Cystathione Beta-synthase Research Articles

Pathogenic Homocystinuria-Associated T236N Mutation Dramatically Alters the Biochemical Properties of Cystathionine Beta-Synthase Protein.

Cystathione beta-synthase (CBS) T236N is a novel mutation associated with pyridoxine non-responsiveness, which presents a significant difficulty in the medical treatment of homocystinuria. Reported severe phenotypes in homocystinuria patients highlight the urgent requirement to comprehend the molecular mechanisms underlying mutation pathogenicity for the advancement of the disease. In this study, we used a multidisciplinary approach to investigate the molecular properties of bacterially expressed and purified recombinant CBST236N protein, which we directly compared to those of the wild-type (CBSWT) protein. Our data revealed a profound impact of the p.T236N mutation on CBS enzymatic activity, with a dramatic reduction of ~96% compared to the CBSWT protein. Circular dichroism (CD) experiments indicated that the p.T236N mutation did not significantly alter the secondary structure of the protein. However, CD spectra unveiled distinct differences in the thermal stability of CBSWT and CBST236N mutant protein species. In addition, chemical denaturation experiments further highlighted that the CBSWT protein exhibited greater thermodynamic stability than the CBST236N mutant, suggesting a destabilizing effect of this mutation. Our findings provide an explanation of the pathogenicity of the p.T236N mutation, shedding light on its role in severe homocystinuria phenotypes. This study contributes to a deeper understanding of CBS deficiency and may improve the development of targeted therapeutic strategies for affected individuals.

P0095MOLECULAR MECHANISMS OF THE CARDIOVASCULAR EFFECTS OF LANTHIONINE, A NEW UREMIC TOXIN, AND ITS INTERACTIONS WITH THE REDOX MICROENVIRONMENT

Abstract Background and Aims The non-proteinogenic amino acid lanthionine is a byproduct of the biosynthesis of hydrogen sulfide (H2S), an endogenously produced gas with cardiovascular properties). Lanthionine concentration is increased in uremia and it has been proposed as a new uremic toxin. In the zebrafish model, lanthionine induces effects on cardiac embriogenesis, arrhythmia, and locomotor alterations. Some effects are counteracted by glutathione, the well known antioxidant. In a human endothelial cell model, lanthionine significantly reduces H2S release, both protein content and glutathionylation of cystathione beta-synthase (CBS), one of the main H2S-producing enzymes, miR-200c and miR-423 levels, as well as vascular endothelial growth factor expression, while it also increases intracellular calcium levels. We investigated in the present abstract the actions of glutathione on some of lanthionine effects on zebrafish and endothelial cells. We utilized what is considered to be a more stable form of glutathione, acetylglutathione (AcGSH). Method Zebrafish behavior and heart size were analyzed by the Danio Vision system and confocal microscopy, respectively. Gene and protein expression were analyzed usimg qPCR and Western Blot, respectively. CBS glutathionylation has been assessed by immunoprecipitation with a magnetic beads-coated anti-GSH. Interleukins levels were evaluated by ELISA. Lanthionine acetylation was evaluated by LC/MS. Results In zebrafish, we demonstrated that AcGSH significantly increases heart size and the expression of atrium specific proteins. Increased heart rate and heart rhythm plasticity determined by AcGSH were partially counteracted by lanthionine. Monitoring of larval movements showed that this behavior was affected by AcGSH supplementation. In endothelial cells, AcGSH increased H2S release, and, utilized in combination with lanthionine, it was able to partially counteract its effects on H2S release. AcGSH was able to increase CBS glutathionylation and to offset lanthionine effects on glutathionylation. In addition, it increased tubulin, histone, and NFKB acetylation and IL-8 and IL-13 levels. We also demonstrated with mass spectrometry that lanthionine was non-enzymatically acetylated by AcGSH, suggesting a possible mechanism. Conclusion In conclusion, our findings support the notion that lanthionine is a uremic toxin, which is strongly modified in its effects by the redox microenvironment.

Attenuation of cardiac ischemia-reperfusion injury by sodium thiosulfate is partially dependent on the effect of cystathione beta synthase in the myocardium.

Our early studies have shown that sodium thiosulfate (STS) treatment attenuated the ischemia-reperfusion (IR)-induced injury in an isolated rat heart model by decreasing apoptosis, oxidative stress, and preserving mitochondrial function. Hydrogen sulfide, the precursor molecule is reported to have similar efficacy. This study aims to investigate the role of endogenous hydrogen sulfide in STS-mediated cardioprotection against IR in an isolated rat heart model. D, L-propargylglycine (PAG), an inhibitor of cystathionine γ-lyase was used as endogenous H2S blocker. In addition, we used the hypoxia-reoxygenation (HR) model to study the impact of STS in cardiomyocytes (H9C2) and fibroblast (3T3) cells. STS treatment to animal and cells prior to IR or HR decreased cell injury. The sensitivity of H9C2 and 3T3 cells towards HR (6 h hypoxia followed by 12 h reoxygenation) challenge varies, where, 3T3 cells exhibited higher cell death (54%). Cells treated with PAG prior to STS abrogate the protective effect in 3T3 (cell viability 61%) but not in H9C2 (cell viability 82%). Further evaluation in rat heart model showed partial recovery (46% RPP) of heart from those hearts pretreated with PAG prior to STS condition. In conclusion, we demonstrated that STS-mediated cardioprotection to IR-challenged rat heart is not fully dependent on endogenous H2S level and this dependency may be linked to higher fibroblast content in rat heart.

Abstract A65: An integromics approach identifies immune escape as a potential mechanism for prostate cancer disparities

Abstract Background: AA men with PCa will die at a rate nearly 2.5 times higher than their EA counterparts. Presently, no mechanism has been described to explain the differences observed between AA and EA men with PCa; yet, many have interrogated the role of social and anthropometric data on PCa outcomes within these populations. However, when these factors are statistically controlled, the differences in incidence, morbidity, and mortality between AA and EA PCa persist. These findings support the notion that biological factors are one of the major driving forces of PCa disparities. Therefore, to obtain better insights into the molecular aspects of PCa disparities, we examined metabolomic and gene expression profiles of PCa and matched adjacent benign tissue from AA and EA men. Metabolomics is defined as the study of all the small molecule metabolites produced by cellular processes in the body. We hypothesize that racially distinct metabolic pathways and the associated bioprocesses, may contribute to PCa health disparities Methodology: A total of 190 polar and mid-polar metabolites were measured using mass spectrometry across 50 and 28 PCa/benign tissue pairs from AA and EA men, respectively. Likewise, gene expression microarray analysis was performed on 48 and 21 PCa/benign tissue pairs from AA and EA men. Ancestry informative markers were genotyped and ancestry estimates were determined. Metabolic profiles of AA and EA PCa and benign adjacent pairs were compared using paired t-tests. FDR corrected p-values were used to detect differential metabolites and genes. Results: Unique biochemical alterations associated with AA tumors were identified. Pathways such as cysteine/methionine, arginine/proline and de novo purine biosynthesis were enhanced in AA PCa. Metabolites such as cystathione, cysteine, and S-Adenosylhomocysteine (SAH), are elevated in AA PCa compared to matched adjacent benign. Consistent with this, transcript and protein levels of DNA Methyl Transferase 1 (DNMT1) and Cystathione Beta Synthase (CBS) were elevated in AA PCa tissues and cell lines. Interestingly, protein and transcript levels of Adenosine Deaminase, an enzyme that converts SAM-derived adenosine to inosine was significantly down regulated in AA PCa, both in tissues and cell lines. Consistent with this, accumulated levels of adenosine were found in AA PCa. Conclusions: Taken together, the metabolomics data alludes to the existence of an efficient immune escape mechanism in AA PCa. Corroborating this, analysis of gene expression data from AA men with PCa also reveals enriched immune escape pathways. Citation Format: Stacy M. Lloyd, Jie Gohlke, Sumanta Basu, Salil Bhowmik, Vasanta Putluri, Rashmi Krishnapurnam, Kimal Rajapakshe, Cristian Coarfa, Nilanjan Guha, SA Deepak, Arunkumar Padmanabhan, Mohammed Sayeeduddin, Patricia Castro, Michael Ittmann, Ganesh Palapattu, Nagireddy Putluri, George Michailidis, Arun Sreekumar. An integromics approach identifies immune escape as a potential mechanism for prostate cancer disparities. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr A65.

Association of cystathionine β-synthase gene polymorphisms with essential hypertension in ethnic Uyghurs and Hans from Xinjiang

To investigate the cystathione beta synthase (CBS) gene T833C, G919A, 844ins68 polymorphisms and plasma homocysteine (Hcy) levels in ethnic Uyghur and Han patients with essential hypertension (EH) in Xinjiang. Four hundred twenty nine cases including 211 Uyghur and 218 Han EH patients were recruited, whilst 410 healthy individuals including 210 Uyghurs and 200 Hans were used as the controls. Amplification refractory mutation system (ARMS) was adopted to analyze the CBS gene polymorphisms including T833C, G919A and 844ins68. Enzymoimmunoassay was applied to determine the plasma level of Hcy. Chemiluminescence was applied to determine the plasma folic acid and vitamin B12. Compared with the controls, the plasma Hcy level was significantly higher in the EH group in both ethnic Uyghurs and Hans (P < 0.05). Plasma levels of Hcy in T833C, G919A genotypes (for both heterozygotes and homozygotes) were statistically higher than wild types (P < 0.05). A significant difference was detected in G919A polymorphism between the EH patients and controls in both Uyghur and [CM(144.5mm] Han ethnics (Uyghur: x² = 10.264, P < 0.01; Han: x² = 23.075, P < 0.01), and in T833C between the EH patients and controls in ethnic Uyghurs (x² = 40.254, P < 0.01). Logistic regression analysis indicated that age (OR=1.151, P=0.047, 95% CI = 1.002-1.323), T833C (CC) (OR = 1.078, P = 0.003, 95% CI = 1.043-1.114), obesity (OR = 1.284, P = 0.021, 95% CI = 1.038-1.590), hyperhomocysteine (OR = 3.296, P = 0.016, 95% CI = 1.244-8.733) were independent risk factors for EH among ethnic Uygurs, while age (OR = 1.162, P = 0.007, 95% CI = 1.042-1.297), obesity (OR = 3.501, P = 0.003, 95% CI = 1.521-8.060), hyperhomocysteine (OR = 1.046, P = 0.031, 95% CI = 1.011-1.459) were independent risk factors for EH in ethnic Hans after adjusting for confounding factors. Plasma level of Hcy is associated with ethnic Uyghur and Han patients with EH in Xinjiang. CBS gene T833C CC genotype may be associated with the EH among Uyghur ethnics.

P104 Inhibition of platelet aggregation in vitro and in vivo by the H2S releasing compound GYY4137

Platelets are key modulators of thrombotic events and a major target in the prevention of conditions including myocardial infarction. The role of H 2 S in regulating platelets remains unclear with contradictory reports. The functional modulation of platelets by H 2 S was investigated using light transmission aggregometry of isolated human platelets and in a mouse model for assessing aggregation of radiolabelled platelets in vivo . Additional biochemical assays determined the expression and activity of H 2 S generating enzymes. Western blotting revealed that human platelets express the H 2 S generating enzyme cystathione-beta synthase (CBS). Cystathione-gamma-lysase (CSE) was present at lower or undetectable levels. CBS activity was significantly higher in platelets compared to cell free samples whereas CSE was not active. Platelets were shown to generate H 2 S using a fluorescent probe and H 2 S production was significantly reduced by the CBS antagonist amino-oxyacetic acid. GYY4137 significantly reduced collagen-induced platelet aggregation in vitro and in vivo compared to control compound lacking the H 2 S donating moiety. GYY4137 also enhanced NO-mediated inhibition of platelet aggregation in vitro , an effect that was shown to be mediated via soluble guanylyl cyclase. The ability of GYY4137 to inhibit platelet aggregation in vivo was attenuated in eNOS −/− mice. Platelets therefore generate H 2 S primarily via CBS. H 2 S inhibits platelet aggregation and enhances the inhibitory actions of NO via cGMP-mediated signaling. H 2 S therefore has potential physiological roles in platelets and as a putative therapeutic strategy in the treatment of platelet-driven disorders. We continue to develop and assess the effects of other novel H 2 S donors including AP67 and AP72.

Mesenteric vascular remodeling in different mouse strains

Inflammatory Bowel Disease (IBD) is a category of inflammatory conditions of the colon and small intestine associated with oxidative stress (OS). Hyperhomocysteinemia (HHcy) is known as the cause of mesenteric vascular remodeling, inducing hypertension and acute abdomen pain. Patients with IBD often have HHcy compare to healthy people. We followed alterations of homocysteine (Hcy) level and mesenteric artery remodeling comparing four different strains of mice: C5BJ/L6 wild type (WT) as a control; cystathione beta synthase heterozygote knockout (CBS+/−) with increased level of Hcy; C3H, which is known as the most resistant to (OS) and double‐transgenic CBS/C3H mice. We suggested that Hcy level will be decreased in CBS/C3H compared to CBS, there will be amelioration of hemodynamics in mesenteric artery of CBS/C3H in comparison with CBS mice and attenuation of mesenteric vascular remodeling in CBS/C3H versus CBS. We used blood flow and mesenteric artery reactivity measurements, immunohistochemistry, electron microscopy of mitochondria, measurement of Dpr‐1 level (mediates mitochondrial fission during OS), measurement of Hcy level in plasma, Masson trichrome, PCR and Western blot (eNOS/iNOS, DDAH‐2, SAHH, CBS, CSE, 3MST, MTHFR, DNMT). Our preliminary data suggests that mesenteric vascular remodeling and HHcy are attenuated and hemodynamic of mesenteric artery is ameliorated in CBS/C3H versus CBS mice.

Clinical study regarding the link between cystathionine β-synthase 844ins68 polymorphism and maternal risk for Down syndrome

Folic acid is commonly used for the primary prevention of severe congenital malformations and Down syndrome (DS). The objective of this study was to evaluate the frequency of 844ins68 polymorphism of the cystathione beta-synthase (CBS) gene and to verify if the occurrence of this polymorphism is associated with trisomy 21. In the present study we analyzed 72 mothers (ages between 20 and 42 years old) in which those children presented or not DS. Mothers of children with DS (26 cases) were included as study group and those with children without DS and who had never suffered a miscarriage were enrolled as control group (46 cases). Genomic deoxyribonucleic acid (DNA) was isolated from whole peripheral blood collected on ethylenediaminetetraacetic acid, using peqGOLD blood DNA mini kit. The common CBS polymorphism that causes an insertion of 68 bp at the 844 position was further identified. Results. The frequencies of CBS 844ins68 genotypes (ins-/ins-, ins+/ins-) among case mothers were 84.6 and 15.4%, respectively. The corresponding frequencies among controls were 91.3 and 8.7%, respectively. In our study, we did not find any significant differences in genotype frequencies between the two analyzed groups. <br/><a href=

Mesenteric vascular remodeling in hyperhomocysteinemia

Remodeling by its very nature implies synthesis and degradation of extracellular matrix components (such as elastin, collagen, and connexins). Most of the vascular matrix metalloproteinase (MMP) are latent because of the presence of constitutive nitric oxide (NO). However, during oxidative stress peroxinitrite (ONOO-) activates the latent MMPs and instigates vascular remodeling. Interestingly, in mesenteric artery, homocysteine (Hcy) decreases the NO bio-availability, and folic acid (FA, an Hcy-lowering agent) mitigates the Hcy-mediated mesentery artery dysfunction. Dimethylarginine dimethylaminohydrolase-2 (DDAH-2) and endothelial nitric oxide synthase (eNOS) increases NO production. The hypothesis was that the Hcy decreased NO bio-availability, in part, activating MMP, decreasing elastin, DDAH-2, eNOS and increased vasomotor response by increasing connexin. To test this hypothesis,the authors used 12-week-old C57BJ/L6 wild type (WT) and hyperhomocysteinemic (HHcy)-cystathione beta synthase heterozygote knockout (CBS+/-) mice. Blood pressure measurements were made by radio-telemetry. WT and MMP-9 knockout mice were administered with Hcy (0.67 mg/ml in drinking water). Superior mesenteric artery and mesenteric arcade were analyzed with light and confocal microscopy. The protein expressions were measured by western blot analysis. The mRNA levels for MMP-9 were measured by RT-PCR. The data showed decreased DDAH-2 and eNOS expressions in mesentery in CBS-/+ mice compared with WT mice. Immuno-fluorescence and western blot results suggest increased MMP-9 and connexin-40 expression in mesenteric arcades of CBS-/+ mice compared with WT mice. The wall thickness of third-order mesenteric artery was increased in CBS-/+ mice compared to WT mice. Hcy treatment increased blood pressure in WT mice. Interestingly, in MMP-9 KO, Hcy did not increase blood pressure. These results may suggest that HHcy causes mesenteric artery remodeling and narrowing by activating MMP-9 and decreasing DDAH-2 and eNOS expressions, compromising the blood flow, instigating hypertension, and acute abdomen pain.

Matrix imbalance by inducing expression of metalloproteinase and oxidative stress in cochlea of hyperhomocysteinemic mice

Clinical study reports hearing loss in patients with low folic acid (FA) and elevated homocysteine (Hcy). We hypothesize that elevated Hcy induces imbalance in matrix turnover and oxidative stress in cochlea. Cystathione beta-synthase heterozygous knockout mice were used as model for hyperhomocysteinemia. Matrix remodeling induced by Hcy resulted from elevated MMP-2, -9, and -14. MMP-2 and -9 showed elevated gelatinase activity in CBS (+/-) cochlea. Tissue inhibitors of matrix metalloproteinase were significantly lower in CBS (+/-) cochlea. The expression analyses for MMPs and TIMPs were equally represented at protein and mRNA levels. Cochlea of CBS mice showed following structural changes; (1) detachment of tectorial membrane lying on hair cells (2) thinner s. vascularis (3) large fibroblast in spiral ligament. Hcy induced higher protein nitrotyrosination and cytosolic NADPHoxidase subunit p22(phox) in cochlea. It is thus suggested that Hcy induced matrix imbalance, structural changes and oxidative stress in cochlea.

Association study between cystathione-beta-synthase gene polymorphism and schizophrenia

Objective To explore the relationship between cystathione-beta-synthase(CBS) gene T833C,G919A polymorphisms and schizophrenia. Methods The CBS gene polymorphism was detected by polymerase chain reaction and DNA sequencing technique in 93 patients with schizophrenia and 102 healthy people as normal control. Results ⑴ No CBS gene T833C ,G919A polymorphisms were found in all subjects,but mutation of G→A was detected at the 33th base in intron 8-9. Both heterozygote G/A and homozygote A/A were found in patient group,only heterozygote G/A was found in control group. ⑵ There was no significant difference of genotype distribution for mutation of G→A between patient group and control group(P>0.05). The frequency of A allele in patient group (8.07%)was higher than that in control group(3.43%) (χ2=3.92,P=0.048,OR=2.47,95%CI=1.01～6.05). ⑶ There was no significant difference of genotype distribution for mutation of G→A between clinic subgroups and control group(all P>0.05). The frequency of A allele in positive subgroup(8.70%) and later first-onset age subgroup(8.93%) were higher than that in control group(χ2=4.35,P=0.037,OR=2.68,95%CI=1.06～6.77; χ2=4.29,P=0.038,OR=2.76,95%CI=1.06～6.43). Conclusion The finding suggests that mutation of CBS gene T833C ,G919A is very low and it may be not correlated with schizophrenia. Mutation of G→A at the 33th base in intron 8-9 of CBS gene may be one of risk factors for schizophrenia with later first-onset age or positive symptom. Key words: Schizophrenia; Cystathione-beta-synthase; Genetics; Polymorphism

Dissecting the pleiotropic consequences of a quantitative trait nucleotide

The downstream consequences of a single quantitative trait polymorphism can provide important insight into the molecular basis of a trait. However, the molecular consequences of a polymorphism may be complex and only a subset of these may influence the trait of interest. In natural isolates of Saccharomyces cerevisiae, a nonsynonymous polymorphism in cystathione beta-synthase (CYS4) causes a deficiency in both cysteine and glutathione that results in rust-colored colonies and drug-dependent growth defects. Using a single-nucleotide allele replacement, we characterized the effects of this polymorphism on gene expression levels across the genome. To determine whether any of the differentially expressed genes are necessary for the production of rust-colored colonies, we screened the yeast deletion collection for genes that enhance or suppress rust coloration. We found that genes in the sulfur assimilation pathway are required for the production of rust color but not the drug-sensitivity phenotype. Our results show that a single quantitative trait polymorphism can generate a complex set of downstream changes, providing a molecular basis for pleiotropy.

Hyperhomocysteinemia induces matrix disruption and oxidative stress in inner ear

Increasing number of clinical data suggests lower folic acid (FA) and high homocysteine (Hcy) causing hearing impairment. We analyzed hyperhomocysteinemia (HHcy) related matrix remodeling, oxidative stress and blood flow which may contribute to impaired hearing. We used wild type (WT) and heterozygous Cystathione beta‐synthase (CBS) mice. CBS () has higher level of plasma Hcy and exhibits some pathological features of HHcy. Expression of matrix metalloprotease (MMPs), their inhibitors, the TIMPs and nitrotyrosine (NT), p22phox and NOX‐3 were analyzed by western blotting and RT‐PCR. Masson Trichrome staining was done for collagen. Western blot analyses revealed increased level of active MMP‐2, MMP‐14 protein in CBS () mice. MMP‐9, MMP‐12 and MMP‐13 mRNA were higher in CBS () than the WT. Nitrotyrosine and p22phox was higher in CBS (), whereas NOX‐3 level showed moderate increase in CBS () as compared to WT. Staining for collagen was higher in WT as compared to CBS (). Hair cells were disorganized in CBS () along with hypertrophy as compared to WT. Our data suggested increased disruption of intercellular matrix resulting in damage and disorganized cellular structure in cochlea. Moreover an increased level of oxidative stress may also contribute to impaired hearing threshold.

Relationship between multiple genetic polymorphisms and hypertensive disorder in pregnancy

Hypertensive disorder of pregnancy has a wide spectrum of clinical manifestations which are controlled by several genes. We genotyped 3 polymorphic sites of cystathione beta-synthase(CBS) G9276A, manganese superoxide dismutase(MnSOD) Val(-9)Ala and vascular endothelial growth factor(VEGF) C936T and examined whether they play a role in the development of hypertension during pregnancy. The subjects were 294 cases complicated by hypertensive disorder of pregnancy and 144 controls without complications. The medical records were reviewed. Cases were categorized according to the classification suggested by National High Blood Pressure Education Program Working Group. Maternal CBS G9276A, MnSOD Val(-9)Ala and VEGF C936T was determined by restriction fragment length polymorphism. Chi-square analysis was used to assess genotype and allele frequency differences. We applied haplotype analysis by HapAnalyzer for association study. All the study subjects were successfully genotyped and met Hardy-Weinberg equilibrium. Genotypes and allele frequencies of each polymorphism did not differ significantly between two groups. We evaluated 4 haplotypes (CBS G9276/MnSOD Val(-9)/VEGF C936, CBS G9276/MnSOD Val(-9)/VEGF T936, CBS A9276/MnSOD Val(-9)/VEGF C936, CBS G9276/ MnSOD Ala(-9)/VEGF C936). However, all showed no significant results. These three polymorphic sites may not be involved in the development of hypertensive disorder during pregnancy.

Raman spectroscopy of the human nail: A potential tool for evaluating bone health?

Dual X-ray absorptiometry (DEXA) is the current gold standard for the diagnosis of osteoporosis. However, patients can suffer osteoporotic fractures despite normal bone mineral density, partly because of unmeasured influences of both the protein and mineral phases of bone that are affected in osteoporosis. There is currently no clinically applicable method of evaluating the health of the protein phase. The proteins in human nail (keratin) and bone (collagen) require sulphation and disulphide bond (S-S) formation for structural integrity and disorders of either sulphur metabolism or cystathione beta-synthase can lead to structural abnormalities in these tissues. Raman protein spectra provide a method of non-invasive measurement of the degree of sulphation of structurally related proteins that may be indicative of bone health. Raman spectroscopy was used to evaluate the disulphide (S-S) content of fingernails. The nail samples came from from 169 women (84 pre- and 85 post-menopausal), of which 39 had a history of osteoporotic fracture. BMD was measured by DXA at the spine. Analyses included parametric and non-parametric tests, dependent on the distribution of the test variable. Mean disulphide content of the nail reduced with age and was slightly higher in pre-, compared to post-menopausal women (P = 0.187). Significantly lower disulphide content was observed in nails obtained from subjects with a history of fracture (P = 0.025). When either disulphide content or BMD was used as a predictor, the odds ratio of these two measures were found to be comparable predictors for fracture status. This suggests that measurements of change in the protein phase of structural proteins such as keratin in the human nail may be correlated with clinically relevant changes in bone proteins that are important in fracture risk.

The Use of Fingernails as a Means of Assessing Bone Health: A Pilot Study

Anecdotally, patients volunteer reports of increasing hardness of their fingernails within months of starting diverse treatments for osteoporosis. The properties of both nail and bone may be linked in a comparable, measurable way. We examined the fingernails of two groups of patients, with (n = 9) and without (n = 13) osteoporosis at either the hip or lumbosacral spine. We performed nanoindentation to assess the degree of nail brittleness and Raman spectroscopy to assess the disulfide bond content of nail. The mean moduli of fingernails of patients with low bone mineral density (BMD) are lower than those of patients with normal BMD. The mean difference in mean modulus between the groups was found to be 0.996 (p = 0.15 between groups). The spectroscopy data also showed differences between the two sets of nails. The disulfide bond content of the nails sourced from osteoporotic patients was lower than that from healthy patients (p = 0.06 between groups). Bone collagen and nail keratin are two distinct structural proteins, and both require protein sulfation and disulfide bond formation, via cysteine, for structural integrity. A disorder of either process may lead to disordered collagen and keratin synthesis. This is reflected in the structural abnormalities seen in clinical syndromes in which there is either protein deficiency, disorders of sulfur metabolism, or cystathione beta-synthase deficiency. The relationship between nail and bone may exist in a measurable way. This pilot study should lead to further work to explore this relationship. Could nail prove to be a valuable adjunct to diagnosis or provide a means of more rapid follow-up after commencement of therapy?

Pretreatment with vitamins E and C prevent the impairment of memory caused by homocysteine administration in rats.

Homocystinuria is a metabolic disorder caused by deficiency of cystathione beta-synthase activity leading to tissue accumulation of homocysteine (Hcy); affected patients present neurological dysfunction. Considering that Hcy induces free radical formation and that memory is impaired by oxidative stress, in the present study we investigated the effect of an acute administration of Hcy on retrieval of step-down inhibitory avoidance in adult rats. The action of vitamins E and C on the effects produced by Hcy was also tested. Adult Wistar rats were pretreated for 1 week with daily i.p. administration of saline (control group) and vitamins E and C (vitamin E 40 mg/kg and vitamin C 100 mg/kg). Hcy (11 mmol/kg) or an equivalent volume of 0.9% saline were administered 1 h before training, 1 h before testing, or immediately after training sessions. Memory was significantly impaired in Hcy-treated group, whereas the rats chronically treated with vitamins E and C had this effect prevented. Present data strongly indicate that Hcy administration impairs memory, an effect probably mediated by oxidative stress since treatment with vitamins E and C prevented amnesia. Assuming the possibility that this might occur in the human condition, reported results may be relevant to explain, at least in part, neurologic dysfunction associated with homocystinuria.

Characteristics and crystal structure of bacterial inosine-5'-monophosphate dehydrogenase.

IMP dehydrogenase (IMPDH) is an essential enzyme that catalyzes the first step unique to GTP synthesis. To provide a basis for the evaluation of IMPDH inhibitors as antimicrobial agents, we have expressed and characterized IMPDH from the pathogenic bacterium Streptococcus pyogenes. Our results show that the biochemical and kinetic characteristics of S. pyogenes IMPDH are similar to other bacterial IMPDH enzymes. However, the lack of sensitivity to mycophenolic acid and the Km for NAD (1180 microM) exemplify some of the differences between the bacterial and mammalian IMPDH enzymes, making it an attractive target for antimicrobial agents. To evaluate the basis for these differences, we determined the crystal structure of the bacterial enzyme at 1.9 A with substrate bound in the catalytic site. The structure was determined using selenomethionine-substituted protein and multiwavelength anomalous (MAD) analysis of data obtained with synchrotron radiation from the undulator beamline (19ID) of the Structural Biology Center at Argonne's Advanced Photon Source. S. pyogenes IMPDH is a tetramer with its four subunits related by a crystallographic 4-fold axis. The protein is composed of two domains: a TIM barrel domain that embodies the catalytic framework and a cystathione beta-synthase (CBS) dimer domain of so far unknown function. Using information provided by sequence alignments and the crystal structure, we prepared several site-specific mutants to examine the role of various active site regions in catalysis. These variants implicate the active site flap as an essential catalytic element and indicate there are significant differences in the catalytic environment of bacterial and mammalian IMPDH enzymes. Comparison of the structure of bacterial IMPDH with the known partial structures from eukaryotic organisms will provide an explanation of their distinct properties and contribute to the design of specific bacterial IMPDH inhibitors.

Novel Risk Factors for Vascular Disease: The Homocysteine Hypothesis of Cardiovascular Disease

The possibility that modest elevations in the level of blood homocysteine (hyperhomocysteinaemia) could contribute to cardiovascular disease arose from investigation of patients with rare, severe homocysteine elevations caused by cystathionine beta-synthase deficiency. Such patients often had thromboembolic events before the age of 30 years. Since the established cardiovascular risk factors could only partly account for the occurrence and severity of vascular disease in the general population, other risk factors had to exist, and homocysteine elevation seemed to be a possible candidate. Australian case-control studies identified an association between mild homocysteine elevation and early-onset coronary disease, and also with chronic renal failure. Patients in the latter group have a high prevalence of unexplained vascular disease and particularly high homocysteine levels. Such elevations in levels of homocysteine in vascular patients could usually be normalised by daily supplementation with folic acid (1-5 mg) while in patients with chronic renal failure 5 mg of folic acid daily markedly reduced the increased concentrations of homocysteine. These initial observations have been confirmed by many investigators and biologically plausible mechanisms for homocysteine-induced vascular dysfunction, and particularly endothelial dysfunction, have been identified. However, associations between hyperhomocysteinaemia and other risk factors, such as smoking and hypertension, have also been documented and need to be controlled for when assessing any increase in risk that homocysteine may independently confer. Although it has been established that lowering the greatly elevated blood homocysteine levels in homocystinuria, due to cystathione beta-synthase deficiency, unquestionably reduces cardiovascular risk, it remains to be determined whether normalising mild homocysteine elevation could reduce cardiovascular risk. Trials to test this possibility have been initiated and others are planned.