned (US Department of Health and Human

Services (DHHS), National Institute for Occupational Safety and Health (NIOSH) Publication









No. 81-106). Additionally, morphological changes of the cells, such as smaller size and nucleus

extrusion, are markers of the terminal stage of differentiation (27).

Zinc Metabolism during Terminal Erythroid Differentiation

Zinc is required for the activities of several proteins related to erythroid differentiation.

One of the most apparent biochemical properties of erythroid zinc is based on its role as a key

structural component of the zinc Einger proteins. Since the CFU-Es are committed to differentiate

into mature erythrocytes by EPO-induction, an EPO-responsive zinc finger transcription factor,

GATA-1, of which binding sites are located in all erythroid-specific genes, initiates its critical

role as the coordinator of multiple events composing the differentiation process by regulating

relevant gene expressions (14, 15). Another red cell-specific zinc finger transcription factor,

erythroid Kruippel-like factor (EKLF), is essential for the transcriptional induction of genes

encoding P-globin, which with heme composes Hb, and ferrochelatase, which facilitates the

incorporation of ferrous ions into protoporphyrin as the final step of heme synthesis (13).

Additionally, both of these zinc finger proteins are known to be involved in the transcription of

ALAS-2 and porphobilinogen deaminase (PBGD) genes (13,14). Their products are the rate-

limiting enzymes of the heme biosynthetic pathway.

The production of the zinc metalloenzyme, carbonic anhydrase (CA), which contains

around 87% of total RBC zinc content (5), occurs during the EPO-mediated terminal

differentiation as well (15). This enzyme is the second most abundant protein in mature

erythrocytes, after Hb, and requires zinc as an essential component for its catalytic activity. The

induction of CA expression during the differentiation of erythroleukemia cells precedes

hemoglobin synthesis (15), which may imply an increased requirement of zinc at the early stage

of terminal erythroid differentiation. Supporting these functional properties of erythroid zinc, a










study by Huber et al. showed that zinc uptake by the bone marrow of zinc-restricted rats

increased during induced-erythropoiesis (12).

Zinc Status and Anemia

Since a case of zinc deficiency associated-anemia was firstly reported by Prasad et al in

1961 (1), clinical studies on the value of zinc supplementation to anemic subj ects have been

performed. For instance, two consecutive studies by Nishiyama et al. were designed to determine

effects of zinc supplementation on anemic middle-aged or pregnant women (17, 18). Conclusive

benefits where shown when adequate iron intake was ensured. Briefly, the concentrations ofHb,

numbers ofRBCs and reticulocytes in anemic patients were significantly increased by the zinc

plus iron treatment, while the values from the other treatment groups, i.e., treated exclusively

with zinc or iron, did not change. In addition, this research group also suggested that normocytic

anemia with low total iron binding capacity (TIBC), which is inversely related to the transferring

saturation status and thus implicates the status of iron deficiency, may serve as an indicator of

zinc deficiency (17).

While zinc supplementation has been reported to have beneficial effects on overcoming

anemic symptoms, adverse effects leading hematological abnormalities introduced by excessive

zinc treatment have been observed as well. Sideroblastic anemia associated with excessive and

prolonged intake of oral zinc in human has been notified by several case reports (19,20). In

addition, increased frequency of abnormal and/or immature erythrocytes in the blood stream has

been observed in zinc intoxicated rats, mallards, and carps (30-32). Although secondary copper

deficiency has been considered as the major reason of these symptoms, Bloomer et al. suggested

that anemia introduced by excessive zinc may be attributed to the increased formation of a

biologically inactive compound, zinc protoporphyrin (ZPP), instead of heme (21). Zinc strongly

competes with iron for ferrochelatase and its product, ZPP, can exert the feedback inhibition of









ALAS-2 produced by heme. Even a slight decrease in iron availability by increased zinc can

cause ZPP accumulation. Supporting this concept, increased formation of ZPP-globin by the

decreased erythroid iron/zinc ratio in the maturing erythrocytes is known to be the reason of

anemic symptoms during iron deficiency (25).

Erythroid Zinc Trafficking System

The intracellular zinc concentration in circulating RBCs is approximately fifteen times

larger than the plasma levels (4). Over 90% of total erythrocyte zinc attributes to the enzyme

activities of carbonic anhydrase and Cu2+/Zn2+-Superoxide dismutase as catalytic and structural

elements, respectively (5). Accordingly, an adequate amount of zinc supply would be essential

even after the erythroid maturation is accomplished. Supportively, impaired carbonic anhydrase

activities, which catalyze the reversible hydration and dehydration of carbon dioxide, have been

observed in subj ects with low zinc diets (33).

The differential and tissue specific expression of zinc transporters have been strongly

related to the homeostatic regulation of exchangeable systemic and cellular zinc pools in

biological systems (3). Zinc transporters are composed of two distinct gene families, ZnT and

Zip, of which proteins facilitate the decrease and increase of cytosolic zinc, respectively, by

expression across plasma or vesicle membranes. The zinc removal by ZnT occurs either by

exporting through the plasma membrane or by sequestering zinc in vesicles, while the influx by

Zip is mediated in the opposite manner. Even though the zinc trafficking system in various

tissues and cell types have been extensively studied during the past decade, little is known about

the specific pathways for ionic zinc transport across RBC membranes. Some classical studies

have suggested that the zinc uptake mechanisms would be mediated by interactions with other

biological compounds. For instance, facilitated diffusion of zinc by chelation with amino acids,

particularly L-histidine and L-cysteine, has been thought to be related the zinc influx in rat and









human RBCs (6). Additionally, two anion-dependent mechanisms related to the zinc influx

system in human erythrocytes were suggested (7). One of these requires bicarbonate ions which

induces the formation of a zinc-anion complex, [Zn(HCO3)2Cl]-, that can be taken up via the Cl-

/HCO3~ aniOn exchanger. The other one, involving thiocyanate or salicylate ions, has been

thought to be accomplished by forming a neutral zinc complex that migrates across the lipid

bilayer. With regard of pathways whereby RBC zinc efflux may occur, a calcium-dependent

mechanism through the Ca2+/Zn2+ exchanger is the only route speculated so far (8).

Despite absence of definitive evidences, it is likely that zinc transporters are involved in

the homeostatic regulation of erythroid zinc metabolism. Animal and human studies have shown

a modulation in the zinc trafficking rate of erythrocytes during zinc deficiency (9-1 1).

Specifically, increased in vitrO 65Zn uptake rates ofRBCs were observed in rat, sheep and human

subj ects when fed zinc-restricted diets. These results would imply the presence of a zinc-

responsive intrinsic factor that enables the homeostatic regulation of erythroid intracellular zinc.

In other words, the regulated activity of this factor would result in a higher zinc uptake rate

which would correct the cellular zinc loss introduce by the zinc-deprived conditions in vivo. This

aspect is quite consistent with the general properties of the homeostatic regulatory mechanism

mediated by zinc transporter expressions in other tissues (3). Consequently, it was of interest to

determine the transporter expressed in erythroid lineage cells. The purpose of the current study is

to identify of zinc transporter thought to be involved in erythroid zinc trafficking system, and

characterize the expression trends during the maturation of RBCs.









CHAPTER 3
MATERIALS AND METHODS

Preparation of RBC Membranes

Whole blood (~ 400 Cll/subj ect) was collected from anesthetized CD-1 mice through

cardiac puncture and aliquoted into EDTA-treated centrifuge tubes. Three volumes of wash

buffer [5 mM Sodium Phosphate (pH 7.4~8), 0. 15M NaCl] was added to the blood sample which

was then centrifuged at 2,000 x g x 10 min, 4oC. After carefully aspirating the supernatant and

buffy coat, the pellet was washed with 2 vol of wash buffer, extensively. Following the final

wash, the presence ofRBCs was confirmed by phase contrast microscopy. Isolated RBCs were

then lysed by washing with 3 vol of a hypotonic lysis buffer [5 mM Sodium Phosphate (pH

7.4~8) with protease inhibitor cocktail (Sigma, St. Louis, MO)]. After centrifugation at 12,000 x

g x 10 min, 4oC, the supernatant was aspirated. Then the tube was tilted and rotated for the

aspiration of the red button, which is beneath the ghost pellet and contains proteases. The cell

lysis step was repeated until the pellet lost its red color (3~7 times). Each final pellet was

dissolved in 200 Cll of storage buffer [5 mM Tris-HC1, 0.5% Triton X-100 with protease inhibitor

cocktail (Sigma)] for solubilization. Finally, the protein concentration of the red cell membrane

preparation was determined colorimetrically with the Dc Protein Assay kit (Bio-Rad, Hercules,

CA). Standards developed with diluted bovine serum albumin solutions and the absorbance was

read at 750 nm using a Beckman DU 640 spectrophotometer (Beckman, Fullerton, CA).

Production of Primary Erythroid Progenitor Cells

Primary erythroid progenitor cells were prepared from spleens of PHZ-treated anemic

mice. CD-1 mice were inj ected with PHZ hydrochloride (Sigma) in 0.9% saline (60 mg/kg bw)

intraperitoneally on day 1 and day 2. On day 5, the PHZ-treated mice were sacrificed and the

spleen was collected. Monocellular splenocyte suspensions were prepared by cutting the spleen









into small fragments in AMEM (Mediatech, Herndon, VA) containing 10% FBS and antibiotic

antimycotic solution (Sigma), and by passing the disrupted spleen through nylon mesh. The

viability and concentration of cells were determined by staining an aliquot of the cell suspension

with Trypan Blue solution (0.4%; Sigma).

Cell Culture

To induce further differentiation of the late stage erythroid progenitors, cells were

incubated with recombinant human EPO (ProSpec-Tany, Rehovot, Israel). The single spleen cell

suspension was diluted into 1.0 x 107 cells/ml with AMEM containing 10% FBS and

antibiotics/antimycotics for treatment. The treatment was conducted by a further 1:2 dilution

with an equal volume of fresh medium containing EPO as 10 IU/ml. With the final concentration

of cells and EPO as 5.0 x 106 CellS/ml and 5 IU/ml, respectively, cells were equally plated in 6-

well culture plates as 1.0 x 107 cells/well and were incubated for up to 48 h at 37oC in 5% CO2-

The control cell cultures were prepared in parallel with identical procedures except for using

EPO-absent medium at the final step of dilution.

o-D~ianisidine Staining

Differentiation of cells was confirmed by comparing the hemoglobin synthesis levels of

cells collected at 0 h and 48 h after EPO-treatment through 0-dianisidine staining. The staining

solution [0.1 mg/ml 0-dianisidine (Sigma D9154), 0.2% (v/v) SDS, 0.3% (v/v) hydrogen

peroxide in PBS] was prepared freshly for each time-point. 500 Cll of cell suspension was washed

with 500 Cll of sterile PBS by centrifugation at 250 x g x 5 min, 4oC. The supernatant was

carefully aspirated and the final pellet was resuspended with 500 Cll of the staining solution.

After incubation at room temperature for 30 min, cells were spun down and washed with 500 Cll

of PB S. Cell suspensions were transferred to wells of a Lab-Tek Permanox Chamber Slide









(Nunc, Rochester, NY) and the staining was examined by light microscopy with an Axiovert

microscope (Zeiss, Oberkochen, Germany).

RNA Isolation

Cells were harvested at certain time-points covering the whole 48 h differentiation. The

time-course was designed to determine the temporal trends in changes of each mRNA level

throughout differentiation and detect the time-point at which maximum EPO-responsiveness

exists. After centrifugation at 400 x g x 5 min, 4oC, cell pellets were resuspended with 1 ml of

TRlzol reagent (Invitrogen, Carlsbad, CA) and stored at -80oC until the entire time-course was

over. When all samples were collected, cell lysates were incubated at room temperature for 5

min after thoroughly thawed. After adding 200 Cll of chloroform, each sample were centrifuged

at 12,000 x g x 10 min, 4oC and the top clear aqueous layer was transferred to a new sterile tube.

The aqueous phase was incubated with 500 Cll of isopropyl alcohol at room temperature for 10

min and centrifuged at 12,000 x g x 15 min, 4oC for RNA precipitation. The supernatant was

discarded and the RNA pellet was washed with 1 ml of 75% (v/v) ethanol by centrifuging at

12,000 x g x 10 min, 4oC. After repeating the wash step at least twice, ethanol was removed and

dried out from the RNA pellet at room temperature. Finally, the RNA pellet was resuspended

with 50 Cll of nuclease-free water and incubated in 370C for complete dissolve. RNA solutions

were treated with DNase I (Ambion, Austin, TX) to avoid any DNA contamination and the

concentrations were determined spectrophotometrically (NanoDrop Technologies, Wilmington,

DE).

cDNA Synthesis and Quantitative RT-PCR

Relative mRNA levels of ALAS-2, Zipl0, ZnT1, MT-1 and MTF-1 were measured by

quantitative real-time PCR (qRT-PCR). Primers for mouse ALAS-2 gene were designed with

PRIMER EXPRESS V2.0 (Applied Biosystems, Foster City, CA) while those for others were









available from previous studies of our lab. Primers for the PCR amplication of ALAS-2 cDNA

were: forward primer, 5 '-CAGAGGGCAGCTCCAGAAGTT-3 '; reverse primer, 5'-

GCTTCGGGTGGTTGAATCC-3 '. cDNA was generated by reverse transcription of total RNA

(100 ng/reaction) using the high capacity cDNA archive kit (Applied Biosystems), and 1:25

diluted to be used as templates of each PCR reaction. The cDNA product of the total RNA

collected at 0 h was used as the standard after 1:2 dilution and further 1:10 serial dilutions. All

qRT-PCR assays were conducted with the Power SYBR Green Master Mix (Applied

Biosystems) and amplification products were fluorometrically measured by the iCycler RT-PCR

detection system (Bio-Rad). The specificity of each primer pair was confirmed after each qRT-

PCR assay by melting curve analyses and the relative quantity was determined by normalization

with respective 18s rRNA values.

Protein Isolation

Cells were treated and cultured as described above. After designated time-points of

incubation, cells were spun down at 400 x g x 7 min, 4oC, and the supernatant was aspirated

carefully. Total cell lysates were prepared by dissolving the cell pellets (~ 1.0 x 107 cells) with

100 Cll of SDS cell lysis buffer [10% (w/v) SDS, 10 mM EDTA, 50 mM Tris-HC1, pH 8.0 with

protease inhibitor cocktail (Sigma)] and following homogenization with brief sonication. Total

membrane fractions of the erythroid splenocytes were prepared by utilizing a commercial

membrane protein extraction kit from BioVision (Mountain View, CA). Cell pellets (~ 9.0 x 107

cells), collected after washing with PBS, were homogenized with a Dounce cell disrupter using

100 up/down strokes in the homogenization buffer provided. After spinning down the

homogenate at 700 x g x 10 min, the supernatant was then further centrifuged at 1000 x g x 30

min, 4oC. The final total cellular membrane protein was dissolved in 50 Cll of solubilization

buffer [5 mM Tris-HC1, 0.5% Triton X-100 with protease inhibitor cocktail (Sigma)] and stored









at -80oC. The protein concentrations were determined colorimetrically with the Dc Protein

Assay kit (Bio-Rad).

Affinity Purification of Antibodies

Rabbits were inj ected with respective antigenic peptides for production of polyclonal

antibody to each Zip and ZnT transporter for previous studies (Table 3-1). Total IgG was

prepared from serum using the Montage Antibody Purification PROSEP-A Kit (Millipore).

Columns for affinity chromatography of each antibody were generated by immobilizing peptide

to a Sulfo-link Coupling Gel (Pierce) bed. The cysteine added to the C-terminus allowed the

peptide to conjugate to the Sulfo-link matrix during this process. 15~20 mg of total IgG was then

incubated in the column overnight at 4oC, and purified by passing Elution Buffer (Pierce)

through the column. The fractions (1 ml) with the highest protein concentrations as determined

spectrophotometrically (NanoDrop Technologies) were pooled. Finally, the affinity purified IgG

solution was desalted into TBS containing 0.02% sodium azide and stored at 4oC.

Western Analysis

Forty Clg of each protein was denatured in a 2x denaturing buffer [125 mM Tris-HC1, pH

6.8, 4% (w/v) SDS, 20% (v/v) glycerol, 10% (v/v) 2-mercaptoethanol, 0.02% (w/v) bromophenol

blue] for 15 min at 55oC. Denatured proteins were separated by 10% sodium dodecyl sulfate-

polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose (Schleicher

und Schuell, Dassel, Germany) or polyvinylidene difluoride membrane (PVDF) (Immobilon;

Millipore, Bedford, MA). Protein transfer and equal protein loading were confirmed by Ponceau

Red staining. After washing out the stain with TB S, membranes were blocked in 5% skim milk

(0.5 g skim milk in 10 ml TBS-T) for 50 min on orbital shaker in room temperature. PVDF

membranes were treated with methanol and Milli-Q water until they were completely wet before

blocking. Blocked membranes were incubated in primary antibody solution (10 Clg IgG/ml of









blocker or 1-2 Clg affinity-purified IgG/ml ofblocker) for additional 1 h and washed with TBS-T

4 times for 5 min each. Membranes were then incubated with anti-rabbit IgG secondary antibody

(1:2000 of antibody in 5% skim milk) conjugated to horseradish peroxidase (GE Healthcare,

Buckinghamshire, UK) or alkaline phosphatase (Amersham Biosciences, Arlington Heights, 1L)

for 50 min. Thereafter, membranes were washed twice with TBS-T and TBS for 5 min each.

Membranes probed with horseradish peroxidase-conjugated antibodies were treated with an

enhanced chemiluminescent substrate (SuperSignal WestPico; Pierce, Rockford, IL), and

exposed to autoradiographic film for detection. Blots treated with alkaline phosphatase-

conjugated antibodies were incubated with ECF chemifluorescent substrate (Amersham

Biosciences) for visualization with a PhosphorImager (Storm 840 Imager; Molecular Dynamics).

Specificity of bands was determined by comparing results to those obtained with primary

antibody preincubated with respective peptide solution (250 Clg peptide/ml of blocker).

Statistical Analysis

Data are expressed as mean + standard deviation and were analyzed by two-way ANOVA

with time and EPO treatment as independent variables. Differences between each treatment

group at respective time-points were determined by a following Bonferroni post-test (Prism 5 for

Windows; GraphPad Software, San Diego, CA). The level of statistical significance was set at P

< 0.05, and differences are annotated as follows: *, P < 0.05; **, P <0.01; ***, P < 0.001.









Table 3-1. Zinc transporters screened in erythrocyte ghosts and peptides used for the production
and affinity purification of antibodies against each respective protein.
Transporter Peptide
ZnT1 CGTRPQVHSGKE
ZnT2 CHAQKDSGSHP
ZnT4 CMQLIPGS SSKWEE
ZnT5 CSPSKRGQKGTLI
ZnT6 CVAPNVLNF SDHHVIP
Zip l CRSGANHEASASG
Zip2 CEEEWGGTHAFGFH
Zip3 CAGLRLRELGRPG
Zip4 CAEETPELLNPETRRL
Zip l0 CKRNHKCDPEKE
*Antigenic peptide sequences were designed by previous and current members of Robert J.
Cousins' lab.









CHAPTER 4
RESULTS

Expression of Zinc Transporters in RBC Membranes

According to the absence of a nucleus in circulating mature erythrocytes, the zinc

transporters expressed in RBCs was determined with an approach in protein levels. Membrane

fractions erythrocytee ghosts) were prepared directly from whole blood of adult male CD-1 mice.

Purity and presence of the RBC population after fractionation were confirmed by light

microscopy to eliminate effects of contamination by any other blood cells. Of the ZnT proteins

tested (Table 3-1), ZnT1 was the only zinc exporter detectable in the erythrocyte membrane

fraction. As the estimated molecular mass at 30 kDa was inconsistent with that reported by

previous studies (34,35), the specifieity of the affinity purified antibody to ZnT1 was further

confirmed by peptide competition. Signals on the blot were successfully competed out when the

antibody was preincubated with the ZnT1 peptide (Fig. 4-1A). Among the Zip proteins screened

(Table 4-1), only the presence of Zipl10 was confirmed with the expected band size suggested by

other research groups (40 kDa) (36,37). The reliability of the Zipl0 western analysis was also

validated by peptide competition, and the single prominent band signal was blocked by the Zipl0

peptide (Fig. 4-1B).

Confirmation of Erythroid Differentiation

Proteins in circulating RBCs are likely to be remnants of the protein components

synthesized in differentiating erythroid progenitor cells. For the characterization of the

responsiveness of the ZnT and Zip transporters, an in vitro model of late stage erythroid

progenitors prepared from splenocytes of PHZ-treated anemic CD-1 mice was utilized. Cells

were collected only when splenomegaly was observed as an indicator of PHZ-induced hemolytic

anemia (Fig. 4-2).









For the confirmation of erythroid maturation, erythroid splenocytes were incubated with 0-

dianisidine prior to and after EPO-stimulation for Hb-staining. An increase in the number of Hb-

positive cells was observed in the cell population incubated with EPO for 48 h (Fig. 4-3A and

Fig. 4-3B). The transcript abundance of an EPO-dependent erythroid specific gene, ALAS-2,

was also measured during the 48 h time-course as a more comprehensive positive marker of

terminal erythroid differentiation. A temporal pattern of ALAS-2 expression, consistent to that

reported by Hodges et al. (27), was detected in the EPO-treated cells. The maximum ALAS-2

mRNA abundance was observed at 24 h, with a 3-fold increase after EPO-stimulation. The

significantly higher levels in differentiating cells lasted until 48 h after addition of EPO (Fig. 4-

3 C).

Effects of EPO on Zipl0 and ZnT1 Transcript Levels

Temporal trends in transporter expression during the late stage erythroid differentiation

were determined by qRT-PCR. Each value was normalized to 18S rRNA and the basal levels

measured at 0 h of incubation. The transcript levels from cells cultured in the presence or

absence of EPO were compared at each time-point to determine the effect of EPO per se. Zipl10

mRNA levels in differentiating cells showed an approximately 2-fold increase after 6h of

incubation (Fig. 4-4). However, the levels were not sustained during subsequent periods and

values became less than 50% of the basal (0 h) levels after 24 h of EPO-treatment. Cells deprived

of EPO failed to show any increase in Zipl0 mRNA levels throughout the entire culture period.

The relative mRNA abundance in differentiating cells was significantly higher than that of

resting cells from the 6 h to 24 h post-incubation, while there was no significant difference

detectable at the following time-points. Expression trends of ZnT1 were distinctively different

with those of Zipl10 when cells were differentiating. A gradual increase of ZnT 1 transcripts was

observed in both differentiating and resting cells until 12 h (Fig. 4-5). However, as the incubation










period reached 24 h, the mRNA abundance in differentiating cells became significantly higher

than that in EPO-deprived cells, and the difference was sustained until the whole time-course

was accomplished. The maximum abundance was also observed at 24 h, with a 3-fold higher

level than the 0 h basal levels.

Protein Expression of Zipl0 and ZnT1 during Differentiation

The effects of EPO-induction on protein levels of Zipl10 and ZnT1 were initially

determined by western analyses with total cell lysates. Decreased Zipl0 protein abundance was

detected after 48 h (Fig. 4-6A and Fig. 4-6B) regardless of the presence or absence of EPO. Even

though the induction of Zipl0 expression by EPO was observed in mRNA levels at early time-

points (Fig. 4-4), these differences were barely detectable in protein levels with the total cell

lysates. Accordingly, a pilot experiment with total cell membrane fractions was conducted to

remove any potential compromising effects by excessive cytosolic proteins. Since the membrane

fraction in protein samples became more concentrated, the effects of EPO on Zipl10 protein

expression could be detected in a consistent manner to the mRNA data (Fig. 4-6C). An increase

in Zipl0 abundance was observed at 9 h by EPO-induction compared to splenocytes not treated

with EPO. The protein expression trend of ZnT1 in resting cells was quite similar to that of

Zipl10 by having a decreased expression at 48 h (Fig. 4-7). However, a different trend was

observed when cells were differentiating. The abundance of ZnT 1 was sustained throughout the

whole time-course by the EPO-mediated terminal erythroid differentiation. Consequently, a

prominent difference between the ZnT1 protein levels in the differentiating and resting cells was

detectable at the final time-point, 48 h (Fig. 4-7A). In agreement with the mRNA data, these

results indicate that the relative ZnT 1 protein levels to those of Zipl10 tend to be higher at the

very late stage of terminal erythroid differentiation than during the preceding periods.









Effects of EPO on MT-1 and MTF-1 Transcript Levels

The temporal expression patterns of two zinc responsive genes involved in zinc

metabolism gene expression were investigated. Little is known about the zinc metabolism during

terminal erythroid differentiation. The effects of EPO-induction on MT-1 mRNA abundance

were determined as this protein has been reported to be a sensitive indicator of the intracellular

zinc availability in various cell types (2). It was hypothesized that MT-1 mRNA abundance

would also be affected either directly or indirectly by the EPO-induced zinc transporter

expressions. Even though the MT-1 transcript levels in both cell groups decreased drastically and

were sustained lower than the basal level during the whole culture period, the relative abundance

in differentiating cells was significantly higher than in resting cells until 24 h of incubation (Fig.

4-8).

Zinc-finger transcription factor MTF-1 is involved in the transcriptional regulation of

numerous zinc-responsive genes including MT-1 and the expression of both Zipl10 and ZnT1. In

differentiating cells, MTF-1 mRNA abundance started to increase by 6 h of EPO-induction, and

then stayed relatively higher than those measured in resting cells (Fig. 4-9). It is of interest that

the expression trend of MTF-1 during differentiation revealed two peak levels, unlike trends

observed in other mRNA levels. Specifically, the mRNA levels reached its first peak at 12 h, and

a subsequent decrease at 18 h followed. These peaks in MTF-1 mRNA coincided with the

periods when the decrease of Zipl0 and the increase of ZnT1 mRNA levels occurred.























1111111111+- 30 kDa


IgG AP AP+Peptide









+- 40 kDa








IgG AP AP+Peptide

Figure 4-1. Zinc transporter expression in mature red blood cells. Erythrocyte ghosts were
prepared for western analyses of zinc transporters. Among the transporters (Zipl-4,
Zipl0, ZnT1-2, ZnT4-6) tested, only A) ZnT1 and B) Zipl0 expression were detected.
The membranes were incubated with either the total IgG, affinity-purified IgG (AP),
or AP that were pre-exposed to the corresponding ZnT1 or Zipl10 peptide. The
molecular mass of ZnT1 and Zipl0 at 30 and 40 kDa, respectively, were determined
with commercial molecular markers. There were no signals developed by antibodies
against other zinc transporters (data not shown).










I I
A B
Figure 4-2. Induction of splenomegaly by phenylhydrazine-inj section. CD-1 mice were treated
with or without PHZ by intraperitoneal inj section on day 1 and 2. Spleens were
collected at day 5. A) A normal spleen and B) an enlarged spleen from PHZ-inj ected
anemic mouse are compared.




























4.5
*** --EPO+
S3.5
E 3.0
2.5
~~2.0


1.0
0.5
0.0
0 6 12 18 24 30 36 42 48
Time (h)

Figure 4-3. Indicators of EPO-mediated terminal erythroid differentiation in vitro. Hemoglobin
staining of cells A) prior to and B) 48 h after EPO-treatment. C) Relative ALAS-2
mRNA abundance in EPO-treated and -deprived cells. Splenocytes were collected
from spleens of two PHZ-inj ected mice and pooled for culture at each experiment.
qRT-PCR assays were performed on duplicate total RNA samples. Values at each
time-point are relative to the basal levels at 0 h. Data are expressed as mean + SD of
four independent experiments (n = 4). Statistically significant differences between
each treatment group are annotated as ***, P < 0.001.












- --*- -EPO-
SEPO+


0 6 12 18 24
Time (h)


30 36 42 48


Figure 4-4. Relative Zipl0 mRNA abundance during terminal erythroid differentiation.
Splenocytes were collected from spleens of two PHZ-inj ected mice and pooled for
culture at each experiment. qRT-PCR assays were performed on duplicate total RNA
samples. Values at each time-point are relative to the basal levels at 0 h. Data are
expressed as mean & SD of four independent experiments (n = 4). Statistically
significant differences between each treatment group are annotated as *, P < 0.05;
***, P < 0.001.


- --*--- EPO-


EPO


0 6 12 18 24
Time (h)


30 36 42 48


Figure 4-5. Relative ZnT1 mRNA abundance during terminal erythroid differentiation.
Splenocytes were collected from spleens of two PHZ-inj ected mice and pooled for
culture at each experiment. qRT-PCR assays were performed on duplicate total RNA
samples. Values at each time-point are relative to the basal levels at 0 h. Data are
expressed as mean & SD of four independent experiments (n = 4). Statistically
significant differences between each treatment group are annotated as **, P <0.01;
***, P < 0.001.


~I~..----------I--------.I





+- 40 kDa


(EPO)
(h)


12


- + +
24 48


B




0 9 27 48 9 27 48


(h)
(EPO)


I- I _~


.40 kOa


Oh EPO-9h EPO+9h

Figure 4-6. Zipl0 protein expression during terminal erythroid differentiation. Cultured cells
were collected at designated time-points. A,B) Western analyses from two
experiments with total cell lysates reveal a decrease in Zipl0 protein expression at 48
h regardless of EPO-treatment. C) EPO-induced Zipl10 expression was only
detectable with total membrane fractions.l A band with estimated molecular mass as
40 kDa was consistently observed in independent experiments.













SResults from the total membrane fraction reflect a pilot experiment conducted (n=1). Further assessments would be
appropriate to affirm the data.















+ + + (EPO)
0 12 24 48 (h)


r~ c30 kDa
0 9 27 48 9 27 48 (h)
+ (EPO)

Figure 4-7. ZnT1 protein expression during terminal erythroid differentiation. Cultured cells
were collected at designated time-points. A,B) Western analyses from two
experiments with total cell lysates from EPO-treated cells imply a constitutive
expression of ZnT 1 during differentiation, while a decrease occurs at 48 h in EPO-
deprived conditions. Only the band with estimated molecular mass as 30 kDA was
consistently observed in independent experiments.












- --*- -EPO-
SEPO+


1.0

S0.8

S0.6

S0.4

0.2


0 6 12 18 24 30 36 42 48
Time (h)

Figure 4-8. Relative MT-1 mRNA abundance during terminal erythroid differentiation.
Splenocytes were collected from spleens of two PHZ-inj ected mice and pooled for
culture at each experiment. qRT-PCR assays were performed on duplicate total RNA
samples. Values at each time-point are relative to the basal levels at 0 h. Data are
expressed as mean & SD of four independent experiments (n = 4). Statistically
significant differences between each treatment group are annotated as ***, P < 0.001.


4.0
3.5 ** -* EPO-


~i
'E--...,........-- ---~-.......~~~--t


0 6 12 18 24
Time (h)


30 36 42 48


Figure 4-9. Relative MTF-1 mRNA abundance during terminal erythroid differentiation.
Splenocytes were collected from spleens of two PHZ-inj ected mice and pooled for
culture at each experiment. qRT-PCR assays were performed on duplicate total RNA
samples. Values at each time-point are relative to the basal levels at 0 h. Data are
expressed as mean & SD (n 2 x 2). 2 Statistically significant differences between
each treatment group are annotated as *, P < 0.05; ***, P < 0.001.


SSamples of the 18 h time-point, at which the fluctuation of MTF-1 mRNA levels was detected, were only available
from two experiments. Thus, the results are represented as mean + SD from n = biological duplicates x analytical
duplicates.









CHAPTER 5
DISCUSSION

Studies with regard of the zinc transport mechanism in various tissues and cell types have

revealed two distinct gene families related to ionic zinc trafficking pathway across cellular

plasma and vesicle membranes (3). Zip and ZnT proteins produced from these genes facilitate

the cytosolic zinc influx and efflux, respectively, and establish the mechanism for the

homeostatic regulation of intracellular zinc. Through the tissue-specific and differential

expression of these transporters, the cellular zinc trafficking system can be modulated in

response to various factors, such as the extracellular zinc availability, intracellular utilization,

and numerous cytokines, growth factors and hormones (3).

Previous studies have consistently reported the zinc-responsiveness of the zinc trafficking

system in circulating erythrocytes of animal and human subj ects (9-1 1). Even though these may

imply regulated transporter activities by dietary zinc, there has been no study to define the

presence of zinc transporters in circulating RBCs. Consequently, the primary purpose of this

study was to determine which transporters are expressed in mature RBCs. Each transporter was

screened at the protein level utilizing the library of antibodies to numerous zinc transporters,

available in our lab. The results from this experiment demonstrate that Zipl10 and ZnT1 are

expressed in circulating RBCs; thus, they are likely to be the zinc transporters directly involved

in the homeostatic regulation of erythroid zinc metabolism. Although the estimated molecular

mass of ZnT 1 in RBCs (~30 kDa) conflicts with the value calculated from the amino acid

composition (55 kDa), inconsistent molecular mass speculated from the migration by SDS-

PAGE analysis has been reported by other ZnT1 studies as well (34,35). Possible explanations

for the discrepancy in the aberrant migration of ZnT 1 are well-delineated in a previous study

utilizing the identical antibody for ZnT 1 detection (34).









One of the most unique characteristics of circulating erythrocytes, compared to other cell

types, is the absence of nucleus. In other words, the protein contents of mature cells are formed

during preceding developmental stages, i.e., erythropoiesis, and the gene expression ability is

deprived after maturation. Thus, the differential activity of the zinc trafficking system observed

in mature RBCs in response to the host' s zinc status (9-11) would be determined during the

differentiation stages of earlier erythroid cell precursors. It is of note that the expression of both

zinc transporters detected in mature RBC membranes have been suggested to be transcriptionally

regulated in a zinc-dependent manner by the zinc-responsive activity of MTF-1; however,

resulting in opposite modes (3). Even though further exploration is required to clarify these zinc

effects on the RBC zinc transporters, it can be suggested that the modulated erythroid zinc

uptake rate during zinc deficiency may be associated with the decreased DNA binding activity of

MTF-1 that results in either the up-regulation of Zipl0, down-regulation of ZnT 1, or both during

preceding erythroid developmental stages.

Among the available cellular models of terminal erythroid differentiation, splenocytes

from PHZ-treated and FVA-infected animals have been suggested to most accurately represent

the physiological aspects of in vivo erythroid progenitor cells (27). Accordingly, the PHZ model

was selected for the characterization of zinc transporter expression during the EPO-mediated

erythroid differentiation in the current study. EPO acts as a key factor for the initiation of further

differentiation of late stage erythroid progenitor cells into reticulocytes either in vivo or in vitro

(22,24). The properties of EPO during the RBC protein production during terminal erythroid

differentiation can be categorized into two general aspects; first, the induction of de novo

synthesis of certain proteins; second, the enhancement of an ongoing production initiated at a

developmental stage prior to terminal erythroid differentiation (3 8). It is likely that the









expression of Zipl10 and ZnT1 are extended by EPO-treatment based on the results shown in the

present study. When the erythroid progenitor cells were deprived of EPO, despite a gradual

increase of ZnT 1 mRNA abundance at 12 h, the respective mRNA levels of Zipl0 and ZnT1

generally decreased throughout the time-course examined. The final measurements, at 48 h, of

both transporter mRNA levels were lower than the basal levels determined at the initial time-

point when the in vitro culture without EPO was started. These results implicate that certain

levels of Zipl0 and ZnT1 mRNA expressed prior to the in vitro EPO-induction during

developmental stages in vivo, could not be sustained when the differentiation process was

discontinued. The temporal trend of ALAS-2 expression is known to be induced exclusively by

EPO during terminal erythroid differentiation (27,29). Because of the absence of background

mRNA levels from preceding differentiation stages, the ALAS-2 mRNA levels were stably

sustained at the basal (0 h) level when further differentiation was blocked by EPO-deprivation.

Previous studies with in vitro erythroid progenitor cell models suggest that the gene

expression patterns during differentiation strongly reflect the functional hierarchy of the

respective protein product activities (15,27,29,38). It was of interest that the mRNA levels of

Zipl10 and ZnT1 revealed different temporal patterns during the EPO-mediated differentiation in

vitro. While the EPO-dependent Zipl10 expression occurred rapidly after the terminal erythroid

differentiation was initiated, the EPO-responsiveness of ZnT 1 gene expression was only

detectable after 24 h of EPO-treatment. These results demonstrate that the zinc transporters

present in mature RBCs are differentially regulated by EPO and, thus, may be involved in the

homeostatic regulation of zinc in differentiating erythroid progenitor cells. The hierarchical

precedence of EPO-dependent Zipl10 expression to that of ZnT1 are in agreement with the zinc

expenditure trend during terminal erythroid differentiation (Fig. 5-1). Specifically, various events









that involve dynamic zinc utilization, such as synthesis of zinc metalloenzymes and zinc finger

transcription factors, have been shown to occur at the early stages of terminal erythropoiesis

(15,27). The earlier EPO-responsiveness of Zipl10 gene expression may be associated with an

increased requirement of zinc supply based on the metabolic use during these events (Fig. 5-1).

However, after the cells reach the very late stage of terminal erythropoiesis, the metabolic needs

of zinc decrease and, additionally, free zinc ions can introduce adverse affect to heme

biosynthesis by interfering with incorporation of ferrous iron into protoporphyrin (15,21). Thus,

the later EPO-dependent expression of ZnT 1 would be a strategic mechanism of differentiating

progenitor cells to remove excessive free zinc ions and, consequently, ensure the normal

hemoglobin biosynthesis at the final step ofRBC maturation (Fig. 5-1).

These expression trends of Zipl0 and ZnT 1 were confirmed at the protein level as well.

Molecular masses of Zipl0 and ZnT1 in the erythroid progenitor cells, speculated from the band

migration, were corresponding to those determined in mature RBCs. The ZnT1 protein

expression examined with total cell lysates revealed a similar trend to that observed in mRNA

levels as expected. However, the EPO-dependent elevation of Zipl10 expression at the early time-

points, observed at the mRNA level, was hardly detectable within these protein samples. In

addition, even though a decrease in mRNA levels occurred rapidly after EPO-deprivation, the

protein levels of Zipl0 observed in the total cell lysates were sustained relatively longer. It is of

note that these discrepancies between the mRNA and protein data were eliminated when the

cytosolic protein fraction were removed from the total cellular protein content by producing a

total cellular membrane fraction. This implicates that effects of certain cytosolic components,

which can be either internalized Zipl0 protein or other cytosolic proteins that are abundant in










erythroid progenitor cells, compromised the detectability of EPO-dependent Zipl0 expression in

the total cell fractions.

MT-1 mRNA levels monitored in the present study also reveal a unique temporal trend in

gene expression during terminal erythroid differentiation. The zinc-responsiveness of MT-1

protein expression in differentiating erythroblasts has been confirmed by a previous study (4,12).

Thus, it was presumed here that MT-1 mRNA levels may partially reflect the intracellular zinc

levels regulated by the differential expression of Zipl0 and ZnT 1 during terminal erythroid

differentiation. Although a rapid decrease occurred in both EPO-treated and -deprived cells

within 6 h, MT-1 mRNA levels was sustained higher in differentiating cells than in resting cells

until 24 h. These periods correspond to the time-points when the EPO-dependent Zipl0 mRNA

induction was observed. Thus, these results may partially indicate that an increased intracellular

zinc level was introduced by the early EPO-mediated Zipl0 expression.

With regard of the EPO-independent down-regulation of MT-1 mRNA abundance,

possible explanations of this phenomenon can be derived from previous studies. Abdel-Mageed

et al. showed that up-regulation of MT-1 expression in erythroid progenitor cells occur during

the proliferation stage that precedes the EPO-mediated terminal erythroid differentiation (39). In

addition, an inhibitory effect of MT-1 on the EPO-derived cell differentiation was indicated (39).

Conclusively, it was proposed that the expression of MT-1 transcripts in proliferating progenitor

cells should be repressed once further erythroid differentiation is committed by EPO. In another

study, the dependency of MT-1 synthesis on proliferation was determined by measuring

decreased MT levels by mitomycin-c treatment to K562 erythroleukemia cells (Huber et al.,

unpublished observation). This may imply the presence of an intrinsic factor that induces MT-1

specifically during the proliferation of erythroid progenitor cells. Thus, the rapid repression of









MT-1 mRNA levels observed in the present study would be related to the remnants from the

abundant MT-1 mRNA level expressed during the proliferation in vivo and the absence of the

proliferation-dependent MT-1 inducing factor in vitro.

As mentioned above, the association of MTF-1 activity with the transcriptional regulation

of Zipl0 and ZnT 1 has been suggested by previous studies. Up-regulation of Zipl0 and

repression of ZnT1 expression has been observed in MTF-1-/- hepatocytes and embryos,

respectively (3). Thus, as both Zipl0 and ZnT 1 are shown to be differentially expressed in

maturing erythroid progenitor cells, it was of interest to determine whether EPO-responsive of

MTF-1 gene expression occurs during terminal erythroid differentiation. The results presented in

the current study reveal certain interrelations of Zipl10 and ZnT 1 transcript levels to EPO-

dependent MTF-1 mRNA abundance. Peaks observed in the temporal pattern of MTF-1

transcription in differentiating cells corresponded to the decrease and increase in Zipl0 and

ZnT1 mRNA levels, respectively. Although the effects of EPO on MTF-1 activity in erythroid

progenitor cells need to be further explored, these results suggest that EPO-dependent

transcription of MTF-1 would be involved in the regulatory mechanism of the differential Zipl10

and ZnT1 expression during erythroid maturation.

Overall, the presence of erythroid zinc transporters, as Zipl0 and ZnT 1, has been

demonstrated in the current study. Furthermore, EPO-mediated expression of these transporters

was confirmed in differentiating erythroid progenitor cells. Several suggestions for future

approaches, particularly, with clinical perspectives can be derived from these results. The zinc

uptake rate of erythrocytes in vitro has been suggested to be a suitable indicator of early dietary,

subclinical zinc deficiency (11). Thus, the differential expression of these transporters in RBCs,

which are likely to be zinc-responsive, could be another candidate parameter for the assessment










of dietary zinc status. In addition, the expression of these zinc transporters could be connected to

the rigorous modulation of RBC intracellular zinc levels during Pla;smodium falciparum

parasitemia (40,41). In other words, the abnormal zinc sequestration in malarial RBCs would be

possibly caused by a transformation in the host cell zinc trafficking system, which may involve

Zipl10 and ZnT1 activities, by the parasite infection. Finally, to some extent, the EPO-responsive

Zipl10 expression observed in this study may support the suggestions from studies related to the

metastasis of breast cancer. Recently, it has been shown that EPO receptors (EPO-R) are highly

expressed in breast carcinoma, while the expression levels in benign mammary tissues are

generally negative (42). Although the functionality of EPO-R on these cancer cells remains

controversial, it has been associated with the stimulatory effect of EPO on the cell migration

activity (43). Expression of Zipl10 in breast carcinoma has been reported to be essential for the

migratory and invasive activity of breast cancer cells (44); however, the molecular mechanism of

Zip l0 induction has not been understood. Based on the results of the present study and evidence

mentioned above, the induction of Zipl10 expression by EPO may be a possible explanation for

the EPO-R mediated metastasis of breast cancer cells.





















Zn2+r IIIU UVV roroporpnynn i Zipl0
S~ Glycine

ZnT1 zinc Finger :M
Transcription Factors ALAS-2
GATA-1, EKLF, MTF-1 j : ciy-o
ZnT1

tNucleus


Early Stage of Terminal = Late Stage of Terminal
Erythroid Differentiation : Erythroid Differentiation

Figure 5-1. Putative model for the contribution of erythroid zinc transporters to the homeostatic
regulation of zinc during terminal erythroid differentiation. EPO binds to EPO-R and
induces the initiation of terminal erythroid differentiation. During the early stage of
terminal erythroid differentiation Zipl0 level is relatively higher than that at the late
stage. Intracellular Zn2+ 1) inhibits Ras-Raf signaling pathway and leads EPO-
mediated differentiation; 2) incorporates into CA and zinc finger transcription factors.
During the hemoglobin biosynthetic pathway, down-regulation of Zipl10 occurs while
ZnT1 level is relatively sustained. Thus, excessive Zn2+ is removed and abnormal
ZPP accumulation is prevented.









LIST OF REFERENCES


1. Prasad AS. Recognition of zinc-deficiency syndrome. Nutrition. 2001;17:67-9.

2. Cousins RJ. Zinc. In: Bowman BA, Russell RM, editors. Present knowledge in nutrition. 9
ed. Washington, D.C.: International Life Sciences Institute; 2006. p. 445-57.

3. Cousins RJ, Liuzzi JP, Lichten LA. Mammalian zinc transport, trafficking, and signals. J
Biol Chem. 2006;281:24085-9.

4. Grider A, Bailey LB, Cousins RJ. Erythrocyte metallothionein as an index of zinc status in
humans. Proc Natl Acad Sci U S A. 1990;87:1259-62.

5. Ohno H, Doi R, Yamamura K, Yamashita K, lizuka S, Taniguchi N. A study of zinc
distribution in erythrocytes of normal humans. Blut. 1985;50: 113-6.

6. Horn NM, Thomas AL, Tompkins JD. The effect of histidine and cysteine on zinc influx
into rat and human erythrocytes. J Physiol. 1995;489 (Pt 1):73-80.

7. Kalfakakou V, Simons TJ. Anionic mechanisms of zinc uptake across the human red cell
membrane. J Physiol. 1990;421:485-97.

8. Simons TJ. Calcium-dependent zinc efflux in human red blood cells. J Membr Biol.
1991;123:73-82.

9. De KJ, Van Der SC, Veldhuizen M, Wolterbeek HT. The uptake of zinc by erythrocytes
under near-physiological conditions. Biol Trace Elem Res. 1993;38:13-26.

10. Sasser LB, Bell MC, Jarboe GE. Influence of acute tissue injury on in vitro incorporation
of Zn by sheep erythrocytes. J Anim Sci. 1975;41:1679-85.

11. Van Wouwe JP, Veldhuizen M, De Goeij JJ, Van den Hamer CJ. Laboratory assessment of
early dietary, subclinical zinc deficiency: a model study on weaning rats. Pediatr Res.
1991;29:391-5.

12. Huber KL, Cousins RJ. Zinc metabolism and metallothionein expression in bone marrow
during erythropoiesis. Am J Physiol. 1993;264:E770-E775.

13. Hodge D, Coghill E, Keys J, Maguire T, Hartmann B, McDowall A, Weiss M, Grimmond
S, Perkins A. A global role for EKLF in definitive and primitive erythropoiesis. Blood.
2006;107:3359-70.

14. Ferreira R, Ohneda K, Yamamoto M, Philipsen S. GATAl function, a paradigm for
transcription factors in hematopoiesis. Mol Cell Biol. 2005;25:1215-27.

15. Welch JJ, Watts JA, Vakoc CR, Yao Y, Wang H, Hardison RC, Blobel GA, Chodosh LA,
Weiss MJ. Global regulation of erythroid gene expression by transcription factor GATA-1.
Blood. 2004; 104:313 6-47.










16. Tomoda T, Nomura I, Kurashige T, Kubonishi I, Miyoshi I, Sukenaga Y, Taniguchi T.
Changes in Cu,Zn-superoxide dismutase gene during induced erythroid and myeloid
differentiation. Acta Haematol. 1991;86:183-8.

17. Nishiyama S, Irisa K, Matsubasa T, Higashi A, Matsuda I. Zinc status relates to
hematological deficits in middle-aged women. J Am Coll Nutr. 1998;17:291-5.

18. Nishiyama S, Kiwaki K, Miyazaki Y, Hasuda T. Zinc and IGF-I concentrations in pregnant
women with anemia before and after supplementation with iron and/or zinc. J Am Coll
Nutr. 1999; 18:261-7.

19. Forman WB, Sheehan D, Cappelli S, Coffman B. Zinc abuse--an unsuspected cause of
sideroblastic anemia. West J Med. 1990; 152: 190-2.

20. Fiske DN, McCoy HE, III, Kitchens CS. Zinc-induced sideroblastic anemia: report of a
case, review of the literature, and description of the hematologic syndrome. Am J Hematol.
1994;46:147-50.

21. Bloomer JR, Reuter RJ, Morton KO, Wehner JM. Enzymatic formation of zinc-
protoporphyrin by rat liver and its potential effect on hepatic heme metabolism.
Gastroenterology. 1983;85:663-8.

22. Kaushansky K. Lineage-specific hematopoietic growth factors. N Engl J Med.
2006;,354:2034-45.

23. Woj chowski DM, Menon MP, Sathyanarayana P, Fang J, Karur V, Houde E, Kapelle W,
Bogachev O. Erythropoietin-dependent erythropoiesis: New insights and questions. Blood
Cells Mol Dis. 2006;36:232-8.

24. Krantz SB. Erythropoietin. Blood. 1991;77:419-34.

25. Labbe RF, Rettmer RL. Zinc protoporphyrin: a product of iron-deficient erythropoiesis.
Semin Hematol. 1989;26:40-6.

26. Alcindor T, Bridges KR. Sideroblastic anaemias. Br J Haematol. 2002; 116:733-43.

27. Hodges VM, Winter PC, Lappin TR. Erythroblasts from friend virus infected- and
phenylhydrazine-treated mice accurately model erythroid differentiation. Br J Haematol.
1999;106:325-34.

28. Cooper MC, Levy J, Cantor LN, Marks PA, Rifkind RA. The effect of erythropoietin on
colonial growth of erythroid precursor cells in vitro. Proc Natl Acad Sci U S A.
1974;71:1677-80.

29. Dolznig H, Boulme F, Stangl K, Deiner EM, Mikulits W, Beug H, Mullner EW.
Establishment of normal, terminally differentiating mouse erythroid progenitors: molecular
characterization by cDNA arrays. FASEB J. 2001;15:1442-4.










30. Piao F, Yokoyama K, Ma N, Yamauchi T. Subacute toxic effects of zinc on various tissues
and organs of rats. Toxicol Lett. 2003;145:28-3 5.

31. Levengood JM, Sanderson GC, Anderson WL, Foley GL, Brown PW, Seets JW. Influence
of diet on the hematology and serum biochemistry of zinc-intoxicated mallards. J Wildl Dis.
2000;36: 111-23.

32. Witeska M, Kosciuk B. The changes in common carp blood after short-term zinc exposure.
Environ Sci Pollut Res Int. 2003;10:284-6.

33. Lukaski HC. Low dietary zinc decreases erythrocyte carbonic anhydrase activities and
impairs cardiorespiratory function in men during exercise. Am J Clin Nutr. 2005;81:1045-
51.

34. McMahon RJ, Cousins RJ. Regulation of the zinc transporter ZnT-1 by dietary zinc. Proc
Natl Acad Sci U S A. 1998;95:4841-6.

35. Kim AH, Sheline CT, Tian M, Higashi T, McMahon RJ, Cousins RJ, Choi DW. L-type
Ca(2+) channel-mediated Zn(2+) toxicity and modulation by ZnT-1 in PC12 cells. Brain
Res. 2000;886:99-107.

36. Kaler P, Prasad R. Molecular cloning and functional characterization of novel zinc
transporter rZipl0 (Slc39al0) involved in zinc uptake across rat renal brush-border
membrane. Am J Physiol Renal Physiol. 2007;292:F217-F229.

37. Pawan K, Neeraj S, Sandeep K, Kanta RR, Raj endra P. Upregulation of Slc39al0 gene
expression in response to thyroid hormones in intestine and kidney. Biochim Biophys Acta.
2007;1769:117-23.

38. Koury MJ, Bondurant MC, Mueller TJ. The role of erythropoietin in the production of
principal erythrocyte proteins other than hemoglobin during terminal erythroid
differentiation. J Cell Physiol. 1986; 126:259-65.

39. Abdel-Mageed AB, Zhao F, Rider BJ, Agrawal KC. Erythropoietin-induced
metallothionein gene expression: role in proliferation of K562 cells. Exp Biol Med
(Maywood ). 2003;228:1033-9.

40. Ginsburg H, Gorodetsky R, Krugliak M. The status of zinc in malaria (Plasmodium
falciparum) infected human red blood cells: stage dependent accumulation,
compartmentation and effect of dipicolinate. Biochim Biophys Acta. 1986;886:337-44.

41. Hiremath GS, Sullivan DJ, Jr., Tripathi AK, Black RE, Sazawal S. Effect of Plasmodium
falciparum parasitemia on erythrocyte zinc protoporphyrin. Clin Chem. 2006;52:778-9.

42. Acs G, Zhang PJ, Rebbeck TR, Acs P, Verma A. Immunohistochemical expression of
erythropoietin and erythropoietin receptor in breast carcinoma. Cancer. 2002;95:969-81.










43. Lester RD, Jo M, Campana WM, Gonias SL. Erythropoietin promotes MCF-7 breast
cancer cell migration by an ERK/mitogen-activated protein kinase-dependent pathway and
is primarily responsible for the increase in migration observed in hypoxia. J Biol Chem.
2005;280:39273-7.

44. Kagara N, Tanaka N, Noguchi S, Hirano T. Zinc and its transporter ZIP10 are involved in
invasive behavior of breast cancer cells. Cancer Sci. 2007;98:692-7.









BIOGRAPHICAL SKETCH

Moon-Suhn Ryu was born on March 28, 1979 in Seoul, South Korea. He attended Yonsei

University in Seoul from 1997 to 2001. Upon graduation with his Bachelor of Science in

Biotechnology, Moon-Suhn joined the Republic of Korea Air Force to fulfill his military service

required by the South Korean government. Since discharging his duty, Moon-Suhn worked for a

beverage company, Lotte Chilsung Beverage Company Limited, in South Korea as an assistant

manager in the overseas business team. He came to the United States in the fall of 2005 to start

his master' s study in nutritional sciences at the University of Florida. Moon-Suhn is planning to

continue his graduate studies in the doctorate program for nutritional sciences at the University

of Florida.





PAGE 1

1 ZINC TRANSPORTER EXPRESSION IN MATURE RED BLOOD CELLS AND DIFFERENTIATING ERYTHROID PROGENITOR CELLS By MOON-SUHN RYU A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2007

PAGE 2

2 2007 Moon-Suhn Ryu

PAGE 3

3 To my grandfather, Hong-Ryol Ryu (1911-1995). The lessons from him have always inspired me to pursue the delight to be wi se with the right insights.

PAGE 4

4 ACKNOWLEDGMENTS First of all, I thank my beloved parents for their endless love and trust on me throughout my life. Their guidance and support have been my greatest source of wisdom and confidence. I also thank my younger sister, Jin-Suhn Ryu, for always sharing her happiness with me through the sweetest and brightest stories from far away in South Korea. Additionally, I express my gratitude to my wonderful lab mates, Juan P. Liuzzi, Louis A. Lichten, Liang Guo, Shou-mei Chang, and Tolunay Beker Aydemir, for their encouragement and advices from the beginning and the end of this study. The suggestions from their own experiences were always reliable and helpful for me, especially, when encountering any f actors that could have led me into frustration. Finally, I render my deep appreciation to my supervisory committee members, Dr. Robert J. Cousins, Dr. Mitchell D. Knutson, and Dr. David R. Allred, who shared their precious time to give me the insights to move forward into th e appropriate direction. Especially, I thank Dr. Robert J. Cousins for being a great model for my future plans as a successful scientist and for giving me the opportunity to continue my doctoral studies under his supervision.

PAGE 5

5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........8 ABSTRACT....................................................................................................................... ..............9 CHAPTER 1 INTRODUCTION..................................................................................................................11 2 LITERATURE REVIEW.......................................................................................................14 Erythropoiesis................................................................................................................. ........14 In Vitro Models of Terminal Erythroid Differentiation..........................................................15 Zinc Metabolism during Terminal Erythroid Differentiation.................................................16 Zinc Status and Anemia......................................................................................................... .17 Erythroid Zinc Trafficking System.........................................................................................18 3 MATERIALS AND METHODS...........................................................................................20 Preparation of RBC Membranes.............................................................................................20 Production of Primary Eryt hroid Progenitor Cells.................................................................20 Cell Culture................................................................................................................... ..........21 o-Dianisidine Staining......................................................................................................... ...21 RNA Isolation.................................................................................................................. .......22 cDNA Synthesis and Quantitative RT-PCR...........................................................................22 Protein Isolation.............................................................................................................. ........23 Affinity Purification of Antibodies.........................................................................................24 Western Analysis............................................................................................................... .....24 Statistical Analysis........................................................................................................... .......25 4 RESULTS........................................................................................................................ .......27 Expression of Zinc Transporters in RBC Membranes............................................................27 Confirmation of Erythroid Differentiation.............................................................................27 Effects of EPO on Zip10 and ZnT1 Transcript Levels...........................................................28 Protein Expression of Zip10 and ZnT1 during Differentiation..............................................29 Effects of EPO on MT-1 and MTF-1 Transcript Levels........................................................30 5 DISCUSSION..................................................................................................................... ....38

PAGE 6

6 LIST OF REFRENCES.............................................................................................................. ...46 BIOGRAPHICAL SKETCH.........................................................................................................50

PAGE 7

7 LIST OF TABLES Table page 3-1 Zinc transporters screened in erythroc yte ghosts and peptides used for the production and affinity purification of antibodi es against each respective protein.............................26

PAGE 8

8 LIST OF FIGURES Figure page 4-1 Zinc transporter expressi on in mature red blood cells.......................................................31 4-2 Induction of splenomegal y by phenylhydrazine-injection.................................................32 4-3 Indicators of EPO-mediated terminal erythroid differentiation in vitro ............................33 4-4 Relative Zip10 mRNA a bundance during terminal erythroid differentiation....................34 4-5 Relative ZnT1 mRNA abundance during terminal erythroid differentiation....................34 4-6 Zip10 protein expression during te rminal erythroid differentiation..................................35 4-7 ZnT1 protein expression during terminal erythroid differentiation...................................36 4-8 Relative MT-1 mRNA abundance during terminal erythroid differentiation....................37 4-9 Relative MTF-1 mRNA abundance during terminal erythroid differentiation..................37 5-1 Putative model for the contri bution of erythroid zinc tran sporters to the homeostatic regulation of zinc during termin al erythroid differentiation..............................................45

PAGE 9

9 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science ZINC TRANSPORTER EXPRESSION IN MATURE RED BLOOD CELLS AND DIFFERENTIATING ERYTHROID PROGENITOR CELLS By MOON-SUHN RYU August 2007 Chair: Robert J. Cousins Major: Food Science and Human Nutrition Animal and human studies have shown that the in vitro uptake rate of 65Zn by red blood cells (RBCs) is inversely related to the subject s zinc status. The capabil ity of RBCs to take up zinc may be a remnant of an earlier developmental stage of erythrocytes as zinc is essential for the activities of several proteins formed dur ing the erythroid diffe rentiation, e.g., carbonic anhydrase, Cu2+/Zn2+-superoxide dismutase, and zinc fi nger transcription factors such as erythroid Krppel-like factor (EKLF) and GATA1. Conversely, excessive intracellular free zinc ions can interfere with incorpora tion of ferrous ions into heme at the final stage of erythroid differentiation. Thus, intracellular zinc homeostas is during erythroid differentiation must be tightly regulated. To examine the hypothesis that the expression of zinc transporters would be involved in the strategic mechanism of erythroid zinc homeost asis, transporters in th e membrane fraction of RBCs were screened by western analyses, and on ly Zip10 and ZnT1 were detectable among the zinc transporters tested. Er ythroid progenitor cells were prepared from spleens of phenylhydrazine (PHZ)-treated anemic mice for the characterization of transporter gene expression during the terminal stag e of erythropoiesis. Differentiati on of cells into reticulocytes was induced by erythropoietin (EPO)-treatment in vitro Hemoglobin (Hb) synthesis and

PAGE 10

10 expression of erythroid -aminolevulinic acid synthase (ALA S-2) mRNA were measured for the confirmation of the ex vivo erythroid differentiati on. Transcript levels of each transporter gene and other genes associated with zinc metabolis m were quantified by quantitative real-time PCR (qRT-PCR). Temporal trends in expression of each gene were observed. Briefly, following addition of EPO, Zip10 mRNA levels peaked pr ior to the time-point when metal-responsive transcription factor-1 (MTF-1) transcripts reached its first peak level, and decreased dramatically afterwards. For ZnT1 mRNA, EPO-dependent expr ession was initiated later than Zip10 and was sustained until the experimental time-course wa s over. Metallothionein-1 (MT-1) transcript abundance decreased rapidly after addition of EPO and stayed lower than the 0 h basal levels until 48 h. Expression trends of Zip10 and ZnT1 were further confirmed by western blots utilizing total cell lysates and membrane fractions of these cells. This is the first study to determine which zinc transporters are expressed in the erythroid system. The results presented here suggest th at Zip10 and ZnT1 expression is induced in response to EPO. Furthermore, they could be the zi nc transporters most directly involved in the regulation of intracellular zinc homeostasis in differentiating erythroid progenitor cells and circulating RBCs. The results may also be a route whereby RBCs accumulate excessive amounts of zinc during malaria.

PAGE 11

11 CHAPTER 1 INTRODUCTION Zinc is an essential elemen t which is ubiquitously distri buted in the human body, and its deficiency is known to cause reduced growth, hypogonadism, visceromegaly, and hematological abnormalities associated with iron deficiency anem ia (1). The functional properties of zinc can be categorized by its catalytic, st ructural and regulatory roles in numerous metabolic processes of the biological system (2). Differential expression of zinc transporters is known to be involved in the regulatory mechanism of the intracellular zinc homeostasis in various tissues and cell types (3). There are two distinct gene families of zinc transporters composed of ten ZnT and fourteen Zip transporter genes, respectivel y. ZnT proteins facilitate the re moval of cytosolic free zinc either by exporting through the plas ma membrane or by sequestering zinc in vesicles, while the Zip transporters function in an opposite manner as a pathway for zinc influx from plasma or vesicles. In mature red blood cells (RBCs), the zinc concen trations is about fifteen times larger than that in the plasma (4), and more than 90% of that is known to function as a component essential for the activity of zinc metall oenzymes, carbonic anhydrase and Cu2+/Zn2+-superoxide dismutase (5). Various routes of circulating RBCs where by zinc influx occurs have been reported by classical studies, of which sugge sted mechanisms involve the Cl-/HCO3anion exchanger activity, a neutral complex formation with thiocyanate, salicylate ions, and the chelation by amino acids (6,7). The calcium-dependent zinc efflux by a Ca2+/Zn2+ exchanger has been considered as the mechanism for the cellular zinc export from circ ulating RBCs (8). Addition ally, there have been animal and human studies with zinc defici ent subjects implying th e expression of zincresponsive intrinsic factors involved in the RBC zi nc transport system (9-11). In these studies, RBCs from zinc deficient groups consistently re vealed higher 65Zn uptake rates than those

PAGE 12

12 collected from normal subjects when cultured in conditions with identical zinc contents in vitro Even though the modulation of zinc uptake rate is likely to be influenced by the zinc transporter expression, there have been no reports related to th e determination of erythroid zinc transporters so far. Proteins involved in the zinc metabolism of mature RBCs are likely to be remnants from earlier developmental stages as the capabil ity for additional gene expression or protein production is deprived by enucleation at the final step of erythr opoiesis. A study showing increased zinc uptake by the bone marrow during induced erythropoiesis in zinc deficiency supports the necessity of minimal amount of zinc during erythroid differentiation (12). One of the most well-studied features of zinc in erythr oid differentiation is its incorporation into zinc finger transcription factors which are responsib le for the expression of essential proteins involved in events of terminal erythroid matu ration (13,14). Additionally, zinc metalloenzymes, such as carbonic anhydrase and Cu2+/Zn2+-superoxide dismutase, are produced during erythropoiesis (15,16). Some clinical studies of zi nc treatment for anemia have shown that it can reverse anemic symptoms by increasing the pro duction of hemoglobin (Hb) and, consequently, facilitate the formation of normal RBCs (17,18). Ho wever, in converse, there have been reports of sideroblastic anemia caused by zinc intoxi cation as well (19,20). This may be due to the interference by excessive free zinc ions with incorporation of fe rrous ions into protoporphyrin during the heme biosynthetic pathway (21). Ba sed on these findings, it seems critical for the erythroid intracellular zinc level to be tightly re gulated during late stag e erythroid differentiation so that any adverse effects introduced by inadeq uate or excessive zinc supply can be avoided. With consideration of these aspe cts related to the erythroid zi nc metabolism, this study was designed upon the following hypotheses:

PAGE 13

13 Hypothesis 1: As in other tissues and cell types, certain ZnT and Zip proteins would be expressed in mature red blood cells for th e maintenance of zinc homeostasis during circulation. Hypothesis 2: Zinc transporters detected in mature RBCs would be remnants from preceding developmental stages. Thus, the expression of respective transporters would occur during the EPO-mediated differentiation of late stage erythroid progenitor cells. Consequently, the major aim of this study was to determine which zinc transporters may be involved in the erythroid zinc trafficking system, and characte rize the transporter expression during RBC maturation. Initially, the zi nc transporters expressed in circulating erythrocytes were identified at the protein level. Thereafter, temporal trends of each respective transporter expression in differentiating erythroid proge nitor cells were determined. For a more comprehensive understanding of the zinc metabol ism during terminal erythroid differentiation, mRNA levels of other zinc metabolism genes, MT-1 and MTF-1 were also measured in differentiating cells.

PAGE 14

14 CHAPTER 2 LITERATURE REVIEW Erythropoiesis Red blood cell (RBC) production in itiates from a pluripotenti al hematopoietic stem cell and sequential differentiation of each intermedia te cell type in the hematopoietic hierarchy depends on the activation by lineage-specific grow th factors (22). Normally, the site of erythropoiesis is the bone marrow and in specif ic conditions, such as anemic subjects and embryos, the spleen and the liver becomes the ma jor site of erythropoie sis, respectively (23). Once pluripotential hematopoietic stem cells become erythroid pr ogenitor cells af ter carrying out several steps of erythropoiesis, they exclusively differentiate into RBCs as a response to a specific glycoprotein termed erythropoietin (EPO ) (24). Erythroid progenitor cells have been classified into two types, the burst-forming un it-erythroid (BFU-E) and the colony-forming uniterythroid (CFU-E). Both types require EPO fo r further differentiation; however, the BFU-E requires other growth factors such as inte rleukin-3 or granulocyte-macrophage colony stimulating factor (GM-CSF) in addition to EPO, while the CFU-E does not (24). During the terminal stage of er ythropoiesis, i.e., CFU-E differ entiation, critical events for normal RBC formation occur. For instance, he moglobin (Hb) biosynthe sis and enucleation happen as a response to EPO-induction, and charact erize the unique properti es of erythrocytes (24). Most adverse effects of nut rient deficiencies or toxicities -related to anemia occur during this stage of RBC production. For example, in iron deficiency, an inadequate supply of ferrous ions yields hypochromic anemia by introducing an increased zinc/iron ra tio in erythroblasts which leads the ferrochelatase-f acilitated reaction to produce zinc protoporphyrin rather than the essential component of Hb, heme (25). Vitamin B6 deficiency re sults in sideroblastic anemia

PAGE 15

15 because pyridoxal 5-phosphate is a cofactor for erythoid -aminolevulinic acid synthase (ALAS2) activity (26). In Vitro Models of Terminal Erythroid Differentiation As normal RBC formation depends on the seque ntial events during the terminal stage of erythropoiesis, several ex vivo models representing this step have been developed for hematological studies. For instance, splenoc ytes and bone marrow cel ls from phenylhydrazine (PHZ)-treated or Friend virus (FVA)-infected anemic animal models, and erythroleukemia (MEL) cell lines have been commonly used (23,27). Because of the absence of nucleus in mature RBCs, the use of these systems is necessary especi ally in approaches for the characterization of erythroid gene expression and th e understanding functi ons of the respective proteins. Hodges et al. suggested that erythroblasts from PHZ-treat ed anemic mice show the highest homology to the in vivo erythroid system by evaluating the responsiveness of several erythroid specific genes in three different types of in vitro models after EPO-induction (27). In this cell model, sequential trends in expression of each gene were de tected and the timepoints of maximum mRNA abundance were shown to be dependent on the meta bolic needs of the relevant protein activity during erythroid maturation. Not only reliable ex vivo systems but also protocols fo r the confirmation of terminal erythroid differentiation are well -developed. Staining Hb, of which peroxidase activity yields a brownish-red product in staining so lutions with o-dianis idine or benzidine, has been used as a classical method for erythroid differentiation assessment (27,28). Evalua ting the expression of genes only induced in differentiati ng erythroids, such as ALAS-2 (27,29), is another reliable and also safer way to determine the differentiation st atus. This is especially relevant when the carcinogenic risks of the above r eagents are concerned (US Depa rtment of Health and Human Services (DHHS), National Instit ute for Occupational Safety a nd Health (NIOSH) Publication

PAGE 16

16 No. 81-106). Additionally, morphologi cal changes of the cells, such as smaller size and nucleus extrusion, are markers of the termin al stage of differentiation (27). Zinc Metabolism during Terminal Erythroid Differentiation Zinc is required for the activiti es of several proteins relate d to erythroid differentiation. One of the most apparent biochemical properties of erythroid zinc is based on its role as a key structural component of the zinc finger proteins Since the CFU-Es are co mmitted to differentiate into mature erythrocytes by EP O-induction, an EPO-responsive zinc finger transcription factor, GATA-1, of which binding sites are lo cated in all erythroid-specific genes, initiates its critical role as the coordinator of multiple events composing the differentiation process by regulating relevant gene expressions (14,15) Another red cell-specific zinc finger transcription factor, erythroid Krppel-like factor (EKLF), is essent ial for the transcripti onal induction of genes encoding -globin, which with heme composes Hb, a nd ferrochelatase, which facilitates the incorporation of ferrous ions into protoporphyrin as the final step of heme synthesis (13). Additionally, both of these zinc finger proteins ar e known to be involved in the transcription of ALAS-2 and porphobilinogen deaminase (PBGD) genes (13,14). Their products are the ratelimiting enzymes of the heme biosynthetic pathway. The production of the zinc metalloenzyme, carbonic anhydrase (CA), which contains around 87% of total RBC zinc content (5), o ccurs during the EPO-mediated terminal differentiation as well (15). This enzyme is the second most abundant protein in mature erythrocytes, after Hb, and require s zinc as an essential component for its catalytic activity. The induction of CA expression during the differen tiation of erythroleukemia cells precedes hemoglobin synthesis (15), which may imply an incr eased requirement of zinc at the early stage of terminal erythroid different iation. Supporting these functional pr operties of erythroid zinc, a

PAGE 17

17 study by Huber et al. showed that zinc uptak e by the bone marrow of zinc-restricted rats increased during induced -erythropoiesis (12). Zinc Status and Anemia Since a case of zinc deficiency associated-a nemia was firstly reporte d by Prasad et al in 1961 (1), clinical studies on the va lue of zinc supplementation to anemic subjects have been performed. For instance, two consec utive studies by Nishiyama et al. were designed to determine effects of zinc supplementation on anemic middl e-aged or pregnant wo men (17,18). Conclusive benefits where shown when adequate iron intake was ensured. Briefly, the concentrations of Hb, numbers of RBCs and reticulocytes in anemic pa tients were significantly increased by the zinc plus iron treatment, while the values from the other treatment groups, i.e., treated exclusively with zinc or iron, did not change. In addition, this research group also suggested that normocytic anemia with low total iron binding capacity (TIBC) which is inversely rela ted to the transferrin saturation status and thus implicat es the status of iron deficienc y, may serve as an indicator of zinc deficiency (17). While zinc supplementation has been reported to have beneficial effects on overcoming anemic symptoms, adverse effects leading hema tological abnormalities introduced by excessive zinc treatment have been observed as well. Side roblastic anemia associated with excessive and prolonged intake of oral zinc in human has b een notified by several cas e reports (19,20). In addition, increased frequency of abnormal and/or immature erythrocytes in the blood stream has been observed in zinc intoxicated rats, mallard s, and carps (30-32). A lthough secondary copper deficiency has been considered as the major reas on of these symptoms, Bl oomer et al. suggested that anemia introduced by excessive zinc may be attributed to the increased formation of a biologically inactive com pound, zinc protoporphyrin (ZPP), instea d of heme (21). Zinc strongly competes with iron for ferrochelatase and its prod uct, ZPP, can exert the feedback inhibition of

PAGE 18

18 ALAS-2 produced by heme. Even a slight decrease in iron availability by increased zinc can cause ZPP accumulation. Supporting this concept, increased formation of ZPP-globin by the decreased erythroid iron/zinc ratio in the maturing erythrocytes is known to be the reason of anemic symptoms during iron deficiency (25). Erythroid Zinc Trafficking System The intracellular zinc concentration in circ ulating RBCs is approximately fifteen times larger than the plasma levels (4). Over 90% of total erythrocyte zinc attributes to the enzyme activities of carboni c anhydrase and Cu2+/Zn2+-superoxide dismutase as catalytic and structural elements, respectively (5). Acco rdingly, an adequate amount of zinc supply would be essential even after the erythroid maturation is accomplis hed. Supportively, impaired carbonic anhydrase activities, which catalyze the reversible hydra tion and dehydration of carbon dioxide, have been observed in subjects with low zinc diets (33). The differential and tissue speci fic expression of zinc trans porters have been strongly related to the homeostatic regul ation of exchangeable systemic and cellular zinc pools in biological systems (3). Zinc transporters are composed of two distinct gene families, ZnT and Zip, of which proteins facilitate the decrease and increase of cytosolic zinc, respectively, by expression across plasma or vesicle membranes. The zinc removal by ZnT occurs either by exporting through the plasma membra ne or by sequestering zinc in vesicles, while the influx by Zip is mediated in the opposite manner. Even though the zinc trafficking system in various tissues and cell types have been extensively studi ed during the past decade little is known about the specific pathways for ionic zinc transpor t across RBC membranes. Some classical studies have suggested that the zinc uptake mechanisms would be mediated by interactions with other biological compounds. For instance, facilitated diffusion of zinc by chelation with amino acids, particularly L-histidine and L-cy steine, has been thought to be re lated the zinc influx in rat and

PAGE 19

19 human RBCs (6). Additionally, tw o anion-dependent mechanisms related to the zinc influx system in human erythrocytes were suggested (7 ). One of these requires bicarbonate ions which induces the formation of a zinc-anion complex, [Zn(HCO3)2Cl]-, that can be taken up via the Cl-/HCO3 anion exchanger. The other one, involving thiocyanate or salicylate ions, has been thought to be accomplished by forming a neutral zinc complex that migrates across the lipid bilayer. With regard of pathways whereby RBC zinc efflux may occur, a calcium-dependent mechanism through the Ca2+/Zn2+ exchanger is the only rout e speculated so far (8). Despite absence of definitive evidences, it is likely that zinc transporters are involved in the homeostatic regulation of erythroid zinc me tabolism. Animal and human studies have shown a modulation in the zinc trafficking rate of erythrocytes during zinc deficiency (9-11). Specifically, increased in vitro 65Zn uptake rates of RBCs were observed in rat, sheep and human subjects when fed zinc-restricted diets. Thes e results would imply the presence of a zincresponsive intrinsic factor that enables the homeost atic regulation of erythr oid intracellular zinc. In other words, the regulated ac tivity of this factor would resu lt in a higher zinc uptake rate which would correct the cellu lar zinc loss introduce by the zinc-deprived conditions in vivo This aspect is quite consistent with the general properties of the homeostatic regulatory mechanism mediated by zinc transporter expressions in other tissues (3). Consequently, it was of interest to determine the transporter expresse d in erythroid lineage cells. Th e purpose of the current study is to identify of zinc tran sporter thought to be involved in eryt hroid zinc trafficking system, and characterize the expression trends during the maturation of RBCs.

PAGE 20

20 CHAPTER 3 MATERIALS AND METHODS Preparation of RBC Membranes Whole blood (~ 400 l/subject) was collect ed from anesthetized CD-1 mice through cardiac puncture and aliquoted in to EDTA-treated centrifuge t ubes. Three volumes of wash buffer [5 mM Sodium Phosphate (pH 7.4~8), 0.15M NaCl] was added to the blood sample which was then centrifuged at 2,000 x g x 10 min, 4oC. After carefully aspirating the supernatant and buffy coat, the pellet was washed with 2 vol of wash buffer, extensively. Following the final wash, the presence of RBCs was confirmed by phase contrast microscopy. Isolated RBCs were then lysed by washing with 3 vol of a hypotoni c lysis buffer [5 mM Sodium Phosphate (pH 7.4~8) with protease inhibitor cocktail (Sigma, St. Louis, MO )]. After centrifugation at 12,000 x g x 10 min, 4oC, the supernatant was aspirated. Then the tube was tilted and rotated for the aspiration of the red button, whic h is beneath the ghost pellet a nd contains proteases. The cell lysis step was repeated until the pellet lost it s red color (3~7 times). Each final pellet was dissolved in 200 l of storage buffer [5 mM Tr is-HCl, 0.5% Triton X-100 w ith protease inhibitor cocktail (Sigma)] for solubilizat ion. Finally, the protein concentr ation of the red cell membrane preparation was determined colorimetrically with the DC Protein Assay kit (Bio-Rad, Hercules, CA). Standards developed with diluted bovine serum albumin solutions and the absorbance was read at 750 nm using a Beckman DU 640 spectrophotometer (Beckman, Fullerton, CA). Production of Primary Er ythroid Progenitor Cells Primary erythroid progenitor cells were prep ared from spleens of PHZ-treated anemic mice. CD-1 mice were injected with PHZ hydrochloride (Sigma) in 0.9% saline (60 mg/kg bw) intraperitoneally on day 1 and day 2. On day 5, the PHZ-treated mice were sacrificed and the spleen was collected. Monocellular splenocyte susp ensions were prepared by cutting the spleen

PAGE 21

21 into small fragments in AMEM (Mediatech, Herndon, VA) containing 10% FBS and antibiotic antimycotic solution (Sigma), and by passing th e disrupted spleen through nylon mesh. The viability and concentration of cells were determ ined by staining an aliquot of the cell suspension with Trypan Blue solution (0.4%; Sigma). Cell Culture To induce further differentiation of the late stage erythroid progenitors, cells were incubated with recombinant human EPO (ProSpecTany, Rehovot, Israel). The single spleen cell suspension was diluted into 1.0 x 107 cells/ml with AMEM containing 10% FBS and antibiotics/antimycotics for tr eatment. The treatment was co nducted by a further 1:2 dilution with an equal volume of fresh medium containing EPO as 10 IU/ml. With the final concentration of cells and EPO as 5.0 x 106 cells/ml and 5 IU/ml, respectively, cells were equally plated in 6well culture plates as 1.0 x 107 cells/well and were incubated for up to 48 h at 37oC in 5% CO2. The control cell cultures were prepared in parall el with identical procedures except for using EPO-absent medium at the final step of dilution. o-Dianisidine Staining Differentiation of cells was confirmed by comp aring the hemoglobin sy nthesis levels of cells collected at 0 h and 48 h after EPO-treat ment through o-dianisidin e staining. The staining solution [0.1 mg/ml o-dianis idine (Sigma D9154), 0.2% (v/v ) SDS, 0.3% (v/v) hydrogen peroxide in PBS] was prepared freshly for each time-point. 500 l of cell suspension was washed with 500 l of sterile PBS by cen trifugation at 250 x g x 5 min, 4oC. The supernatant was carefully aspirated and the final pellet was re suspended with 500 l of the staining solution. After incubation at room temperature for 30 mi n, cells were spun down and washed with 500 l of PBS. Cell suspensions were transferred to wells of a La b-Tek Permanox Chamber Slide

PAGE 22

22 (Nunc, Rochester, NY) and the staining was exam ined by light microscopy with an Axiovert microscope (Zeiss, Oberkochen, Germany). RNA Isolation Cells were harvested at cert ain time-points covering the whole 48 h differentiation. The time-course was designed to determine the temp oral trends in changes of each mRNA level throughout differentiation and detect the timepoint at which maximum EPO-responsiveness exists. After centrifugation at 400 x g x 5 min, 4oC, cell pellets were resuspended with 1 ml of TRIzol reagent (Invitrogen, Carl sbad, CA) and stored at -80oC until the entire time-course was over. When all samples were collected, cell lysa tes were incubated at room temperature for 5 min after thoroughly thawed. After adding 200 l of chloroform, each sample were centrifuged at 12,000 x g x 10 min, 4oC and the top clear aqueous layer was transferred to a new sterile tube. The aqueous phase was incubated with 500 l of isopropyl alcohol at ro om temperature for 10 min and centrifuged at 12,000 x g x 15 min, 4oC for RNA precipitation. The supernatant was discarded and the RNA pellet was washed with 1 ml of 75% (v/v) ethanol by centrifuging at 12,000 x g x 10 min, 4oC. After repeating the wash step at least twice, ethanol was removed and dried out from the RNA pellet at room temper ature. Finally, the RNA pellet was resuspended with 50 l of nuclease-free water and incubated in 37oC for complete dissolve. RNA solutions were treated with DNase I (Ambion, Austin, TX) to avoid any DNA contamination and the concentrations were determined spectrophotom etrically (NanoDrop Technologies, Wilmington, DE). cDNA Synthesis and Quantitative RT-PCR Relative mRNA levels of ALAS-2, Zip10, ZnT 1, MT-1 and MTF-1 were measured by quantitative real-time PCR (qRT-P CR). Primers for mouse ALAS-2 gene were designed with PRIMER EXPRESS V2.0 (Applied Biosystems, Fost er City, CA) while those for others were

PAGE 23

23 available from previous studies of our lab. Pr imers for the PCR amplication of ALAS-2 cDNA were: forward primer, 5-CAGAGGGCAGCT CCAGAAGTT-3; reverse primer, 5GCTTCGGGTGGTTGAATCC-3. cDNA was generated by reverse transcription of total RNA (100 ng/reaction) using the high capacity cDNA archive kit (Applied Biosystems), and 1:25 diluted to be used as templates of each PCR reaction. The cDNA product of the total RNA collected at 0 h was used as the standard after 1:2 dilution and further 1:10 serial dilutions. All qRT-PCR assays were conducted with th e Power SYBR Green Master Mix (Applied Biosystems) and amplification products were fl uorometrically measured by the iCycler RT-PCR detection system (Bio-Rad). The specificity of each primer pair was confirmed after each qRTPCR assay by melting curve analyses and the re lative quantity was determined by normalization with respective 18s rRNA values. Protein Isolation Cells were treated and cultured as describe d above. After designated time-points of incubation, cells were spun down at 400 x g x 7 min, 4oC, and the supernatant was aspirated carefully. Total cell lysates were prepared by dissolving the cell pellets (~ 1.0 x 107 cells) with 100 l of SDS cell lysis buffer [10% (w/v) SD S, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0 with protease inhibitor cockta il (Sigma)] and following homogeni zation with brief sonication. Total membrane fractions of the eryt hroid splenocytes were prepar ed by utilizing a commercial membrane protein extraction kit from BioVisi on (Mountain View, CA). Cell pellets (~ 9.0 x 107 cells), collected after washing with PBS, were homogenized with a Doun ce cell disrupter using 100 up/down strokes in the homogenization bu ffer provided. After spinning down the homogenate at 700 x g x 10 min, the supernatant was then further centrifuged at 1000 x g x 30 min, 4oC. The final total cellular membrane protei n was dissolved in 50 l of solubilization buffer [5 mM Tris-HCl, 0.5% Triton X-100 with pr otease inhibitor cocktail (Sigma)] and stored

PAGE 24

24 at -80oC. The protein concentrations were determined colorimetrically with the DC Protein Assay kit (Bio-Rad). Affinity Purification of Antibodies Rabbits were injected with respective an tigenic peptides for production of polyclonal antibody to each Zip and ZnT transporter for pr evious studies (Table 3-1). Total IgG was prepared from serum using the Montage Anti body Purification PROSEP -A Kit (Millipore). Columns for affinity chromatography of each antibody were generated by immobilizing peptide to a Sulfo-link Coupling Gel (Pierce) bed. The cy steine added to the C-terminus allowed the peptide to conjugate to the Sulf o-link matrix during this process. 15~20 mg of total IgG was then incubated in the column overnight at 4oC, and purified by passing Elution Buffer (Pierce) through the column. The fractions (1 ml) with the highest protein concentrations as determined spectrophotometrically (NanoDrop Technologies) were pooled. Finall y, the affinity purified IgG solution was desalted into TBS containi ng 0.02% sodium azide and stored at 4oC. Western Analysis Forty g of each protein was denatured in a 2x denaturing buffer [125 mM Tris-HCl, pH 6.8, 4% (w/v) SDS, 20% (v/v) glycerol, 10% (v /v) 2-mercaptoethanol, 0.02% (w/v) bromophenol blue] for 15 min at 55oC. Denatured proteins were sepa rated by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose (Schleicher und Schuell, Dassel, Germany) or polyvinylid ene difluoride membrane (PVDF) (Immobilon; Millipore, Bedford, MA). Protein transfer and equal protein load ing were confirmed by Ponceau Red staining. After washing out the stain with TB S, membranes were blocked in 5% skim milk (0.5 g skim milk in 10 ml TBS-T) for 50 min on orbital shaker in room temperature. PVDF membranes were treated with methanol and Milli-Q water until they were completely wet before blocking. Blocked membranes were incubated in primary antibody solution (10 g IgG/ml of

PAGE 25

25 blocker or 1-2 g affinity-purif ied IgG/ml of blocker) for additi onal 1 h and washed with TBS-T 4 times for 5 min each. Membranes were then inc ubated with anti-rabbit IgG secondary antibody (1:2000 of antibody in 5% skim milk) conjugated to horseradish peroxidase (GE Healthcare, Buckinghamshire, UK) or alkalin e phosphatase (Amersham Biosci ences, Arlington Heights, IL) for 50 min. Thereafter, membranes were washed twice with TBS-T and TBS for 5 min each. Membranes probed with horseradi sh peroxidase-conjugated antibodi es were treated with an enhanced chemiluminescent substrate (SuperSig nal WestPico; Pierce, Rockford, IL), and exposed to autoradiographic film for detec tion. Blots treated with alkaline phosphataseconjugated antibodies were incubated with ECF chemifluorescent substrate (Amersham Biosciences) for visualization with a PhosphorImager (Storm 840 Imager; Mo lecular Dynamics). Specificity of bands was determined by compar ing results to those obtained with primary antibody preincubated with respective peptide solution (250 g peptide/ml of blocker). Statistical Analysis Data are expressed as mean standard de viation and were anal yzed by two-way ANOVA with time and EPO treatment as independent va riables. Differences between each treatment group at respective time-points were determined by a following Bonferroni post-test (Prism 5 for Windows; GraphPad Software, San Diego, CA). The level of statistical significance was set at P < 0.05, and differences are annotated as follows: *, P < 0.05; **, P <0.01; ***, P < 0.001.

PAGE 26

26 Table 3-1. Zinc transporters sc reened in erythrocyte ghosts and peptides used for the production and affinity purification of antibodi es against each respective protein.* Transporter Peptide ZnT1 CGTRPQVHSGKE ZnT2 CHAQKDSGSHP ZnT4 CMQLIPGSSSKWEE ZnT5 CSPSKRGQKGTLI ZnT6 CVAPNVLNFSDHHVIP Zip1 CRSGANHEASASG Zip2 CEEEWGGTHAFGFH Zip3 CAGLRLRELGRPG Zip4 CAEETPELLNPETRRL Zip10 CKRNHKCDPEKE *Antigenic peptide sequences were designed by previous and current members of Robert J. Cousins lab.

PAGE 27

27 CHAPTER 4 RESULTS Expression of Zinc Transporters in RBC Membranes According to the absence of a nucleus in circulating mature er ythrocytes, the zinc transporters expressed in RBCs wa s determined with an approach in protein levels. Membrane fractions (erythrocyte ghosts) we re prepared directly from whol e blood of adult male CD-1 mice. Purity and presence of the RBC population af ter fractionation were confirmed by light microscopy to eliminate effects of contaminati on by any other blood cells. Of the ZnT proteins tested (Table 3-1), ZnT1 was the only zinc expo rter detectable in the erythrocyte membrane fraction. As the estimated molecular mass at 30 kDa was inconsistent with that reported by previous studies (34,35), the sp ecificity of the affinity purif ied antibody to ZnT1 was further confirmed by peptide competition. Signals on the bl ot were successfully competed out when the antibody was preincubated with th e ZnT1 peptide (Fig. 4-1A). Am ong the Zip proteins screened (Table 4-1), only the presence of Zip10 was conf irmed with the expected band size suggested by other research groups (40 kDa) (36,37). The reliability of the Zip10 western analysis was also validated by peptide competition, and the single prominent band signal was blocked by the Zip10 peptide (Fig. 4-1B). Confirmation of Erythroid Differentiation Proteins in circulating RBCs are likely to be remnants of the protein components synthesized in differentiating erythroid progenitor cells. For the characterization of the responsiveness of the ZnT and Zip transporters, an in vitro model of late stage erythroid progenitors prepared from splenocytes of PHZtreated anemic CD-1 mice was utilized. Cells were collected only when splenomegaly was observe d as an indicator of PHZ-induced hemolytic anemia (Fig. 4-2).

PAGE 28

28 For the confirmation of erythroid maturation, er ythroid splenocytes we re incubated with odianisidine prior to and after EPO-stimulation for Hb-staining. An increase in the number of Hbpositive cells was observed in th e cell population incubated with EPO for 48 h (Fig. 4-3A and Fig. 4-3B). The transcript abundance of an EP O-dependent erythroid specific gene, ALAS-2, was also measured during the 48 h time-course as a more comprehensive positive marker of terminal erythroid differentiation. A temporal pattern of ALAS-2 expression, consistent to that reported by Hodges et al. (27), was detected in the EPO-treated cells The maximum ALAS-2 mRNA abundance was observed at 24 h, with a 3-fold increase afte r EPO-stimulation. The significantly higher levels in differentiating cells lasted until 48 h after addition of EPO (Fig. 43C). Effects of EPO on Zip10 a nd ZnT1 Transcript Levels Temporal trends in transporter expression dur ing the late stage erythroid differentiation were determined by qRT-PCR. Each value was normalized to 18S rRNA and the basal levels measured at 0 h of incubation. The transcript levels from cells cultu red in the presence or absence of EPO were compared at each time-point to determine the effect of EPO per se. Zip10 mRNA levels in differentiating cells showed an approximately 2-fold increase after 6h of incubation (Fig. 4-4). However, the levels were not sustained during su bsequent periods and values became less than 50% of the basal (0 h) le vels after 24 h of EPO-treatment. Cells deprived of EPO failed to show any increase in Zip10 mR NA levels throughout the entire culture period. The relative mRNA abundance in differentiating cells was significantly higher than that of resting cells from the 6 h to 24 h post-inc ubation, while there was no significant difference detectable at the following time-points. Expression trends of ZnT1 were distinctively different with those of Zip10 when cells were differentia ting. A gradual increase of ZnT1 transcripts was observed in both differentiating and resting cells until 12 h (Fig. 4-5). However, as the incubation

PAGE 29

29 period reached 24 h, the mRNA abundance in di fferentiating cells became significantly higher than that in EPO-deprived cells, and the diffe rence was sustained until the whole time-course was accomplished. The maximum abundance was also observed at 24 h, with a 3-fold higher level than the 0 h basal levels. Protein Expression of Zip10 an d ZnT1 during Differentiation The effects of EPO-induction on protein le vels of Zip10 and ZnT1 were initially determined by western analyses with total cell lysates. Decreased Zip10 protein abundance was detected after 48 h (Fig. 4-6A and Fig. 4-6B) regard less of the presence or absence of EPO. Even though the induction of Zip10 expr ession by EPO was observed in mRNA levels at early timepoints (Fig. 4-4), these differences were barely detectable in protein levels with the total cell lysates. Accordingly, a pilot experiment with total cell membrane fractions was conducted to remove any potential compromising effects by exce ssive cytosolic proteins Since the membrane fraction in protein samples became more conc entrated, the effects of EPO on Zip10 protein expression could be detected in a consistent manner to the mRNA data (Fig. 4-6C). An increase in Zip10 abundance was observed at 9 h by EPO-i nduction compared to splenocytes not treated with EPO. The protein expression trend of ZnT1 in resting cells was quite similar to that of Zip10 by having a decreased expres sion at 48 h (Fig. 4-7). Howe ver, a different trend was observed when cells were differentiating. The abundance of ZnT1 was sustained throughout the whole time-course by the EPO-mediated termin al erythroid differentiation. Consequently, a prominent difference between the ZnT1 protein leve ls in the differentiating and resting cells was detectable at the final time-poi nt, 48 h (Fig. 4-7A). In agreement with the mRNA data, these results indicate that the relative ZnT1 protein le vels to those of Zip10 te nd to be higher at the very late stage of terminal erythroid differentiation than during the preceding periods.

PAGE 30

30 Effects of EPO on MT-1 and MTF-1 Transcript Levels The temporal expression patterns of two zinc responsive genes involved in zinc metabolism gene expression were investigated. Little is known about the zinc metabolism during terminal erythroid differentiation. The effect s of EPO-induction on MT-1 mRNA abundance were determined as this protein has been reported to be a sensitive indicator of the intracellular zinc availability in various cell types (2). It was hypothesized that MT-1 mRNA abundance would also be affected either directly or indirectly by the EPO-i nduced zinc transporter expressions. Even though the MT-1 transcript levels in both cell groups decreased drastically and were sustained lower than the basal level duri ng the whole culture period, the relative abundance in differentiating cells was significan tly higher than in resting cells until 24 h of incubation (Fig. 4-8). Zinc-finger transcription fact or MTF-1 is involved in the transcriptional regulation of numerous zinc-responsive genes including MT-1 and the expression of both Zip10 and ZnT1. In differentiating cells, MTF-1 mRNA abundance star ted to increase by 6 h of EPO-induction, and then stayed relatively higher than those measured in resting cells (Fig. 4-9). It is of interest that the expression trend of MTF-1 during differentia tion revealed two peak levels, unlike trends observed in other mRNA levels. Sp ecifically, the mRNA levels reach ed its first peak at 12 h, and a subsequent decrease at 18 h followed. These peaks in MTF-1 mRNA coincided with the periods when the decrease of Zip10 and the increase of ZnT1 mRNA levels occurred.

PAGE 31

31 IgG AP AP+Peptide IgG AP AP+Peptide Figure 4-1. Zinc transporter expression in mature red blood cells. Erythrocyte ghosts were prepared for western analyses of zinc tr ansporters. Among the transporters (Zip1-4, Zip10, ZnT1-2, ZnT4-6) tested, only A) ZnT1 and B) Zip10 expre ssion were detected. The membranes were incubated with either the total IgG, affinity-purified IgG (AP), or AP that were pre-exposed to the corresponding ZnT1 or Zip10 peptide. The molecular mass of ZnT1 and Zip10 at 30 and 40 kDa, respectively, were determined with commercial molecular markers. Ther e were no signals developed by antibodies against other zinc transp orters (data not shown). 30 kDa 40 kDa A B

PAGE 32

32 A B Figure 4-2. Induction of splenomegaly by phenyl hydrazine-injection. CD-1 mice were treated with or without PHZ by in traperitoneal inj ection on day 1 and 2. Spleens were collected at day 5. A) A normal spleen and B) an enlarged spleen from PHZ-injected anemic mouse are compared.

PAGE 33

33 A B *** *** ***0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 0612182430364248 Time (h)Relative ALAS-2 mRNA /18S rRNA Level EPOEPO+ C Figure 4-3. Indicators of EPO-mediat ed terminal erythroid differentiation in vitro Hemoglobin staining of cells A) prior to and B) 48 h after EPO-treatment. C) Relative ALAS-2 mRNA abundance in EPO-treated and -dep rived cells. Splenocytes were collected from spleens of two PHZ-injected mice a nd pooled for culture at each experiment. qRT-PCR assays were performed on duplicat e total RNA samples. Values at each time-point are relative to the basal levels at 0 h. Data are expressed as mean SD of four independent experiments (n = 4). St atistically significant differences between each treatment group are annotated as ***, P < 0.001.

PAGE 34

34 *** *** ***0.0 0.5 1.0 1.5 2.0 2.5 0612182430364248 Time (h)Relative Zip10 mRNA /18S rRNA Level EPOEPO+ Figure 4-4. Relative Zip10 mRNA abundance during terminal erythroid differentiation. Splenocytes were collected from spleens of two PHZ-injected mice and pooled for culture at each experiment. qRT-PCR assa ys were performed on duplicate total RNA samples. Values at each time-point are relati ve to the basal levels at 0 h. Data are expressed as mean SD of four indepe ndent experiments (n = 4). Statistically significant differences between each trea tment group are annotated as *, P < 0.05; ***, P < 0.001. *** *** **0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 0612182430364248 Time (h)Relative ZnT1 mRNA /18S rRNA Level EPOEPO+ Figure 4-5. Relative ZnT1 mRNA abundance du ring terminal erythroid differentiation. Splenocytes were collected from spleens of two PHZ-injected mice and pooled for culture at each experiment. qRT-PCR assa ys were performed on duplicate total RNA samples. Values at each time-point are relati ve to the basal levels at 0 h. Data are expressed as mean SD of four indepe ndent experiments (n = 4). Statistically significant differences between each treatment group are annotated as **, P <0.01; ***, P < 0.001.

PAGE 35

35 + + + (EPO) 0 12 24 48 (h) 0 9 27 48 9 27 48 (h) + (EPO) 0 h EPO 9 h EPO+ 9 h Figure 4-6. Zip10 protein expres sion during terminal erythroi d differentiation. Cultured cells were collected at designated time-poi nts. A,B) Western analyses from two experiments with total cell lysates reveal a decrease in Zip10 protein expression at 48 h regardless of EPO-treatment. C) EPO-induced Zip10 expression was only detectable with total membrane fractions.1 A band with estimated molecular mass as 40 kDa was consistently observed in independent experiments. 1 Results from the total membrane fraction reflect a pilot experiment conducted (n=1). Further assessments would be appropriate to affirm the data. 40 kDa 40 kDa 40 kDa A B C

PAGE 36

36 + + + (EPO) 0 12 24 48 (h) 0 9 27 48 9 27 48 (h) + (EPO) Figure 4-7. ZnT1 protein expres sion during terminal erythroi d differentiation. Cultured cells were collected at designated time-poi nts. A,B) Western analyses from two experiments with total cell lysates from EPO-treated cells imply a constitutive expression of ZnT1 during di fferentiation, while a decrease occurs at 48 h in EPOdeprived conditions. Only the band with estimated molecular mass as 30 kDA was consistently observed in independent experiments. 30 kDa 30 kDa A B

PAGE 37

37 *** *** ***0.0 0.2 0.4 0.6 0.8 1.0 1.2 0612182430364248 Time (h)Relative MT-1 mRNA /18S rRNA Level EPOEPO+ Figure 4-8. Relative MT-1 mRNA abundance du ring terminal erythroid differentiation. Splenocytes were collected from spleens of two PHZ-injected mice and pooled for culture at each experiment. qRT-PCR assa ys were performed on duplicate total RNA samples. Values at each time-point are relati ve to the basal levels at 0 h. Data are expressed as mean SD of four indepe ndent experiments (n = 4). Statistically significant differences between each treatment group are annotated as ***, P < 0.001. *** ***0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 0612182430364248 Time (h)Relative MTF-1 mRNA /18S rRNA Level EPOEPO+ Figure 4-9. Relative MTF-1 mRNA abundance during terminal erythroid differentiation. Splenocytes were collected from spleens of two PHZ-injected mice and pooled for culture at each experiment. qRT-PCR assa ys were performed on duplicate total RNA samples. Values at each time-point are relative to the basal levels at 0 h. Data are expressed as mean SD (n = 2 x 2). 2 Statistically significant differences between each treatment group are annotated as *, P < 0.05; ***, P < 0.001. 2 Samples of the 18 h time-point, at which the fluctuation of MTF-1 mRNA levels was detected, were only available from two experiments. Thus, the results are represented as mean SD from n = biologic al duplicates x analytical duplicates.

PAGE 38

38 CHAPTER 5 DISCUSSION Studies with regard of the zi nc transport mechanism in vari ous tissues and cell types have revealed two distinct gene families related to ionic zinc trafficking pathway across cellular plasma and vesicle membranes (3). Zip and ZnT pr oteins produced from these genes facilitate the cytosolic zinc influx and efflux, respect ively, and establish the mechanism for the homeostatic regulation of intr acellular zinc. Through the tissue-specif ic and differential expression of these transporters the cellular zinc trafficking system can be modulated in response to various factors, such as the extracell ular zinc availability, intracellular utilization, and numerous cytokines, growth factors and hormones (3). Previous studies have consiste ntly reported the zinc-responsive ness of the zinc trafficking system in circulating erythrocytes of animal and human subjects (9-11). Even though these may imply regulated transporter activ ities by dietary zinc, there has been no study to define the presence of zinc transporters in circulati ng RBCs. Consequently, the primary purpose of this study was to determine which transporters are e xpressed in mature RBCs. Each transporter was screened at the protein level ut ilizing the library of antibodies to numerous zinc transporters, available in our lab. The result s from this experiment demonstrate that Zip10 and ZnT1 are expressed in circulating RBCs; thus they are likely to be the zinc transporters directly involved in the homeostatic regulation of erythroid zi nc metabolism. Although the estimated molecular mass of ZnT1 in RBCs (~30 kDa) conflicts with the value calculated from the amino acid composition (55 kDa), inconsistent molecula r mass speculated from the migration by SDSPAGE analysis has been reported by other ZnT1 studies as well (34,35). Possible explanations for the discrepancy in the aberra nt migration of ZnT1 are well -delineated in a previous study utilizing the identical anti body for ZnT1 detection (34).

PAGE 39

39 One of the most unique characteristics of circ ulating erythrocytes, compared to other cell types, is the absence of nucleus. In other words, the protein contents of mature cells are formed during preceding developmental stages, i.e., eryt hropoiesis, and the gene expression ability is deprived after maturation. Thus, the differential ac tivity of the zinc trafficking system observed in mature RBCs in response to the hosts zinc status (9-11) would be determined during the differentiation stages of earlier er ythroid cell precursors. It is of note that the expression of both zinc transporters detected in mature RBC membrane s have been suggested to be transcriptionally regulated in a zinc-dependent manner by the zinc-responsive activity of MTF-1; however, resulting in opposite modes (3). Even though further exploration is required to clarify these zinc effects on the RBC zinc transporters, it can be suggested that the modul ated erythroid zinc uptake rate during zinc deficiency may be associated with the decreased DNA binding activity of MTF-1 that results in either the up-regulation of Zip10, down-regul ation of ZnT1, or both during preceding erythroid developmental stages. Among the available cellular models of term inal erythroid differentiation, splenocytes from PHZ-treated and FVA-infected animals have been suggested to most accurately represent the physiological aspects of in vivo erythroid progenitor cells ( 27). Accordingly, the PHZ model was selected for the characterization of zinc transporter expression dur ing the EPO-mediated erythroid differentiation in the cu rrent study. EPO acts as a key factor for the initiation of further differentiation of late stage erythroid pr ogenitor cells into reticulocytes either in vivo or in vitro (22,24). The properties of EPO during the RBC pr otein production during terminal erythroid differentiation can be categori zed into two general aspects; first, the induction of de novo synthesis of certain proteins; second, the enhan cement of an ongoing production initiated at a developmental stage prior to terminal erythroi d differentiation (38). It is likely that the

PAGE 40

40 expression of Zip10 and ZnT1 are extended by EPOtreatment based on the results shown in the present study. When the erythroi d progenitor cells were deprived of EPO, despite a gradual increase of ZnT1 mRNA abundance at 12 h, th e respective mRNA levels of Zip10 and ZnT1 generally decreased throughout th e time-course examined. The final measurements, at 48 h, of both transporter mRNA levels were lower than the basal levels determined at the initial timepoint when the in vitro culture without EPO was started. These results implicate that certain levels of Zip10 and ZnT1 mR NA expressed prior to the in vitro EPO-induction during developmental stages in vivo could not be sustained when the differentiation process was discontinued. The temporal tre nd of ALAS-2 expression is know n to be induced exclusively by EPO during terminal erythroid differentiation (27,29). Because of the absence of background mRNA levels from preceding differentiation stag es, the ALAS-2 mRNA levels were stably sustained at the basal (0 h) level when furt her differentiation was bl ocked by EPO-deprivation. Previous studies with in vitro erythroid progenitor cell mode ls suggest that the gene expression patterns during differentiation strongl y reflect the functional hierarchy of the respective protein product activit ies (15,27,29,38). It was of interest that the mRNA levels of Zip10 and ZnT1 revealed different temporal patterns during the EPO-mediated differentiation in vitro While the EPO-dependent Zip10 expression occurred rapidly after the terminal erythroid differentiation was initiated, the EPO-respons iveness of ZnT1 gene expression was only detectable after 24 h of EPO-treatment. These re sults demonstrate that the zinc transporters present in mature RBCs are differentially regu lated by EPO and, thus, may be involved in the homeostatic regulation of zinc in differentiating eryt hroid progenitor cells. The hierarchical precedence of EPO-dependent Zip10 expression to th at of ZnT1 are in agreement with the zinc expenditure trend during terminal erythroid differentiation (Fig. 5-1) Specifically, various events

PAGE 41

41 that involve dynamic zinc utiliza tion, such as synthesis of zinc metalloenzymes and zinc finger transcription factors, have been shown to occur at the early stages of terminal erythropoiesis (15,27). The earlier EPO-responsiveness of Zip10 ge ne expression may be associated with an increased requirement of zinc supply based on th e metabolic use during these events (Fig. 5-1). However, after the cells reach the very late stage of te rminal erythropoiesis, the metabolic needs of zinc decrease and, additionally, free zinc ions can introduce adverse affect to heme biosynthesis by interfering with incorporation of ferrous iron into prot oporphyrin (15,21). Thus, the later EPO-dependent expressi on of ZnT1 would be a strategi c mechanism of differentiating progenitor cells to remove excessive free zinc ions and, consequentl y, ensure the normal hemoglobin biosynthesis at the final step of RBC maturation (Fig. 5-1). These expression trends of Zip10 and ZnT1 were confirmed at the protein level as well. Molecular masses of Zip10 and ZnT1 in the eryt hroid progenitor cells, sp eculated from the band migration, were corresponding to those dete rmined in mature RBCs. The ZnT1 protein expression examined with total cell lysates reveal ed a similar trend to that observed in mRNA levels as expected. However, the EPO-dependent elevation of Zip10 expr ession at the early timepoints, observed at the mRNA level, was hardly detectable within these protein samples. In addition, even though a decrease in mRNA levels occurred rapi dly after EPO-deprivation, the protein levels of Zip10 observed in the total cell lysates were sustaine d relatively longer. It is of note that these discrepancies between the mRNA and protein data were eliminated when the cytosolic protein fraction were removed from th e total cellular protein content by producing a total cellular membrane fraction. Th is implicates that effects of certain cytosolic components, which can be either internalized Zip10 protein or other cytosolic proteins that are abundant in

PAGE 42

42 erythroid progenitor cells, compromised the dete ctability of EPO-depende nt Zip10 expression in the total cell fractions. MT-1 mRNA levels monitored in the present st udy also reveal a unique temporal trend in gene expression during terminal erythroid differentiation. The zinc-responsiveness of MT-1 protein expression in differentia ting erythroblasts has been conf irmed by a previous study (4,12). Thus, it was presumed here that MT-1 mRNA leve ls may partially reflect the intracellular zinc levels regulated by the differential expression of Zip10 and ZnT1 during terminal erythroid differentiation. Although a rapid decrease occurred in both EPO-treated and -deprived cells within 6 h, MT-1 mRNA levels was sustained highe r in differentiating cells than in resting cells until 24 h. These periods correspond to the timepoints when the EPO-dependent Zip10 mRNA induction was observed. Thus, these results may pa rtially indicate that an increased intracellular zinc level was introduced by the early EPO-mediated Zip10 expression. With regard of the EPO-independent dow n-regulation of MT-1 mRNA abundance, possible explanations of this phenomenon can be derived from previous studies. Abdel-Mageed et al. showed that up-regulation of MT-1 expr ession in erythroid progenitor cells occur during the proliferation stage that pre cedes the EPO-mediated terminal erythroid differentiation (39). In addition, an inhibitory effect of MT-1 on the EP O-derived cell differentia tion was indicated (39). Conclusively, it was proposed that the expression of MT-1 transc ripts in proliferating progenitor cells should be repressed once further erythroid differentiation is committed by EPO. In another study, the dependency of MT-1 synthesis on pr oliferation was determined by measuring decreased MT levels by mitomycin-c treatment to K562 erythroleukemia cells (Huber et al., unpublished observation). This may imply the presen ce of an intrinsic factor that induces MT-1 specifically during the prolifera tion of erythroid progenitor cells Thus, the rapid repression of

PAGE 43

43 MT-1 mRNA levels observed in the present study would be rela ted to the remnants from the abundant MT-1 mRNA level expr essed during the proliferation in vivo and the absence of the proliferation-dependent MT-1 inducing factor in vitro As mentioned above, the associ ation of MTF-1 activity with the transcriptional regulation of Zip10 and ZnT1 has been suggested by pr evious studies. Up-re gulation of Zip10 and repression of ZnT1 expression has been obser ved in MTF-1-/hepatocytes and embryos, respectively (3). Thus, as both Zip10 and ZnT1 are shown to be differentially expressed in maturing erythroid progenitor cells it was of interest to determ ine whether EPO-responsive of MTF-1 gene expression occurs during terminal er ythroid differentiation. Th e results presented in the current study reveal certain interrelations of Zip10 and ZnT1 transcript levels to EPOdependent MTF-1 mRNA abundance. Peaks observ ed in the temporal pattern of MTF-1 transcription in differentiating cells corresponde d to the decrease and increase in Zip10 and ZnT1 mRNA levels, respectively. Although the e ffects of EPO on MTF-1 activity in erythroid progenitor cells need to be further explore d, these results suggest that EPO-dependent transcription of MTF-1 would be involved in the regulatory mech anism of the differential Zip10 and ZnT1 expression duri ng erythroid maturation. Overall, the presence of er ythroid zinc transporters, as Zip10 and ZnT1, has been demonstrated in the current study. Furthermore, EP O-mediated expression of these transporters was confirmed in differentiating erythroid proge nitor cells. Several suggestions for future approaches, particularly, with cl inical perspectives can be deri ved from these results. The zinc uptake rate of erythrocytes in vitro has been suggested to be a su itable indicator of early dietary, subclinical zinc deficiency (11) Thus, the differential expression of these transporters in RBCs, which are likely to be zinc-res ponsive, could be another candida te parameter for the assessment

PAGE 44

44 of dietary zinc status. In addition, the expression of these zinc transporters could be connected to the rigorous modulation of RBC in tracellular zinc levels during Plasmodium falciparum parasitemia (40,41). In other words, the abnormal zinc sequestration in malarial RBCs would be possibly caused by a transformation in the host ce ll zinc trafficking syst em, which may involve Zip10 and ZnT1 activities, by the parasite infecti on. Finally, to some extent, the EPO-responsive Zip10 expression observed in this study may support the suggestions from studies related to the metastasis of breast cancer. Recently, it has been shown that EPO recepto rs (EPO-R) are highly expressed in breast carcinoma, while the expression levels in benign mammary tissues are generally negative (42). Alt hough the functionality of EPO-R on these cancer cells remains controversial, it has been associated with the stimulatory effect of EPO on the cell migration activity (43). Expression of Zip10 in breast carcinoma has been reported to be essential for the migratory and invasive activity of breast cancer cells (44); howeve r, the molecular mechanism of Zip10 induction has not been unders tood. Based on the results of th e present study and evidence mentioned above, the induction of Zip10 expressi on by EPO may be a possible explanation for the EPO-R mediated metastasis of breast cancer cells.

PAGE 45

45 Figure 5-1. Putative model for the contribution of erythr oid zinc transporters to the homeostatic regulation of zinc during terminal erythr oid differentiation. EPO binds to EPO-R and induces the initiation of terminal erythroi d differentiation. During the early stage of terminal erythroid differentiation Zip10 level is relatively higher than that at the late stage. Intracellular Zn2+ 1) inhibits Ras-Raf signa ling pathway and leads EPOmediated differentiation; 2) incorporates into CA and zinc finger transcription factors. During the hemoglobin biosynthetic pathwa y, down-regulation of Zip10 occurs while ZnT1 level is relatively sustained. Thus, excessive Zn2+ is removed and abnormal ZPP accumulation is prevented.

PAGE 46

46 LIST OF REFRENCES 1. Prasad AS. Recognition of zinc-deficiency syndrome. Nutrition. 2001;17:67-9. 2. Cousins RJ. Zinc. In: Bowman BA, Russell RM, editors. Present knowledge in nutrition. 9 ed. Washington, D.C.: International Li fe Sciences Institute; 2006. p. 445-57. 3. Cousins RJ, Liuzzi JP, Lichten LA. Mammalia n zinc transport, trafficking, and signals. J Biol Chem. 2006;281:24085-9. 4. Grider A, Bailey LB, Cousins RJ. Erythrocyte me tallothionein as an index of zinc status in humans. Proc Natl Acad Sci U S A. 1990;87:1259-62. 5. Ohno H, Doi R, Yamamura K, Yamashita K, Iizuka S, Taniguchi N. A study of zinc distribution in erythrocytes of normal humans. Blut. 1985;50:113-6. 6. Horn NM, Thomas AL, Tompkins JD. The effe ct of histidine and cy steine on zinc influx into rat and human erythrocytes J Physiol. 1995;489 (Pt 1):73-80. 7. Kalfakakou V, Simons TJ. Anionic mechanis ms of zinc uptake across the human red cell membrane. J Physiol. 1990;421:485-97. 8. Simons TJ. Calcium-dependent zinc e fflux in human red blood cells. J Membr Biol. 1991;123:73-82. 9. De KJ, Van Der SC, Veldhuizen M, Wolterb eek HT. The uptake of zinc by erythrocytes under near-physiological conditions. Bi ol Trace Elem Res. 1993;38:13-26. 10. Sasser LB, Bell MC, Jarboe GE. Influence of acute tissue injury on in vitro incorporation of Zn by sheep erythrocytes. J Anim Sci. 1975;41:1679-85. 11. Van Wouwe JP, Veldhuizen M, De Goeij JJ, Van den Hamer CJ. Laboratory assessment of early dietary, subclinical zi nc deficiency: a model study on weaning rats. Pediatr Res. 1991 ;29:391-5. 12. Huber KL, Cousins RJ. Zinc metabolism a nd metallothionein expr ession in bone marrow during erythropoiesis. Am J Physiol. 1993;264:E770-E775. 13. Hodge D, Coghill E, Keys J, Maguire T, Hartmann B, McDowall A, Weiss M, Grimmond S, Perkins A. A global role for EKLF in defi nitive and primitive er ythropoiesis. Blood. 2006;107:3359-70. 14. Ferreira R, Ohneda K, Yamamoto M, Philipsen S. GATA1 function, a paradigm for transcription factors in hematopoi esis. Mol Cell Biol. 2005;25:1215-27. 15. Welch JJ, Watts JA, Vakoc CR, Yao Y, Wang H, Hardison RC, Blobel GA, Chodosh LA, Weiss MJ. Global regulation of erythroid gene expression by transcription factor GATA-1. Blood. 2004;104:3136-47.

PAGE 47

47 16. Tomoda T, Nomura I, Kurashige T, Kubonish i I, Miyoshi I, Sukena ga Y, Taniguchi T. Changes in Cu,Zn-superoxide dismutase ge ne during induced er ythroid and myeloid differentiation. Acta Haematol. 1991;86:183-8. 17. Nishiyama S, Irisa K, Matsubasa T, Higa shi A, Matsuda I. Zinc status relates to hematological deficits in middle-ag ed women. J Am Coll Nutr. 1998;17:291-5. 18. Nishiyama S, Kiwaki K, Miyazaki Y, Hasuda T. Zinc and IGF-I concentrations in pregnant women with anemia before and after supplem entation with iron and/or zinc. J Am Coll Nutr. 1999;18:261-7. 19. Forman WB, Sheehan D, Cappelli S, Coffman B. Zinc abuse--an unsuspected cause of sideroblastic anemia. West J Med. 1990;152:190-2. 20. Fiske DN, McCoy HE, III, Kitchens CS. Zinc -induced sideroblastic anemia: report of a case, review of the literature, and descripti on of the hematologic syndrome. Am J Hematol. 1994;46:147-50. 21. Bloomer JR, Reuter RJ, Morton KO, We hner JM. Enzymatic formation of zincprotoporphyrin by rat liver and its potentia l effect on hepatic heme metabolism. Gastroenterology. 1983;85:663-8. 22. Kaushansky K. Lineage-specific hemat opoietic growth factors. N Engl J Med. 2006 ;354:2034-45. 23. Wojchowski DM, Menon MP, Sathyanarayana P, Fang J, Karur V, Houde E, Kapelle W, Bogachev O. Erythropoietin-dependent eryt hropoiesis: New insights and questions. Blood Cells Mol Dis. 2006;36:232-8. 24. Krantz SB. Erythropoietin. Blood. 1991;77:419-34. 25. Labbe RF, Rettmer RL. Zinc protoporphyrin : a product of iron-defi cient erythropoiesis. Semin Hematol. 1989;26:40-6. 26. Alcindor T, Bridges KR Sideroblastic anaemias. Br J Haematol. 2002;116:733-43. 27. Hodges VM, Winter PC, Lappin TR. Erythr oblasts from friend virus infectedand phenylhydrazine-treated mice accurately model erythroid differentiation. Br J Haematol. 1999;106:325-34. 28. Cooper MC, Levy J, Cantor LN, Marks PA, Rifkind RA. The effect of erythropoietin on colonial growth of erythroid precursor cel ls in vitro. Proc Natl Acad Sci U S A. 1974;71:1677-80. 29. Dolznig H, Boulme F, Stangl K, Deiner EM, Mikulits W, Beug H, Mullner EW. Establishment of normal, terminally differen tiating mouse erythroid progenitors: molecular characterization by cDNA arrays. FASEB J. 2001;15:1442-4.

PAGE 48

48 30. Piao F, Yokoyama K, Ma N, Yamauchi T. S ubacute toxic effects of zinc on various tissues and organs of rats. Toxicol Lett. 2003;145:28-35. 31. Levengood JM, Sanderson GC, Anderson WL, Foley GL, Brown PW, Seets JW. Influence of diet on the hematology and serum biochemist ry of zinc-intoxicated mallards. J Wildl Dis. 2000;36:111-23. 32. Witeska M, Kosciuk B. The changes in co mmon carp blood after shortterm zinc exposure. Environ Sci Pollut Res Int. 2003;10:284-6. 33. Lukaski HC. Low dietary zinc decreases erythrocyte carbonic a nhydrase activities and impairs cardiorespiratory function in men during exercise. Am J Clin Nutr. 2005;81:104551. 34. McMahon RJ, Cousins RJ. Regulation of the zi nc transporter ZnT-1 by dietary zinc. Proc Natl Acad Sci U S A. 1998;95:4841-6. 35. Kim AH, Sheline CT, Tian M, Higashi T, McMahon RJ, Cousins RJ, Choi DW. L-type Ca(2+) channel-mediated Zn(2+) toxicity and modulation by ZnT-1 in PC12 cells. Brain Res. 2000;886:99-107. 36. Kaler P, Prasad R. Molecular cloning and functional characterization of novel zinc transporter rZip10 (Slc39a10) involved in zinc uptake across rat renal brush-border membrane. Am J Physiol Renal Physiol. 2007;292:F217-F229. 37. Pawan K, Neeraj S, Sandeep K, Kanta RR, Rajendra P. Upregulation of Slc39a10 gene expression in response to thyroid hormones in intestine and kidney. Biochim Biophys Acta. 2007;1769:117-23. 38. Koury MJ, Bondurant MC, Mueller TJ. The role of erythropoietin in the production of principal erythrocyte protei ns other than hemoglobin during terminal erythroid differentiation. J Cell Ph ysiol. 1986;126:259-65. 39. Abdel-Mageed AB, Zhao F, Rider BJ, Agrawal KC. Erythropoietin-induced metallothionein gene expression: role in proliferation of K562 cells. Exp Biol Med (Maywood ). 2003;228:1033-9. 40. Ginsburg H, Gorodetsky R, Krugliak M. Th e status of zinc in malaria (Plasmodium falciparum) infected human red blood cells: stage dependent accumulation, compartmentation and effect of dipicolin ate. Biochim Biophys Acta. 1986;886:337-44. 41. Hiremath GS, Sullivan DJ, Jr., Tripathi AK, Black RE, Sazawal S. Effect of Plasmodium falciparum parasitemia on erythrocyte zi nc protoporphyrin. Clin Chem. 2006;52:778-9. 42. Acs G, Zhang PJ, Rebbeck TR, Acs P, Verma A. Immunohistochemical expression of erythropoietin and erythropoiet in receptor in breast car cinoma. Cancer. 2002;95:969-81.

PAGE 49

49 43. Lester RD, Jo M, Campana WM, Gonias SL. Erythropoietin promotes MCF-7 breast cancer cell migration by an ERK/mitogen-activat ed protein kinase-dependent pathway and is primarily responsible for the increase in migration observed in hypoxia. J Biol Chem. 2005;280:39273-7. 44. Kagara N, Tanaka N, Noguchi S, Hirano T. Zinc and its transporter ZIP10 are involved in invasive behavior of breast cance r cells. Cancer Sci. 2007;98:692-7.

PAGE 50

50 BIOGRAPHICAL SKETCH Moon-Suhn Ryu was born on March 28, 1979 in Se oul, South Korea. He attended Yonsei University in Seoul from 1997 to 2001. Upon gra duation with his Bachelor of Science in Biotechnology, Moon-Suhn joined the Republic of Ko rea Air Force to fulfill his military service required by the South Korean government. Since discharging his duty, Moon-Suhn worked for a beverage company, Lotte Chilsung Beverage Compa ny Limited, in South Korea as an assistant manager in the overseas business team. He came to the United States in the fall of 2005 to start his masters study in nutritional sciences at the University of Florida. Moon-Suhn is planning to continue his graduate studies in the doctorate program for nutriti onal sciences at the University of Florida.