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Am J Physiol Renal Physiol 293: F594-F600, 2007. First published May 30, 2007; doi:10.1152/ajprenal.00171.2007
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Testosterone regulation of renal cystathionine beta-synthase: implications for sex-dependent differences in plasma homocysteine levels

Victor Vitvitsky,1,2 Anna Prudova,1 Sally Stabler,3 Sanjana Dayal,4 Steven R. Lentz,4,5 and Ruma Banerjee1

1Redox Biology Center and the Biochemistry Department, University of Nebraska, Lincoln, Nebraska, 2National Research Center for Hematology, Russian Academy of Medical Sciences, Moscow, Russia; 3Department of Medicine, University of Colorado Health Sciences Center, Denver Colorado; and 4University of Iowa Carver College of Medicine and 5Veterans Affairs Medical Center, Iowa City, Iowa

Submitted 11 April 2007 ; accepted in final form 29 May 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Elevated plasma total homocysteine (tHcy) is an independent risk factor for ischemic heart disease and stroke. Epidemiological studies reveal that men have higher tHcy levels than women, but the mechanism underlying this sex-dependent difference is unknown. One route for intracellular disposal of homocysteine is catalyzed by cystathionine beta-synthase (CBS). Renal function is known to be an important determinant of tHcy, and, in this study, we demonstrate that renal CBS expression and activity in mice diminished approximately twofold after castration, whereas ovariectomization was without effect. The higher renal CBS activity in males (22.7 ± 3.1 mmol cystathionine·h–1·kg kidney–1) vs. females (8.4 ± 3.4 mmol cystathionine·h–1·kg kidney–1, P ≤ 10–6) in C57Bl/6J mice was associated with lower plasma tHcy levels in males vs. females, and this difference was exacerbated in Cbs+/– mice (7.7 ± 1.9 µmol/l in males vs. 13.8 ± 6.4 µmol/l in females, P = 0.005). Surprisingly, mammals exhibit a diversity of regulatory patterns for kidney CBS, with females exhibiting lower CBS activity in mice, higher in rats and humans, and being indistinguishable from males in rabbit, hamster, and guinea pig. Our data suggest that testosterone-dependent regulation of human CBS in kidney may contribute to sex-dependent differences in homocysteine transsulfuration.

total homocysteine; S-adenosylmethionine

HOMOCYSTEINE IS a rogue sulfur-containing amino acid that, at elevated concentrations, is correlated with an increased risk for cardiovascular disease (7, 8, 11), neural tube defects (31, 50), and Alzheimer's disease (12). Homocysteine is a byproduct of methionine metabolism, and its intracellular concentrations are kept low by efficient disposal through the transmethylation and transsulfuration pathways (Fig. 1). In liver and kidney, parallel transmethylation reactions are catalyzed by methionine synthase and betaine homocysteine methyltransferase, whereas cystathionine beta-synthase (CBS) catalyzes the first step in the transsulfuration pathway. Mutations in CBS are the single most common cause of congenital hyperhomocysteinemia (33). Age, sex, and renal function are each determinants of total plasma homocysteine (tHcy) levels in humans (1, 6, 27, 35, 56). The molecular mechanisms underlying these associations are not known.


Figure 1
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  		Fig. 1. Homocysteine metabolism in mammals. BHMT, betaine homocysteine methyltransferase; AdoMet, S-adenosylmethionine; AdoHcy, S-adenosylhomocysteine; {gamma}-Glu-Cys, {gamma}-glutamylcysteine.

 
Plasma homocysteine levels are significantly lower in women than in men. This difference narrows with age, since tHcy levels increase after menopause (1, 27, 35, 57). It has been suggested that the higher serum concentration of 17 beta-estradiol in premenopausal women may account in part for the lower tHcy in this group (57). On the other hand, the increase in tHcy with age has also been attributed to lower B-vitamin status in the elderly (51).

Although liver is prominently involved in buffering methionine levels in plasma (29), kidney appears to be the major organ responsible for plasma homocysteine clearance and metabolism (5, 10, 18, 22, and see Ref. 19 for an overview). Renal insufficiency is strongly associated with elevated plasma tHcy (49). Mild-to-moderate hyperhomocysteinemia is observed in >85% of dialysis patients, who have up to a 30-fold higher risk of death from a cardiovascular event compared with the general population (16, 17). Because homocysteine is largely reabsorbed by the kidney and its urinary excretion is low, the metabolic effect of renal disease on plasma tHcy appears to be mediated via decreased homocysteine clearance through the transsulfuration pathway (49). Recently, elevated renal and plasma homocysteine levels following injury due to ischemia and reperfusion in rats was reported to result from diminished CBS activity in this organ (38).

CBS condenses homocysteine and serine to produce cystathionine, which is subsequently converted to cysteine via the action of cystathionine {gamma}-lyase. Cysteine is the limiting substrate in the synthesis of glutathione, the major cellular redox buffer, and it is estimated that ~50% of the cysteine in the intracellular glutathione pool can be derived via this pathway (3, 32). Therefore, in addition to providing a route for detoxification of homocysteine, the transsulfuration pathway intimately links it to redox homeostasis (54).

We have previously described tissue- and sex-specific differences in murine CBS activity (54). In kidney, CBS activity is approximately twofold higher in male vs. female mice and is correlated with an increase in steady-state CBS protein levels (54). In this study, we demonstrate that the higher CBS activity in male mice is associated with lower levels of plasma tHcy. We further report that renal CBS expression and activity decrease after castration of male mice, which suggests that testosterone is involved in the regulation of renal CBS, and this is related to higher tHcy levels in female vs. male mice. However, the situation is reversed in humans where the higher plasma tHcy level in males is correlated with lower CBS activity in male vs. female kidney cortical tissue. These studies suggest that testosterone may be an important determinant of sex-dependent differences in tHcy levels.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. The studies on mice were approved by the Institutional Animal Care and Use Committees at the University of Nebraska-Lincoln (UNL) and the University of Iowa. Breeding colonies of CBS-deficient mice (in a C57BL/6J background) were maintained at the University of Iowa. Ovariectomized female, castrated or sham-operated males, and control BALB/c mice were ordered from Charles River Laboratories. Following arrival, they were housed at the animal facility at UNL and killed 2, 4, or 6 wk postsurgery. Freshly harvested kidneys from Black Swiss mice were generously provided by Dr. Vadim Gladyshev (University of Nebraska, Lincoln). Mice were killed by exsanguination under ketamine/xylazine narcosis (100 µl ip), and plasma and kidney were removed rapidly as described previously (54). Analyses of metabolite levels and CBS expression and activity were performed on 6- to 10-wk-old mice. Age-matched males and females were killed and studied at the same time and in no particular order.

Rats (3-mo-old Sprague Dawley) were obtained from the animal facility at UNL and killed by CO2 asphyxiation. The kidneys were removed rapidly, snap-frozen in liquid nitrogen, and stored at –80°C until further use. Drosophila melanogaster (Canton-S strain; 5–12 days old) were obtained from the Department of Biology, UNL.

Rabbits, hamsters, and guinea pigs were obtained from the laboratory animal facility "Stolbovaya" (Moscow, Russia). All animals were sexually mature. Upon delivery, animals were held for several days in the animal facility at the National Research Center for Hematology (Moscow, Russia) and provided unlimited access to food and water. The animals were killed by decapitation under ether narcosis, and kidneys were removed rapidly, snap-frozen in liquid nitrogen, and stored at –80°C until use. The experimental protocol was approved by the Scientific Council of the National Research Center for Hematology (Russian Academy of Medical Sciences).

Human kidney samples. Human kidney samples were obtained from the Tissue Banks at the University of Nebraska Medical Center-Omaha and the University of Iowa (Holden Comprehensive Cancer Center Tissue Procurement Facility), following Institutional Review Board guidelines at each institution. Only normal kidney cortical samples established by histological examination at each institution were employed in this study.

Tissue sample preparation. Frozen rodent and human kidney samples were ground in liquid nitrogen using a porcelain mortar and pestle and prepared as described (54). Each sample of Drosophila used for analysis contained 120–180 flies in a tube that was cooled in liquid nitrogen until all the flies dropped to the bottom. The flies were then pulverized under liquid nitrogen with a mortar and pestle, and the whole body extract was prepared as described previously (54).

Northern Blot analysis. Total RNA was isolated using an RNAqueous kit (Ambion). RNA concentration was estimated spectrophotometrically, and 5–10 µg total RNA from each sample were used per analysis. A probe for CBS was generated by PCR amplification of an ~1-kb fragment using the expression construct pGEXCBS as a template and the following primers: 5'-GCCAAGGAGCCCCTGTGCATGCGGCCCGATGCTCCG-3' (sense) and 5'-GTCACCATTCCCAGGATTACCCC-3' (antisense). The probe was labeled with [{alpha}-32P]dCTP (sp act ~3,000 Ci/mmol; Amersham) using a Rediprime II Random Prime Labeling kit (Amersham Biosciences). Northern analyses were performed using NorthernMax reagents (Ambion) as recommended by the manufacturer. Quantification was performed by densitometric analysis using Quantity One BioRad image software. To account for variations in loading, the band intensities were normalized to the 18S ribosomal RNA in each sample visualized by ethidium bromide staining.

Western blot analysis. Western blot analysis of CBS protein levels in kidney samples was performed as described previously (54).

CBS activity and metabolite levels. CBS activity for all kidney samples was measured at 37°C and for Drosophila samples at 30°C using the ninhydrin assay as described (54). Tissue metabolite levels were determined as described previously (40). Plasma tHcy was assayed by stable isotope dilution capillary gas chromatography/mass spectrometry as previously described (48).

Statistical analysis. Statistical analysis was performed using the one-way ANOVA test included in Origin 7.0 software. The values reported in this study represent means ± SD. A P value of <0.05 was used to define statistical significance.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Sex-specific differences in renal CBS activity in mice. We examined whether the sex-specific difference in renal CBS levels that we have previously reported in C57BL/6J mice (54) is specific to this strain by measuring CBS activity in two other common laboratory strains of mice (Table 1). A statistically significant difference in CBS activity was observed in kidneys from BALB/c and Black Swiss mice with females having lower activity compared with their age-matched male littermates. Although the absolute values for renal CBS activity differ, the three mouse strains exhibited a similar 2- to 2.7-fold higher renal CBS activity in males vs. females.


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  		Table 1. Sex-specific differences in renal CBS activity in different animals

 
Effects of sex organs on CBS expression in mice. Lower kidney CBS activity in female BALB/c mice correlated with an approximately twofold lower steady-state level of CBS protein (Fig. 2A and Table 2). To determine whether male or female sex organs contribute to the observed differences in CBS expression, CBS activity and protein levels were measured in BALB/c mice after surgical castration or ovary removal. Ovariectomization had no effect on renal CBS activity and protein levels (Table 2), excluding a major role for estrogen in this regulation. In contrast, castrated mice exhibited significantly lower kidney CBS expression levels than control males. Levels of CBS expression in castrated males were comparable to CBS levels in control females (Fig. 2, B and C, and Table 2). The lower level of CBS protein expression was paralleled by a corresponding decrease in renal CBS activity in castrated mice. Western analysis revealed that the drop in CBS protein levels was fully manifested at 2 wk postoperation and did not change further 4 or 6 wk after surgery (data not shown). The sham-operated animals were indistinguishable from untreated males with respect to CBS protein levels and activity, indicating that the observed decrease in CBS expression in castrated mice is not induced by stresses associated with the surgical procedure (Fig. 2, B and C). These results are consistent with the reported approximately twofold increase in renal CBS activity in female mice treated with testosterone (28).


Figure 2
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  		Fig. 2. Hormonal regulation of kidney cystathionine beta-synthase (CBS) levels in BALB/c mice. A: CBS and actin (used as an equal loading control) in kidney extracts of control male and female BALB/c mice were detected by Western blot analysis as described under MATERIALS AND METHODS and are representative of at least 3 independent experiments. B: effect of castration on CBS expression in mouse kidney. Western blot analysis of renal CBS and actin levels in BALB/c mice, which had undergone castration, sham operation, or no treatment. The surgical procedures were performed 2 wk before the animals were killed. Data are representative of at least 3 independent experiments performed with samples from 4–5 animals from each category. C: quantitative analysis of the data representing CBS protein levels in kidney of normal male and female mice and in castrated and sham-operated males. For details, see Table 2.

 

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  		Table 2. Effect of castration on CBS activity and protein and mRNA levels in BALB/c mice

 
To determine whether castration alters CBS mRNA levels, Northern analysis was performed. The steady-state CBS mRNA was approximately twofold lower in female vs. male kidney (Fig. 3A and Table 2). Castration resulted in diminished CBS mRNA levels (Fig. 3, B and C, and Table 2) that paralleled the changes in protein levels. These results are consistent with the effect of testosterone on CBS being exerted at the level of transcription and/or mRNA stability.


Figure 3
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  		Fig. 3. Transcriptional regulation of murine kidney CBS expression by testosterone. A: renal CBS mRNA levels were determined by Northern blot analysis in female and male BALB/c mice. Equal loading of samples was ensured by ethidium bromide staining of total rRNA levels in each sample. B: Northern blot analysis of CBS mRNA in kidneys of BALB/c mice that had undergone castration, sham operation, or no treatment. The surgical procedures were performed 2 wk before animals were killed. Data are representative of at least 4 independent experiments performed with samples from 4–10 animals from each category. C: quantitative analysis of the data representing CBS mRNA level in kidney of normal male and female mice and in castrated and sham-operated males. For details, see Table 2.

 
Effect of CBS upregulation on renal glutathione and S-adenosylmethionine levels. Kidney glutathione concentrations ([GSH]) and the [GSH]-to-oxidized glutathione concentration ratio did not exhibit a sex dependence (Table 3). In contrast, females and castrated males exhibited ~40% lower S-adenosylmethionine (AdoMet) levels than control males, consistent with the reported increase in kidney AdoMet levels upon treatment of female mice with testosterone (28).


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  		Table 3. Limited metabolite profiling in kidney of BALB/c mice

 
Sex-dependent differences in tHcy in mice. To assess the relationship, if any, between renal CBS activity and plasma tHcy levels, we compared tHcy concentrations in Cbs+/+ and Cbs+/– mice (Table 4). As reported previously (54, 55), heterozygous disruption of the Cbs gene resulted in the expected decrease in kidney CBS activity and in an approximately twofold increase in plasma tHcy concentrations. Plasma tHcy levels were 49% (P = 0.05) and 79% (P = 0.005) higher in female than male mice with the Cbs+/+ and Cbs+/– genotypes, respectively.


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  		Table 4. Total plasma homocysteine in C57BL/6J mice

 
Renal CBS levels and activity in various organisms. We examined CBS activity in several organisms to assess the species specificity and direction of sex-specific differences (Table 1). A small but significant difference, in the opposite direction from mice, was observed in rat kidney, with females exhibiting an ~1.3-fold higher CBS activity than males. In contrast, Syrian hamsters, guinea pigs, and rabbits did not exhibit this sexual dimorphism (Table 1). CBS activity was also measured in Drosophila (whole body), and an approximately threefold higher activity was observed in female flies (31.8 ± 8.5 mmol cystathionine·h–1·kg body wt–1, n = 4) vs. males (10.9 ± 2.5 mmol cystathionine·h–1·kg body wt–1, n = 4, P = 0.03).

CBS activity in human kidney. To assess the relevance of sex-dependent regulation of renal CBS levels and tHcy seen in humans, where males rather than females have higher plasma tHcy (1, 27, 35, 56), we measured CBS activity in human kidney samples (Table 1 and Fig. 4). Because the kidney is an anatomically complex organ, histological examination was performed to confirm that only renal cortical tissue was employed in this analysis. A small but significant sex-dependent difference (P = 0.03) in renal CBS activity was observed, with females (5. 1 ± 1.5 mmol·h–1·kg tissue–1) exhibiting a higher activity than males (3.8 ± 1.3 mmol·h–1·kg tissue–1). Although the difference in CBS activity as a function of age did not reach statistical significance (P = 0.167, r = –0.291), most likely because of the small sample size, it exhibited a trend toward decline with increasing age (Fig. 4).


Figure 4
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  		Fig. 4. Distribution of CBS activity in human male and female kidney cortex with age. CBS activity was measured as described under MATERIALS AND METHODS.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we have tested the hypothesis that a sex-dependent disparity in renal CBS activity underlies the known sex-dependent differences in plasma tHcy levels. Earlier studies in our laboratory had revealed that renal CBS levels and activity are higher in male than in female C57BL/6J mice (54). In the present study, we demonstrate that this difference is also seen in two other mouse strains, is regulated at the mRNA level, and is responsive to testosterone rather than estrogen. Thus the difference in renal CBS activity and mRNA and protein levels between male and female mice is lost upon castration but is unaffected by ovariectomization (Fig. 2 and Table 2). A role for testosterone in regulation of renal CBS is further supported by the observation that injection of this androgen to female mice resulted in a twofold increase in renal CBS activity (28).

A comprehensive analysis of sex-specific gene expression has identified kidney as the somatic organ exhibiting the highest sexual dimorphism (43). The differentially expressed genes predominantly encode enzymes involved in drug and steroid metabolism or regulation of osmotic pressure. Within the proximal tubule of the kidney, testosterone is converted to a more potent derivative, 5{alpha}-dihydrotestosterone, through the activity of 5{alpha}-reductase. Both forms of the hormone serve as ligands for the androgen receptor, and the resulting complex is a transcription factor that binds to androgen response elements (ARE) in the promoter region of target genes, with the consensus sequence 5'-GGA/TACAnnnTGTTCT-3'. Fine tuning of the transcriptional response is achieved through the interaction of the androgen-receptor complex with various cis- and trans-acting factors (9, 44). Alternatively, target genes may also be regulated indirectly via androgen-dependent signaling pathways. The promoter sequence upstream of the murine Cbs gene does not harbor a perfectly matched ARE sequence, but several putative AREs can be identified with ≥75% homology to the consensus sequence. ARE sequences with 75% homology to the canonical sequence appear to suffice for a functional androgen response (20, 58).

A decrease in renal CBS levels in females and castrated males is associated with lower AdoMet concentrations in these mice (Table 3). The mechanism underlying the lower AdoMet concentration in females and castrated males could be related to AdoMet synthetase, whose activity, together with the concentration of AdoMet, was reported to increase in females in response to androgen treatment (28). We have previously reported that an ~14-fold decrease in the intracellular AdoMet concentration in liver leads to a decrease in CBS protein stability and, consequently, to a decrease in steady-state CBS levels (39). In contrast, the approximately twofold difference in AdoMet concentrations between male mice on the one hand (with ~100 µM AdoMet; Table 3) and female mice or castrated males (with ~60 µM AdoMet) on the other is unlikely to significantly change the level of AdoMet saturation of CBS, and therefore its stability or activity, based on the reported KD(AdoMet) value of 7 µM for CBS (52). Furthermore, the lower renal CBS protein levels in females and castrated males are clearly associated with differences at the mRNA level (Fig. 3). These results reveal complex organ-specific regulation of CBS and are consistent with transcriptional regulation of renal CBS levels by androgens in mice.

Three metabolic routes are available for kidney homocysteine clearance, represented by methionine synthase, betaine homocysteine methyltransferase, and CBS (Fig. 1). In response to testosterone, renal methionine synthase activity is unaltered (28), whereas betaine homocysteine methyltransferase mRNA is downregulated in mice (43). However, the difference in betaine homocysteine methyltransferase activity is not statistically significant (28). Assuming that renal methionine metabolism is an important determinant of circulating homocysteine levels, we posited that males, with higher CBS levels, clear homocysteine more efficiently and have a lower tHcy level. A sex-related difference in tHcy is observed in both wild-type mice (P = 0.05) and heterozygous knockout mice (P = 0.005; Table 4). Our results support the hypothesis that testosterone-dependent CBS upregulation is associated with lower tHcy in male mice.

The situation in mice is the converse of what is seen in humans where males rather than females have higher tHcy (1, 24, 27, 35, 56), and high methionine intake increases the risk for acute coronary events in males (53). To investigate this apparent anomaly between mice and men, we first examined kidney CBS activity broadly, in four other rodents and in Drosophila, in which a microarray analysis of development had previously revealed sexual dimorphism in CBS gene expression (2). An array of CBS activity patterns was observed, with all three strains of mice showing higher CBS activity in males, rat and Drosophila exhibiting higher CBS activity in females, and rabbit, Syrian hamster, and guinea pig showing no difference between the sexes (Table 1). This wide range of pan-organism CBS regulation even within the rodent family itself is interesting but not unprecedented. A disparate pattern of gene regulation by testosterone has been demonstrated for ornithine decarboxylase (ODC). ODC is regulated by testosterone in reproductive organs, salivary glands, and kidneys of mice and rats. Upregulation of ODC in rat and murine kidneys is accompanied by a transient increase in mRNA levels in rats and a sustained elevation in mice (13, 23, 25, 26, 36, 41, 46, 47). A detailed analysis of ODC expression in species across the Mus genus has identified intra- and interspecies variations in the androgenic response. In fact, out of eight species examined, six exhibited upregulation of renal ODC protein levels in response to testosterone, whereas two showed decreased levels of ODC (41). Furthermore, laboratory strains of mice representing a single species, Musca domesticus, exhibit a wide, 5- to 20-fold variation in the magnitude of the ODC androgenic response, which did not correlate with the levels of serum testosterone or androgen receptor expression (30). Given the importance of ODC to polyamine synthesis for cell cycle control and proliferation (15, 37) and of CBS in homocysteine and redox homeostasis (54), it is surprising to find such disparate patterns in their regulation, and the physiological significance of these divergent responses to testosterone is not known. We have previously speculated that, in mice, sulfur-containing catabolites formed downstream of the transsulfuration pathway may be used for territorial markings by males as an explanation for their higher CBS activity (54).

A sex-related difference in CBS expression in humans has not been reported previously. However, a possible sex-dependent modulation of homocystinuria due to CBS deficiency is suggested by the report of siblings who display a wide range of clinical phenotypes from severe (in two sisters) to benign (in the brother) despite sharing an identical genotype at the CBS locus (14). We were unable to determine whether estrogen or testosterone affects CBS expression in human kidney because of our inability to find an appropriately responsive cortical or epithelial cell line. However, testosterone downregulates CBS in a human prostate cancer cell line (A. Prudova and R. Banerjee, unpublished observations), which is the opposite effect seen in mice, and would be consistent with our observation that CBS activity is lower in male vs. female human kidney samples (Table 1). The role of sex hormones in modulating tHcy levels in humans is supported by a clinical study of cross-sex hormone administration to transsexuals (21). Female-to-male transsexuals who received androgen therapy for 4 mo showed a statistically significant increase in plasma tHcy. Conversely, administration of antiandrogens and estradiol to male-to-female transsexuals resulted in decreased plasma tHcy.

Gender effects have been noted in other aspects of renal function in humans. Thus a difference in graft survival based on donor gender has been reported with inferior functional prognosis when female kidney is transplanted into male patients. Interestingly, the donor gender effect was less pronounced for older donors, i.e., females >45 yr. Although the mechanistic basis of the donor gender effect is not known, the influence of sex hormones has been considered (42, 59).

The results of our study together with the literature discussed above lead us to suggest that testosterone-mediated upregulation of renal CBS levels in mice contributes to lower tHcy levels in males. Despite the relatively low number of human kidney samples available for this study, a difference in CBS activity is nonetheless apparent between the sexes (Table 1). We suggest that this difference in human renal CBS activity may contribute to the known difference in tHcy levels between the sexes. A trend in declining human renal CBS activity with age is observed in both sexes (Fig. 4).

Epidemiological studies on coronary heart disease indicate that, in all age groups (34, 45), men are at higher risk than women for myocardial infarction, whereas clinical studies indicate that it is not estrogen that protects women (4). Rather, it has been suggested that testosterone levels may underlie a gender-based difference in the propensity for developing unstable coronary atherosclerotic plaques (4). Our study suggests that testosterone-dependent regulation of kidney CBS may contribute to sex-dependent differences in plasma tHcy, a risk factor for ischemic heart disease and stroke.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants DK-64959 to R. Banerjee and HL-63943 to S. R. Lentz and by a predoctoral fellowship from the Heartland Affiliate of the American Heart Association to A. Prudova.


   ACKNOWLEDGMENTS
 
We thank Hadise Kabil (University of Nebraska, Lincoln) for providing the fruit flies used in this study.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Banerjee, Redox Biology Center and the Biochemistry Dept., Univ. of Nebraska, Lincoln, NE 68588-0664 (e-mail: rbanerjee1{at}unl.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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S. Dayal and S. R. Lentz
Murine Models of Hyperhomocysteinemia and Their Vascular Phenotypes
Arterioscler. Thromb. Vasc. Biol., September 1, 2008; 28(9): 1596 - 1605.
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