Lipoamidase (lipoyl-X hydrolase) from pig brain.

Although the optimum substrate for lipoamidase (lipoyl-X hydrolase) has not yet been determined, it is known that lipoamidase activity, as determined by hydrolysis of the synthetic substrate lipoyl 4-aminobenzoate (LPAB), is widely distributed in pig brain tissues, i.e. in the cerebrum, cerebellum and medulla. Over 95% of the enzyme activity is present in the membrane subfractions, indicating that brain lipoamidase is an integral membrane protein enzyme. To elucidate the chemical nature and the optimum substrate of the abundant lipoamidase in the brain, we isolated it from the membrane subfractions. After an 8-step purification procedure, brain lipoamidase was purified 601-fold and identified as a 140 kDa glycoprotein by SDS/PAGE. A mechanistic study to determine Km and Vmax, values was carried out using various synthetic compounds. Lipoyl-lysine, which is generally believed to be a naturally occurring substrate of lipoamidase, was first compared with biotinyl-lysine, because these two vitamins have reactive sulphur atoms and are similar in molecular mass and structure. The Km for lipoyl-lysine was 333 microM, whereas biotinyl-lysine was not hydrolysed. Stringent specificity for the lipoyl moiety is demonstrated, as expected. Dipeptides of amino acid-lysine structures were studied, and dipeptides of aspartyl- and glutamyl-lysine hydrolysis occurred at high Km (3 mM) values. Thus lysine in the moiety is not very effective as an optimum substrate. The chemical bond structures of the amide bond (lipoyl-lysine) and peptide bond (aspartyl-lysine) were hydrolysed. Next, the ester bond compound was tested, and it was observed that lipolylmethyl ester was hydrolysed at high specificity. These findings indicate that this enzyme has broad specificities with respect to bond structure; it therefore is a unique hydrolase having stringent specificity for lipoic acid and relatively broad specificity for the chemical bond and the X moiety. Various inhibitors were tested; a few reagents, such as organic mercurials, di-isopropylfluorophosphate, 1,10-phenanthroline, sodium azide and angiotensin-converting enzyme inhibitor exhibited some inhibition (not more than 60%). Thus the active centre of this enzyme is a complex type. Although ATP is not hydrolysed and the lowest Km value is exhibited by the synthetic substrate reduced from LPAB (12 microM), some other compounds may still be expected to be hydrolysed by this unique and abundant brain lipoamidase.     

Interaction of metals at the reaction center of lipoamidase

Various metals have been shown to inhibit porcine brain lipoamidase activity at 0.1 mM, but not ferrous and ferric ions. However, in the presence of ethylenediamine tetraacetic acid (EDTA) 0.1 mM iron ions did inhibit the activity. No other metals exhibited this type of increased inhibition with the addition of EDTA. The ferric- and ferrous-EDTA compounds were equally effective. Various Fe-containing compounds also inhibited the enzyme activity, the order of inhibition being: EDTA greater than o-phenanthroline greater than azide greater than citrate. Hemin also inhibited the enzyme activity strongly. However, Fe-proteins, e.g. cytochrome c, transferrin and peroxidases, were not inhibitory. These results indicate the importance of Fe ion chelates with structural and molecular size differences for interaction with the reaction center of this enzyme.     

Molecular and cellular requirements for enhanced antigen cross-presentation to CD8 cytotoxic T lymphocytes

MHC class I-mediated cross-priming of CD8 T cells by APCs is critical for CTL-based immunity to viral infections and tumors. We have shown previously that tumor-secreted heat shock protein gp96-chaperoned peptides cross prime CD8 CTL that are specific for genuine tumor Ags and for the surrogate Ag OVA. We now show that tumor-secreted heat shock protein gp96-chaperoned peptides enhance the efficiency of Ag cross-priming of CD8 CTL by several million-fold over the cross-priming activity of unchaperoned protein alone. Gp96 also acts as adjuvant for cross-priming by unchaperoned proteins, but in this capacity gp96 is 1000-fold less active than as a peptide chaperone. Mechanistically, the in situ secretion of gp96-Ig by transfected tumor cells recruits and activates dendritic cells and NK cells to the site of gp96 release and promotes CD8 CTL expansion locally. Gp96-mediated cross-priming of CD8 T cells requires B7.1/2 costimulation but proceeds unimpeded in lymph node-deficient mice, in the absence of NKT and CD4 cells and without CD40L. Gp96-driven MHC I cross-priming of CD8 CTL in the absence of lymph nodes provides a novel mechanism for local, tissue-based CTL generation at the site of gp96 release. This pathway may constitute a critically important, early detection, and rapid response mechanism that is operative in parenchymal tissues for effective defense against tissue damaging antigenic agents.     

Discovery and structure–activity relationship of thienopyridine derivatives as bone anabolic agents

A cell-based assay was performed for the discovery of novel bone anabolic agents. Alkaline phosphatase (ALPase) activity of ST2 cells was utilized as an indicator of osteoblastic differentiation, and thienopyridine derivative 1 was identified as a hit compound. 3-Aminothieno[2,3-b]pyridine-2-carboxamide was confirmed to be a necessary core structure for the enhancement of ALPase activity, and then optimization of the C4-substituent on the thienopyridine ring was carried out. Introduction of cyclic amino groups to the C4-position of the thienopyridine ring improved the activity. Especially, N-phenyl-homopiperazine derivatives were found to be strong enhancers of ALPase among this new series. Furthermore, 3-amino-4-(4-phenyl-1,4-diazepan-1-yl)thieno[2,3-b]pyridine-2-carboxamide (15k) was orally administered to ovariectomized (OVX) rats over 6 weeks for evaluating the effects on areal bone mineral density (aBMD), and statistically significant improvements in aBMD were observed from the dosage of 10 mg/kg/day.     

Effects of chronic treatment with a novel angiotensin converting enzyme inhibitor, CS622, and a vasodilator, hydralazine, on atrial natriuretic factor (ANF) in spontaneously hypertensive rats (SHR)

We studied the effects of chronic treatment with a novel angiotensin converting enzyme inhibitor, alpha-[(2S,6R)-6-[(1S)-1-ethoxycarbonyl-3-phenylpropyl]amino-5-oxo-2- (2-thienyl)perhydro-1,4-thiazepin-4-yl]acetic acid.HCl (CS622), and a vasodilator, hydralazine, on plasma atrial natriuretic factor (ANF) levels and kidney ANF receptors in spontaneously hypertensive rats (SHR). Plasma ANF level was decreased and cardiac hypertrophy reduced in CS622 treated SHR, but not in hydralazine treated SHR, although blood pressure was lowered similarly in both SHR groups. The binding capacity of kidney ANF receptors increased and the affinity decreased in CS622 treated SHR compared to untreated SHR. These results suggest that decrease of plasma ANF results from decreased cardiac load but not from lowered blood pressure, and that changes in ANF receptors result from increased plasma ANF.     

Large-scale search of SNPs for type 2 DM susceptibility genes in a Japanese population

The etiology of type 2 diabetes (DM) is polygenic. We investigated here genes and polymorphisms that associate with DM in the Japanese population. Single-nucleotide polymorphisms (SNPs) of 398 derived from 120 candidate genes were examined for association with DM in a population-based case-control study. The study group consisted of 148 cases and 227 controls recruited from Funagata, Japan. No evident subpopulation structure was detected for the tested population. The association tests were conducted with standard allele positivity tables (chi(2) tests) between SNP genotype frequency and case-control status. The independent association of the SNPs from serum triglyceride levels and body mass index was examined by multiple logistic regression analysis. A value of P<0.01 was accepted as statistically significant. Six genes (met proto-oncogene, ATP-binding cassette transporter A1, fatty acid binding protein 2, LDL receptor defect C complementing, aldolase B, and sulfonylurea receptor) were shown to be associated with DM.     

Association of the ABCA1 gene polymorphisms with type 2 DM in a Japanese population

To examine the association of the ATP-binding cassette transporter 1 (ABCA1) gene with type 2 diabetes (DM), we studied genetic polymorphisms of the ABCA1 gene including its linkage disequilibrium (LD) and haplotype analyses using a Japanese population. A sample set (DM:72, IGT:75, and NGT:227) was genotyped with 34 SNPs distributed from the promoter region to the last exon of the ABCA1 gene. LD between SNPs was assessed in pairwise manner. Among 13 LD blocks constructed, an LD block at the 5'-region showed a significant difference in the haplotype distribution between the study groups (NGT vs. IGT + DM: overall p = 0.0180; NGT vs. DM: 0.0001). Fisher's exact probability test (NGT vs. DM) showed a significant association of the haplotype 2 of the LD block (p = 0.0001), with an odds ratio (OR) of 2.53 (95%CI:1.62-4.12). Diplotype analysis also showed a significant association of the diplotypes with the haplotype 2 (OR:2.59, 95%CI:1.48-4.54, p = 0.0013).     

Association of the Ser326Cys polymorphism in the OGG1 gene with type 2 DM

The association of the Ser326Cys polymorphism of the 8-oxoguanine glycosylase 1 (OGG1) gene with type 2 diabetes was examined using a Japanese population (n (M/W): 4585 (2085/2500); age: 62.6 ± 10.9 years). HbA1c levels and frequency of diabetic subjects were significantly higher in subjects with genotypes with Cys allele than in those without (p = 0.032 and 0.037, respectively). Multiple logistic regression analysis showed that genotypes with Cys allele were significantly associated with diabetes (OR: 1.32, p = 0.0289). In subjects whose glucose tolerance was classified by FPG and 2-h PG (n = 1.634), the association was more substantial (genotypes with Cys allele vs. without, OR: 1.70, p = 0.0059; genotypes Cys/Cys vs. Ser/Ser, OR: 2.19, p = 0.0008). In subjects with genotype Ser/Ser, the insulin secretion index, HOMA-β, increased in the subjects with glucose intolerance and decreased in the subjects with diabetes, while, in subjects with genotypes Ser/Cys + Cys/Cys, HOMA-β decreased as the glucose tolerance progressed (p for trend = 0.010).     

Multivariate meta-analysis of the association of G-protein beta 3 gene (GNB3) haplotypes with cardiovascular phenotypes

The objective of the present study was to review previous investigations on the association of haplotypes in the G-protein β3 subunit (GNB3) gene with representative cardiovascular risk factors/phenotypes: hypertension, overweight, and variation in the systolic and diastolic blood pressures (SBP and DBP, respectively) and as well as body mass index (BMI). A comprehensive literature search was undertaken in Pubmed, Web of Science, EMBASE, Biological Abstracts, LILACS and Google Scholar to identify potentially relevant articles published up to April 2011. Six genetic association studies encompassing 16,068 participants were identified. Individual participant data were obtained for all studies. The three most investigated GNB3 polymorphisms (G-350A, C825T and C1429T) were considered. Expectation–maximization and generalized linear models were employed to estimate haplotypic effects from data with uncertain phase while adjusting for covariates. Study-specific results were combined through a random-effects multivariate meta-analysis. After carefully adjustments for relevant confounding factors, our analysis failed to support a role for GNB3 haplotypes in any of the investigated phenotypes. Sensitivity analyses excluding studies violating Hardy–Weinberg expectations, considering gender-specific effects or more extreme phenotypes (e.g. obesity only) as well as a fixed-effects “pooled” analysis also did not disclose a significant influence of GNB3 haplotypes on cardiovascular phenotypes. We conclude that the previous cumulative evidence does not support the proposal that haplotypes formed by common GNB3 polymorphisms might contribute either to the development of hypertension and obesity, or to the variation in the SBP, DBP and BMI.