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Food Science and Human Nutrition Department and Center for Nutritional Sciences and * Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville FL 32611-0370
3To whom correspondence should be addressed. E-mail: rjcousins{at}mail.ifas.ufl.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • zinc deficiency • thymus • cDNA array • FACS • murine
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Classic symptoms of human zinc deficiency include diarrhea and dermatitis, attributable to failure of the barrier component of innate immunity, and in addition, thymic atrophy, lymphopenia and decreased host resistance to infectious disease, attributable to failure of cell-mediated immunity (2
). Early animal studies demonstrated that subsequent to the thymic cortical involution and atrophy induced by zinc deficiency, T-lymphocyte–mediated responses, both cytotoxic and helper activities, were severely impaired (3
,4
). Furthermore, these changes were reversed by restoration of adequate zinc status (5
). Loss of precursor cells in bone marrow and thymus is believed to result in host inability to replenish peripheral lymphocytes (lymphopenia) and to be responsible for the increased susceptibility to infectious disease observed secondarily to zinc deficiency (1
). Although flow cytometric work with bone marrow cells from zinc-deficient mice has suggested that apoptosis may be responsible for the preferential loss of precursor B cells (6
), molecular mechanisms for the effects of zinc deficiency on the thymus and T-lymphocyte compartment remain to be defined.
Zinc is an established mediator of gene expression through both defined direct and, as yet poorly understood, indirect mechanisms (7
). The last decade has heralded tremendous technological advances in gene sequence and expression analysis (structural and functional genomics), which have provided approaches to answer questions about the influence of zinc on a more global level. In particular, within the last few years, mRNA differential display has been used to characterize the gene expression profile of rat small intestine during zinc deficiency (8
,9
). These results with mRNA differential display support the idea that zinc deficiency alters the expression of multiple mRNA species with functional physiologic significance. For example, this research identified, among many expressed sequence tags, preprouroguanylin mRNA, precursor to the intestinal hormone uroguanylin, as upregulated in zinc-deficient rats (10
). Subsequently, prouroguanylin protein levels were found to be elevated in the villi of zinc-deficient rat intestine (11
), substantiating this finding as a potential molecular mechanism contributing to diarrhea symptoms associated with human zinc deficiency. Additional research will further define the role of dietary zinc in modulating mammalian gene expression and its influence on host health and disease.
The advent of DNA array technology now permits high throughput, parallel expression profiling of hundreds to thousands of genes or expressed sequence tags simultaneously (12
). Greatly facilitated by the influx of structural information provided from the various sequencing initiatives, cDNA arrays permit functional interpretation of the transcriptome state of any given cell or tissue type at a particular physiologic state. These cellular mRNA expression profiles yield global genomic fingerprints that identify the biological state of that cell (12
).
In this paper we report the first expression profile analysis of zinc-deficient murine thymus, utilizing arrays with cDNAs from genes with characterized roles in cellular physiology. The rationale for choosing a mouse model was the wealth of available immunological markers and known murine sequence information. In deciding to explore differential gene expression in zinc-deficient thymus, our objective was to observe initial changes in gene expression rather than consequential changes resulting from the deficient state itself. In the model of moderate zinc deficiency developed for these experiments, mice do not exhibit either the altered eating behavior or altered growth patterns associated with severe zinc deficiencies, nor do they demonstrate changes in thymocyte populations expressing the surface markers CD3, CD4, or CD8. However, our results with array screening and post-hoc confirmations demonstrate that this moderate level of dietary zinc deficiency is, nonetheless, sufficient to alter thymic mRNA and protein abundances in vivo.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Young adult (30 ± 3 g, 
6 wk old) male outbred CD-1 mice (Charles River, Wilmington, MA) were maintained individually in hanging stainless steel cages on a 12-h light:dark cycle with free access to distilled, deionized water. Mice were initially fed an AIN-76a–based (13
) pelleted diet containing 5 mg Zn/kg diet (Research Diets, New Brunswick, NJ) for 3–5 d of acclimation. Then mice were randomly assigned to one of three dietary groups, i.e., zinc deficient (-Zn,4
<1 mg Zn/kg), zinc adequate, ad libitum consumption (ZnN, 30 mg Zn/kg), or zinc adequate, pair-fed to the zinc-deficient group (PF, 30 mg Zn/kg). After a 3-wk feeding period, between 0900–1200 h, mice were anesthetized with methoxyflurane, killed by exsanguination and blood collected for subsequent serum zinc measurement by flame atomic absorption spectrophotometry. All animal studies were approved by the University of Florida Institutional Animal Care and Use Committee.
Metallothionein protein measurement and RNA isolation.
Pancreas was homogenized in 4 volumes of 10 mmol/L Tris containing a protease inhibitor cocktail without EDTA (Sigma Chemical, St. Louis, MO) with a Potter Elvehjem homogenizer. Pancreas metallothionein (MT) levels were measured by the cadmium/hemoglobin affinity assay (14
) as described before (15
). Whole thymus (250 mg) was homogenized in 4 mL TRIpure reagent (Boehringer Mannheim, Indianapolis, IN) and total RNA isolated. RNA concentration was determined by spectrophotometry, and RNA integrity confirmed by UV visualization of EtBr-stained ribosomal bands after electrophoresis in a 1% Agarose/1X MOPS/2.2 mol/L formaldehyde gel (8
).
Fluorescent activated cell sorting (FACS).
In separate experiments, single-cell thymocyte suspensions (1–2 x 109 cells/L) from individual mice (n = 9–10/treatment) were triple stained with phycoerythrin conjugated anti-CD3, fluorescein isothiocyanate conjugated anti-CD4, and Cy-Chrome (CyC) conjugated anti-CD8 monoclonal antibodies (BD PharMingen, San Diego, CA). Background fluorescence was established using the appropriate fluorochrome conjugated immunoglobulin G isotype standards (BD PharMingen). All analyses were performed on a FACScan (BD Immunocytometry Systems, San Diego, CA) instrument at the University of Florida ICBR Flow Cytometry Core.
cDNA array analysis.
Equal amounts of total RNA from either -Zn or ZnN mice (n = 7/treatment group) were pooled and DNase-treated (Boehringer Mannheim) in 40 mmol/L Tris-HCL (pH 7.5), 10 mmol/L NaCl and 6 mmol/L MgCl for 30 min at 37°C. The reaction was terminated with 10 mmol/L EDTA and 100 mg/L glycogen, and RNA extracted with phenol/chloroform/isoamyl (25:24:1, pH 4.5) and precipitated with 2 mol/L NaOAc and 95% ethanol. After resuspension, poly A+ RNA was isolated with Qiagen Oligotex spin columns (Valencia, CA). Recovered poly A+ RNA was ethanol precipitated, resuspended, quantified and assessed for quality as above.
Atlas Mouse 1.2 nylon membrane arrays (Clontech, Palo Alto, CA) containing 1185 partial cDNAs, from genes with known functions in cellular physiology, spotted individually at 10 ng/spot were used for these experiments. Complex probe syntheses and array hybridizations were performed precisely according to manufacturer’s protocol. Briefly, for first-strand cDNA probe synthesis, 1 µg of poly A+ RNA, either -Zn or ZnN, was incubated with cDNA synthesis primer mix (1.6 µL) and converted to cDNA using Moloney murine leukemia virus reverse transcriptase in a reaction with >2500 Ci/mmol [
-33P]dATP (>92.5 TBq; NEN, Boston, MA). Probes were purified by column chromatography, and radioactivity measured by liquid scintillation counting. Probe incorporation levels were within 1–5 x 106 dpm for each cDNA population and, for three separate hybridizations, probe incorporation levels measured between 25 and 50 x 106 dpm. Arrays were prehybridized with ExpressHyb (Clontech) containing sheared salmon testes DNA (Sigma) before addition of denatured probe. Arrays were hybridized overnight (16–20 h) at 68°C, washed according to Clontech’s specifications and exposed to a phosphorimaging screen.
Phosphorimages were scanned on a Storm Imager (Molecular Dynamics, Piscataway, NJ) and densitometries analyzed with AtlasImage software (Clontech). Signal intensities between arrays were normalized by global summation. In this method, a normalization coefficient is calculated from the summation of adjusted intensities (intensity minus background) for all genes on one array (in this case -Zn) divided by the summation of adjusted intensities for all genes on the second array (ZnN). This coefficient was then applied to adjusted intensities of the individual genes on the second array. After normalization, adjusted intensities were exported into Excel (Microsoft, Redmond, WA) for further statistical analyses.
Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR).
Pooled (n = 5), DNase-treated total RNA (1.1 µg), isolated from mice distinct from those used for array hybridizations, was incubated with 500 ng oligo (dT)12–18 primers (GibcoBRL, Gaithersburg, MD) for 10 min, followed by reverse transcription with SUPERSCRIPT II RNase H- Reverse Transcriptase (GibcoBRL). Primer sequences used for PCR amplification were obtained from Clontech, and oligonucleotides synthesized by Gemini Biotech (Alachua, FL). Primers used were for the following (Table 1
): glyceraldehyde-3-phosphate-dehydrogenase (GAPDH); myeloid cell leukemia sequence-1 (MCL-1); lymphocyte-specific protein tyrosine kinase (LCK); DNA damage repair and recombination protein 23B (RAD23B); and mouse laminin receptor (MLR). Using Clontech’s recommended protocol, PCR was performed with Taq DNA polymerase (Boehringer Mannheim) with aliquots removed at 22, 27, 32 and 37 cycles. PCR products were electrophoresed on a 1.5% agarose gel, which was subsequently stained for 60 min with SYBR Green I (Molecular Probes, Eugene, OR) and scanned on the Storm Imager.
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All primers and the TaqMan probe were designed using Primer Express software version 1.0 (PE Applied Biosystems, Foster City, CA) and designed to overlap gene regions amplified by Clontech’s primers. Primers were synthesized by Applied Biosystems and were for the following (Table 1)
: mouse metallothionein-1 (MT1); MCL-1; LCK; RAD23B; and MLR. Primers and TaqMan probe for 18S rRNA gene were purchased from PE Biosystems and used as the endogenous control for initial, total RNA abundance normalizations.
All assays were performed using one-step RT-PCR reagents and a GeneAmp 5700 Sequence Detection System, all from PE Applied Biosystems, and relative quantitation was computed from a 4–5 log range standard curve generated from 1:10 serial dilutions of total RNA. Samples were run in triplicate, and amplicon specificity for the SYBR assays confirmed by the presence of a single peak in the first derivative of primer melt curve analysis for each assay. Total RNA (1–3 ng), isolated from individual -Zn or ZnN mice, again distinct from those used in array and semiquantitative PCR experiments, was used for these confirmation experiments. The MT1 TaqMan assay was performed using 900 nmol/L each of the forward and reverse primers and 250 nmol/L of specific MT1 TaqMan probe, whereas the 18S rRNA TaqMan assay utilized 50 nmol/L forward and reverse primers and 200 nmol/L TaqMan probe, and all SYBR assays used 50 nmol/L forward and reverse primers.
Western analysis.
Whole thymus, isolated from either -Zn or ZnN mice, was immediately homogenized in 20 mmol/L HEPES, pH 7.4, 500 mmol/L EDTA, 300 mmol/L mannitol and 5% protease inhibitor cocktail (P2714; Sigma). Samples were centrifuged for 20 min at 100,000 x g, and the membrane pellet was resuspended in the HEPES buffer, pH 7.4. After a second brief (2 min) centrifugation at 250 x g, supernatant was taken and the protein concentration was determined (16
). An equal amount of membrane fraction from each individual mouse was pooled within treatment groups (n = 7–10), resolved on a 10% SDS-PAGE gel and subsequently electroblotted to Immobilon-P as previously described (17
). A mouse monoclonal anti-LCK antibody (Upstate Biotechnology, Lake Placid, NY) was the primary antibody (1 mg/L), and anti-mouse IgG horseradish peroxidase conjugate (Sigma) was the secondary antibody. Detection was by fluorescence imaging using ECF (Amersham Pharmacia Biotech, Piscataway, NJ) and the Storm Imager.
Statistical analysis.
Food intake and body weight data were analyzed by repeated-measures ANOVA using mixed model methodology (18
). Comparisons between -Zn and ZnN treatment groups were by two-tailed Student’s t test (Instat, Graphpad, San Diego, CA), with significance established at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Initial diet studies included a zinc-adequate group pair-fed (PF) to zinc-deficient (-Zn) mice; however, three replicate diet studies (n = 5/treatment group) demonstrated that 3 wk of dietary zinc restriction in these young adult outbred mice did not permute food intake (Fig. 1A
) or body weight (Fig. 1B
). Consequently, this group was dropped from later experiments in which comparison periods were of 3 wk duration. Zinc homeostasis was assessed by three separate biochemical indices, i.e., serum zinc, thymic MT1 mRNA and pancreas MT protein levels, all of which were significantly (P < 0.004) depressed in -Zn mice (Table 2
) after 3 wk of diet treatment. However, there were no differences between the thymic weights of -Zn mice and ZnN mice (Table 2)
.
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An additional consideration for the dietary time frame was whether thymic atrophy had begun. Because our objective was to observe zinc-mediated changes in mRNA levels rather than those resulting from alterations in cell populations occurring during severe thymic involution, we used FACS analysis to examine the thymocyte populations expressing the surface glycoproteins CD3, CD4 and CD8 (Fig. 2
). FACS analysis established that there was no cellular loss in the -Zn mice nor were there any perturbations in the primary developmental populations examined, which further substantiated 3 wk of zinc deficiency in these mice as modest in effect (Fig. 2)
. After 4 wk of deficiency, there was no significant difference in the total number of CD3+ cells (data not shown). These data gave us confidence that observed changes in mRNA abundance were due to zinc deficiency and not gross alterations in thymocyte populations.
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Array hybridizations resulted in detection of 230 cDNAs with adjusted intensities (total signal minus background) above twofold (200%) of background levels (Fig. 3
). Linear regression of normalized cDNA intensities from both groups (Fig. 4
) established a trendline equation of y = 1.087x + 10 and a correlation coefficient of R2 = 0.988, further highlighting the similarity of expression levels between -Zn and ZnN mice. This strong correlation also demonstrated lack of experimental noise in our animal model, which increased the probability that cDNAs deviating from the line of normality were not spurious. Those cDNAs demonstrating greater than a 1.5-fold change were considered as candidates for post-hoc confirmation. Those confirmed as zinc modulated are circled (Figs. 3
and 4)
, i.e., 1) MCL-1; 2) LCK; 3) MLR; 4) RAD23B.
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Our initial confirmation of zinc-regulated candidates identified by cDNA array analysis was by semiquantitative RT-PCR using the same Clontech primers that were used to produce the cDNA fragment spotted on the array. However, RT-PCR was performed using pooled total RNA derived from -Zn and ZnN mice from a separate diet study rather than the RNA used for the array experiments. This qualitative assessment confirmed the upregulation of LCK, RAD23B and MLR, compared with identical expression of GAPDH (Fig. 5
). Subsequently, we designed our own primers for use in real-time quantitative RT-PCR (Q-PCR), which permits more accurate quantification from the exponential phase of PCR amplification and the use of individual rather than pooled samples, to accurately quantify these perturbations in expression. Our results from Q-PCR (Fig. 6
), performed using RNA from individual mice (n = 5–10/treatment) independent of those used in previous experiments, confirmed zinc-mediated modulation for all four genes, with the greatest intragroup variation, expected as a heterogeneous response to a pathology, seen in the zinc-deficient mice.
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The previous identification of the zinc-binding LCK protein as elevated in zinc-deficient peripheral lymphocytes (19
) prompted our further investigation of the protein status in developing thymocytes. Western analysis of LCK protein after 3 wk of deficiency demonstrated an 80% increase in LCK protein abundance in the -Zn mice (Fig. 7
) relative to ZnN mice.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Having established the dietary framework for this transcriptional analysis of zinc-deficient murine thymus, we chose to use cDNA arrays containing cDNAs from genes with identified roles in cellular physiology. The moderate dietary treatment was underscored by the very close correlation between the expression profiles of the -Zn and ZnN mice. Nonetheless, at this level of in vivo zinc deficiency, array screening identified several potential zinc-regulated candidates, which we confirmed by RT-PCR subsequently, not only using two different techniques but in two independent animal populations. Identified by array screening and confirmed as zinc responsive were the following (relative to ZnN): myeloid cell leukemia sequence-1, found to be depressed (0.6-fold); DNA damage repair and recombination protein-23B, found to be elevated (1.8-fold); the mouse laminin receptor, also elevated (2.3-fold); and last, the lymphocyte-specific protein tyrosine kinase, LCK, also found to be elevated (1.5-fold).
Several factors prompted us to examine genes with less than a twofold change. First, a twofold designation is arbitrary and often dictated by signal-to-noise ratio, which in our system was extremely high. Second, previous research (7
–10
,20
) has demonstrated that nutritional effects on gene expression in vivo are smaller in nature than those associated with development or oncogenic transformation. This is further supported by the statistical significance of the 40% reduction in thymic MT1 mRNA observed in this study. Third, our approach to array analysis was based also on our previous research with differential display (8
). Specifically, we consider the display/array as a primary screening tool, and only mRNAs whose zinc modulation was confirmed independently are reported as differentially expressed. However, perhaps the most compelling reason for us to examine cDNAs showing less than a twofold difference was the array identification of a 1.5-fold increase in LCK mRNA in our zinc-deficient mice. This lymphocyte-specific protein tyrosine kinase associates with the cytoplasmic tail of the CD4 receptor through thiol-mediated tetrahedral coordination of a Zn2+ ion (21
). In addition, the LCK protein was previously identified by Lepage and co-workers (19
) as upregulated in murine splenic lymphocytes during dietary zinc deficiency. These two aspects of LCK expression/function suggested that the 1.5-fold increase observed in our system was worthy of further investigation.
Our array confirmation data demonstrate that smaller fold changes in mRNA populations can be reproducible, and support the hypothesis that not exploring a less than twofold difference in expression may preclude changes with functional importance from consideration. Indeed, Tusher et al. (22
), in developing a method for statistical analysis of microarray data, articulated inadequacies with "fold change" analysis. They demonstrated that the low signal-to-noise ratio seen at low levels of expression, where most genes are expressed, means that twofold changes occur randomly for a large percentage of genes and are associated with a false discovery rate of 81%. Furthermore, they propose that for genes expressed at higher abundance, stoichiometrically smaller changes in gene expression are likely to be significant but are rejected from consideration by a twofold cut-off value. In the array experiments described here, genes detected were predominantly those exhibiting medium-to-high expression levels and very low noise was encountered.
Although the functional importance associated with these observed, reproducible changes in mRNA and protein abundances in zinc-deficient thymus will be further defined by additional research, currently several intriguing observations can be made regarding identified roles for these genes and the potential for a zinc-mediated interaction. Clearly, because LCK is dependent upon zinc for its cytosolic binding, and therefore downstream signal transduction of both the CD4 and CD8 coreceptors (21
,23
), it emerges as a candidate likely to be influenced directly by zinc supply. As a lymphocyte-specific protein tyrosine kinase, LCK not only plays an essential role in the T-cell receptor-linked signal transduction pathways associated with peripheral T-lymphocyte activation (24
), but also is expressed in the thymus at all stages of thymocyte development. Furthermore, from studies in LCK knockout mice, it appears critical for the selection and maturation of developing thymocytes (25
).
Dietary zinc insufficiency has already been reported to increase expression of LCK in peripheral splenic T-lymphocytes (19
); in this report, we provide evidence for its modulation at both the mRNA and protein levels by dietary zinc supply in developing thymocytes. It is plausible that a disruption of the zinc-mediated interaction between LCK and the CD4/CD8 coreceptors in the cytosol results in a feedback signal to upregulate LCK mRNA expression. Such a signal disruption provides a potential mechanism for the zinc deficiency–associated loss of developing thymocytes, and warrants future study. Alternatively, the recent identification of a Kruppel-type zinc finger protein, mtß, as the essential transcriptional activator required for thymus-specific expression of LCK (26
) provides another potential mechanistic avenue to explain the observed zinc-mediated increase in LCK mRNA levels reported here. Of particular relevance is that a reduction in thymic MT expression is concomitant with other alterations observed. Metallothionein, acting as a zinc donor/acceptor, could be a factor in decreasing the availability of zinc necessary for LCK/CD4, LCK/CD8 binding, as has been proposed (23
), or for zinc necessary for structure/function of the mtß transcription factor [a function envisioned from the experiments of Roesijadi et al. (27
)].
For the remainder of the zinc-modulated transcripts identified in this study, a direct zinc interaction is not immediately apparent. MCL-1 is a member of the apoptosis-related BCL-2 family of proteins, originally isolated as an early induction gene from human myeloid cells (28
). It forms heterodimers preferentially with proapoptotic BCL-2 family members (29
), and induction of MCL-1 is associated with a rapid and transient increase in cell viability, suggestive of a permissive environment for hematopoietic differentiation and an antiapoptotic function for MCL-1 (30
). Recently, however, two independent investigations have demonstrated that alternative splicing of the mcl-1 human gene, skipping exon 2, results in a protein variant having proapoptotic function (31
,32
). Because both sets of primers were designed for an amplicon within the 3' untranslated region of the MCL-1 transcript, which variant is affected here cannot be predicted. However, the depression of MCL-1 mRNA observed in these experiments supports a role for apoptosis in zinc deficiency–associated lymphopenia, paralleling data regarding the halt of B-lymphocyte development through apoptotic mechanisms in the bone marrow of zinc-deficient mice (6
).
Although there is relatively little known about the mouse laminin receptor (MLR), it is relevant to note that increased protein expressions of both MLR and MCL-1 have been defined as immunohistochemical markers of tumor metastatic potential. Moreover, MLR is associated with preleukemic thymuses (33
) and MCL-1 with thymic carcinomas (34
,35
). However, MLR, a 67-kDa protein expressed on the cell surface, is formed through the dimerizing of its cytoplasmic precursor, a 37- to 40-kDa protein found tightly associated with the 40S ribosome, i.e., the LBP/p40 (laminin binding protein precursor p40) (36
). Highly conserved, the yeast homologs Rps0A and Rps0B are essential components of the 40S ribosomal subunit (37
), implicating a role for the LBP/p40 in translation.
Similarly, RAD23B (also MHR23B for mouse homolog to RAD23B), identified as a nucleotide excision repair gene product in Saccharomyces cerevisiae, has evolved additional functions in mammals (38
). Expressed constitutively in all tissues, RAD23B interacts with the regulatory S5a subunit domain of the 26S proteasome through its N-terminal ubiquitin-like domain (38
,39
). In addition, very recent work has demonstrated a RAD23B interaction with ubiquitin, which is mediated by its duplicated, highly conserved, C-terminal ubiquitin-associated domain (40
). This supports other work indicating that RAD23B inhibited multiubiquitin formation and proteolytic degradation (41
). Although a direct zinc-mediated interaction is not immediately apparent, in light of zinc’s structural role for so many proteins and our previous data identifying the proteasomal ATPase as increased in zinc-deficient small intestine (9
), it is nonetheless interesting to identify the RAD23B transcript as upregulated in this context.
In summary, this work identifies, by cDNA analysis and subsequent RT-PCR confirmations, four mRNA transcripts significantly modulated by a moderate level of in vivo zinc deficiency. In particular, one of these, LCK, has been previously shown to mediate signal transduction through the CD4 and CD8 receptors through a cytosolic, zinc-dependent interaction. These data show that both LCK mRNA and protein levels are elevated in zinc-deficient murine thymus before the onset of thymic involution as detectable by FACS analysis. The thymic genes, found dysregulated here, may be factors in initiation of the lymphopenia and thymic atrophy associated with severe zinc deficiency and are worthy of future investigation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by National Institutes of Health grant DK 31127, Boston Family Endowment Funds of the University of Florida and by the Florida Agricultural Experiment Station and approved for publication as Journal Series No. R-08373.
4 Abbreviations used: CyC, Cy-Chrome; FACS, fluorescence-activated cell sorting; LCK, lymphocyte-specific protein tyrosine kinase; MCL-1, myeloid cell leukemia sequence-1; MLR, mouse laminin receptor; MT, metallothionein; PF, pair-fed; Q-PCR, quantitative real-time PCR; RAD23B, DNA damage repair and recombination protein 23B; RT-PCR, reverse transcription-polymerase chain reaction; -Zn, zinc-deficient; ZnN, zinc-normal.
Manuscript received July 13, 2001. Initial review completed August 17, 2001. Revision accepted September 18, 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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