Human genome project: pharmacogenomics and drug development

Now that all 30,000 or so genes that make up the human genome have been deciphered, pharmaceutical industries are emerging to capitalize the custom based drug treatment. Understanding human genetic variation promises to have a great impact on our ability to uncover the cause of individual variation in response to therapeutics. The study of association between genetics and drug response is called pharmacogenomics. The potential implication of genomics and pharmacogenomics in clinical research and clinical medicine is that disease could be treated according to the interindividual differences in drug disposition and effects, thereby enhancing the drug discovery and providing a stronger scientific basis of each patient's genetic constitution. Sequence information derived from the genomes of many individuals is leading to the rapid discovery of single nucleotide polymorphisms or SNPs. Detection of these human polymorphisms will fuel the discipline of pharmacogenomics by developing more personalized drug therapies. A greater understanding of the way in which individuals with a particular genotype respond to a drug allows manufacturers to identify population subgroups that will benefit most from a particular drug. The increasing emphasis on pharmacogenomics is likely to raise ethical and legal questions regarding, among other things, the design of research studies, the construction of clinical trials and the pricing of drugs.     

Characterization of a galactose specific adhesin of enteroaggregative Escherichia coli

A fimbrial adhesin was identified from an enteroaggregative Escherichia coli strain. The adhesin was purified to 740-fold by sequential chromatography on an affinity matrix and gel filtration column in the FPLC system. The homogeneity of the purified protein was established by analytical isoelectrofocussing (pI 7.25). The native adhesin appeared as a high-molecular-weight aggregative protein as revealed by gel filtration chromatography on Superose 12HR10/30 column. However, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis the molecular weight of the adhesin was found to be 18 kDa and this was further confirmed by gel filtration chromatography on Superose 6HR 10/30 column presence of 6 M guanidine hydrochloride. The N-terminal 15-amino-acid sequence of the adhesin did not show homology with any of the previously reported fimbrial adhesins. The purified adhesin showed adhesion to human erythrocytes in the presence of Ca(2+) (5 mM). The optimum temperature and pH for the hemadhesion activity was found to be 25 degrees C and 6.5, respectively. The inhibition study clearly suggested that the binding site of the adhesin could recognize galactose as the specific sugar. The fluorescence of 4-methylumbelliferyl-alpha-D-galactopyranoside was quenched on binding to the adhesin and maximum reversal of fluorescence quenching was observed by competitive substitution titration with raffinose. The adhesin was found to contain one binding site per monomer for its specific sugar residue. The association constant and the free energy of binding were obtained as 3.98 x 10(5) M(-1) and -31.97 kJ/mol, respectively. The adherence of the bacteria to HEp-2 monolayer was inhibited in presence of galactose and this was further supported by a significant reduction in the bacterial adherence to the HEp-2 cells, pretreated with beta-D-galactosidase.     

Downregulation of K(+) channel genes expression in type I diabetic cardiomyopathy

Type I diabetic cardiomyopathy has consistently been shown to be associated with decrease of repolarising K(+) currents, but the mechanisms responsible for the decrease are not well defined. We investigated the streptozotocin (STZ) rat model of type I diabetes. We utilized RNase protection assay and Western blot analysis to investigate the message expression and protein density of key cardiac K(+) channel genes in the diabetic rat left ventricular (LV) myocytes. Our results show that message and protein density of Kv2.1, Kv4.2, and Kv4.3 are significantly decreased as early as 14 days following induction of type I diabetes in the rat. The results demonstrate, for the first time, that insulin-deficient type I diabetes is associated with early downregulation of the expression of key cardiac K(+) channel genes that could account for the depression of cardiac K(+) currents, I(to-f) and I(to-s). These represent the main electrophysiological abnormality in diabetic cardiomyopathy and is known to enhance the arrhythmogenecity of the diabetic heart. The findings also extend the extensive list of gene expression regulation by insulin.     

Immunological defect in leprosy patients: altered T-lymphocyte signals

The early events of activation were studied in paucibacillary (TT/BT) and multibacillary (BL/LL) leprosy patients by stimulation of their lymphocytes with mitogenic agents (calcium ionophore A23187/PMA) and Micobacterium leprae antigen (PGL-1). Maximum proliferation in response to PMA/A23187 and PGL-1 was observed in the BT/TT patients and the control group, respectively. Inositol triphosphate (IP3) and calcium were constitutively elevated in BT/TT and LL/BL patients. PMA/A23187 caused an increase in both IP3 and [Ca2+]i in BT/TT patients and controls. PGL-1 marginally increased IP3 levels in BT/TT patients. In the LL/BL patients, although PMA/A23187 increased IP3 levels, but no change was seen in [Ca2+]i, PGL-1 had no effect. Protein kinase C levels were seen to be associated with particulate fractions in BT/TT patients and were found to increase further in response to PMA/A23187. PGL-1 did not increase translocation of protein kinase C in controls or LL/BL patients. A preactivated and sensitised state of T-lymphocytes was observed in BT/TT patients, responsive to antigen and mitogens, whereas the cells of LL/BL patients were unresponsive to PGL-1. The altered signal transduction events characterised in the MB patients thus correlate well with the anergic state of their cells.     

Molecular heterogeneity among north Indian isolates of Group A Streptococcus

AIM: To monitor molecular heterogeneity among the clinical isolates of group A Streptococcus (GAS) from north India by Vir and emm typing. METHODS AND RESULTS: GAS isolates, 31 from pharyngitis and nine from rheumatic fever (RF)/rheumatic heart disease (RHD) patients were differentiated into 16 Vir types (VT). These isolates were further discriminated into 23 emm types. Most of emm types were Vir type specific, except few (7.5%), which revealed different Vir types within same emm type. The most prevalent emm type found was emm 49 (15%) followed by 7.5% of emm 69, emm 71 and emm 75 which were different from emm type distribution reported from south India. CONCLUSIONS: Analysis of data revealed 40% heterogeneity by Vir typing and 57.5% by emm typing among GAS isolates which is significant in view of small number of isolates studied. SIGNIFICANCE OF IMPACT OF THE STUDY: The molecular study for the first time demonstrates different emm types prevalent and circulating in northern region of India and such data may help in selection of types for vaccine development.     

A biologically active lectin of enteroaggregative Escherichia coli

The pathogenesis of enteroaggregative Escherichia coli, a major contributor to paediatric diarrhoea, is still not clearly understood. A complex carbohydrate specific lectin was identified from the culture supernatant of an enteroaggregative E. coli strain. The lectin was purified to 660-fold by a combination of sequential saturated ammonium sulphate precipitation and gel filtration chromatography in the FPLC system. The homogeneity of the purified lectin was established by analytical isoelectrofocusing [pI 6.75]. Hemagglutination of rabbit erythrocytes by the purified lectin was best inhibited by fetuin. The N-terminal sequence of the 41.7 kDa subunit showed homology to the outermembrane porins and the 23.4 kDa subunit showed homology to a hypothetical protein of Yersinia pestis and secreted Hcp protein. This protein could induce extensive morphological changes in HEp-2 cells and significant amount of fluid accumulation in rabbit ileal loop. GM1 showed maximum binding to the lectin among all other gangliosides. This purified protein showed cross-reactivity to the binding subunit of cholera toxin in western immunoblot. The presence of this toxin in some of the clinical isolates of enteroaggregative E. coli was also observed. The structural and functional characteristics of the toxin revealed that it is a novel virulence determinant of aggregative E. coli.     

Suggestive evidence of association of C-159T functional polymorphism of the <Emphasis Type="Italic">CD14</Emphasis> gene with atopic asthma in northern and northwestern Indian populations

CD14 is a lipopolysaccharide receptor known to be an important modulator of Th1–Th2 response during early childhood. Genetic association studies of the CD14 gene with asthma and atopic disorders have shown positive as well as negative results in different ethnic populations. The aim of this study was to test for association of C-159T functional promoter polymorphism with atopic asthma and serum IgE levels in northern and northwestern Indian populations. DNA was assayed for the CD14 C-159T polymorphism in a case-control study involving atopic asthmatics (n=187) and healthy normal controls (n=227), and in a family-based association study of 106 trios. The case-control study showed an association at the genotypic (P=0.0146) as well as the allelic level (P=0.0048). Moreover, we observed a deviation of allelic transmission from random proportions (P=0.024) in the transmission disequilibrium test analysis. When we analyzed our results for serum total IgE levels, against this polymorphism, we observed a difference at the genotypic (P=0.0026) as well as at the allelic level (P=0.0016) in a case-control study, whereas no association in the quantitative transmission disequilibrium test analysis was obtained. These findings provide suggestive evidence of association of the CD14 gene locus with atopic asthma in northern and northwestern Indian populations.     

A point mutation at the C-terminal half of the repressor of temperate mycobacteriophage L1 affects its binding to the operator DNA

The wild-type repressor CI of temperate mycobacteriophage L1 and the temperature-sensitive (ts) repressor CIts391 of a mutant L1 phage, L1cIts391, have been separately overexpressed in E. coli. Both these repressors were observed to specifically bind with the same cognate operator DNA. The operator-binding activity of CIts391 was shown to differ significantly than that of the CI at 32 to 42 degrees C. While 40-95% operator-binding activity was shown to be retained at 35 to 42 degrees C in CI, more than 75% operator-binding activity was lost in CIts391 at 35 to 38 degrees C, although the latter showed only 10% less binding compared to that of the former at 32 degrees C. The CIts391 showed almost no binding at 42 degrees C. An in vivo study showed that the CI repressor inhibited the growth of a clear plaque former mutant of the L1 phage more strongly than that of the CIts391 repressor at both 32 and 42 degrees C. The half-life of the CIts391-operator complex was found to be about 8 times less than that of the CI-operator complex at 32 degrees C. Interestingly, the repressor-operator complexes preformed at 0 degrees C have shown varying degrees of resistance to dissociation at the temperatures which inhibit the formation of these complexes are inhibited. The CI repressor, but not that of CIts391, regains most of the DNA-binding activity on cooling to 32 degrees C after preincubation at 42 to 52 degrees C. All these data suggest that the 131(st) proline residue at the C-terminal half of CI, which changed to leucine in the CIts391, plays a crucial role in binding the L1 repressor to the cognate operator DNA, although the helix-turn-helix DNA-binding motif of the L1 repressor is located at its N-terminal end.     

Enhancement of catalase activity by repetitive low-grade H2O2 exposures protects fibroblasts from subsequent stress-induced apoptosis

Exposure of Chinese hamster V79 fibroblasts to mild and repetitive H2O2 doses in culture for 15 weeks produced no change in lipid peroxidation status, GSH/GSSG ratio and glutathione peroxidase activity of these cells (VST cells). In contrast, in VST cells catalase levels underwent a prominent increase which could be significantly inhibited and brought down to control levels after treatment with the catalase inhibitor 3-aminotriazole (3-AT). When control (VC) cells were exposed to UV radiation (UVC 5 J/m2) or H2O2 (7.5mM, 15 min), intracellular reactive oxygen species (ROS) levels rose prominently with significant activation of caspase-3. Marked nuclear fragmentation and lower cell viability were also noted in these cells. In contrast, VST cells demonstrated a significantly lower ROS level, an absence of nuclear fragmentation and an unchanged caspase-3 activity after exposure to UVC or H2O2. Cell viability was also significantly better preserved in VST cells than VC cells after UV or H2O2 exposures. Following 3-AT treatment of VST cells, UVC radiation or H2O2 brought about significantly higher elevations in intracellular ROS, increases in caspase-3 activity, significantly lowered cell viability and marked nuclear fragmentation, indicating the involvement of high catalase levels in the cytoprotective effects of repetitive stress. Therefore, upregulation of the antioxidant defense after repetitive oxidative stress imparted a superior ability to cope with subsequent acute stress and escape apoptotic death and loss of viability.     

Crystal structure of the 14-3-3zeta:serotonin N-acetyltransferase complex. a role for scaffolding in enzyme regulation

Serotonin N-acetyltransferase (AANAT) controls the daily rhythm in melatonin synthesis. When isolated from tissue, AANAT copurifies with isoforms epsilon and zeta of 14-3-3. We have determined the structure of AANAT bound to 14-3-3zeta, an association that is phosphorylation dependent. AANAT is bound in the central channel of the 14-3-3zeta dimer, and is held in place by extensive interactions both with the amphipathic phosphopeptide binding groove of 14-3-3zeta and with other parts of the central channel. Thermodynamic and activity measurements, together with crystallographic analysis, indicate that binding of AANAT by 14-3-3zeta modulates AANAT's activity and affinity for its substrates by stabilizing a region of AANAT involved in substrate binding.