A high-throughput amenable colorimetric assay for enantioselective screening of nitrilase-producing microorganisms using pH sensitive indicators

Based on the color change of an indicator due to the release of hydrogen ion from a nitrilase-catalyzed reaction, a rapid colorimetric method was established for the enantioselective screening of nitrilase-producing microorganisms. The formation of acids due to the nitrilase-mediated hydrolysis of nitriles causes a drop in the pH, which in turn results in a change of color of the solution (containing indicator) that can be observed visually. The buffer (0.01 M phosphate, pH 7.2) and indicator (Bromothymol blue, 0.01%) were selected in such a way that both have the same affinity for the released protons. The enantioselectivity of nitrilases was estimated by comparing the hydrolysis of (R)-mandelonitrile with that of racemate under the same conditions. The method was used to screen a library of nitrilase-producing microorganisms, isolated in the authors' laboratory for their ability to enantioselectively hydrolyze mandelonitrile to mandelic acid, an important chiral building block.     

Frailty modeling for spatially correlated survival data,with application to infant mortality in Minnesota

The use of survival models involving a random effect or 'frailty' term is becoming more common. Usually the random effects are assumed to represent different clusters, and clusters are assumed to be independent. In this paper, we consider random effects corresponding to clusters that are spatially arranged, such as clinical sites or geographical regions. That is, we might suspect that random effects corresponding to strata in closer proximity to each other might also be similar in magnitude. Such spatial arrangement of the strata can be modeled in several ways, but we group these ways into two general settings: geostatistical approaches, where we use the exact geographic locations (e.g. latitude and longitude) of the strata, and lattice approaches, where we use only the positions of the strata relative to each other (e.g. which counties neighbor which others). We compare our approaches in the context of a dataset on infant mortality in Minnesota counties between 1992 and 1996. Our main substantive goal here is to explain the pattern of infant mortality using important covariates (sex, race, birth weight, age of mother, etc.) while accounting for possible (spatially correlated) differences in hazard among the counties. We use the GIS ArcView to map resulting fitted hazard rates, to help search for possible lingering spatial correlation. The DIC criterion (Spiegelhalter et al., Journal of the Royal Statistical Society, Series B 2002, to appear) is used to choose among various competing models. We investigate the quality of fit of our chosen model, and compare its results when used to investigate neonatal versus post-neonatal mortality. We also compare use of our time-to-event outcome survival model with the simpler dichotomous outcome logistic model. Finally, we summarize our findings and suggest directions for future research.     

Effects of age on antibody affinity maturation

The elderly are more susceptible to infectious diseases. Mortality and morbidity from infections increase sharply over the age of 65 years. At the same time, the efficacy of vaccinations in the elderly is decreased. The elderly also have an increased incidence of cancer and inflammatory diseases. All the above indicate an age-related dysregulation of the immune system. Evidence suggests that the change in the humoral immune response with age is a qualitative rather than a quantitative one, i.e. it is the affinity and specificity of the antibody that changes, rather than the quantity of antibody produced. There are a number of possible causes of this failure, one of which is a defect in the mechanism of hypermutation of immunoglobulin genes. We have studied individual clonal responses within germinal centres of spleen and Peyer's patches in young and old patient groups. Our results indicate that there is no difference in the actual mechanism of hypermutation with age. There are, however, differences that are due either to a change in selection processes or to a change in the founder cells available for activation.     

The role of pilin glycan in neisserial pathogenesis

The pilus of pathogenic Neisseria is a polymer composed mainly of the glycoprotein, pilin. Recent investigations significantly enhanced characterization of pilin glycan (Pg) from N. gonorrhoeae (gonococcus, GC) and N. meningitidis (meningococcus, MC). Several pilin glycosylation genes were discovered recently from these bacteria and some of these genes transfer sugars previously unknown to be present in neisserial pili. Due to these findings, glycans of GC and MC pilin are now considered more complex. Furthermore, various Pg can be expressed by different strains and variants of GC, as well as MC. Intra-species variation of Pg between different groups of GC or MC can partly be due to polymorphisms of glycosylation genes. In pilus of pathogenic Neisseria, alternative glycoforms are also produced due to phase-variation (Pv) of pilin glycosylation genes. Most remarkably, the pgtA (pilin glycosyl transferase A) gene of GC can either posses or lack the ability of Pv. Many GC strains carry the phase-variable (Pv+) pgtA, whereas others carry the allele lacking Pv (Pv–). Mostly, the GC isolates from disseminated gonococcal infection (DGI) carry Pv+ pgtA but organisms from uncomplicated gonorrhea (UG) contain the Pv– allele. This data suggests that Pv of pgtA facilitates DGI, whereas constitutive expression of the Pv– pgtA may promote UG. Additional implications of Pg in various physiological and pathogenic mechanisms of Neisseria can also be envisaged based on various recent data.     

Chemomodulation of carcinogen metabolising enzymes,antioxidant profiles and skin and fore-stomach papillomagenesis by Spirulina platensis

Numerous reports have revealed an inverse association between consumption of some selective natural products and risk of developing cancer. In the present study the effect of 250 and 500 mg/kg body weight of Spirulina was examined on drug metabolising phase I and phase II enzymes, antioxidant enzymes, glutathione content, lactate dehydrogenase and lipid peroxidation in the liver of 7-week-old Swiss albino mice. The implications of these biochemical alterations have been further evaluated adopting the protocol of benzo(a)pyrene induced forestomach and 7,12 dimethylbenz(a)anthracene (DMBA) initiated and croton oil promoted skin papillomagenesis. Our primary findings reveal the Monofunctional nature of Spirulina as deduced from its potential to induce only the phase II enzyme activities associated mainly with carcinogen detoxification. The glutathione S-transferase and DT-diaphorase specific activities were induced in hepatic and all the extrahepatic organs examined (lung, kidney and forestomach) by Spirulina pretreatment (significance level being from p < 0.05 to p < 0.005) except for the low dose treatment in forestomach. With reference to antioxidant enzymes viz., superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase and reduced glutathione were increased significantly by both the chosen doses of Spirulina from p < 0.01 to p < 0.005. Chemopreventive response was quantitated by the average number of papillomas per effective mouse (tumor burden) as well as percentage of tumor bearing animals. There was a significant inhibition of tumor burden as well as tumor incidence in both the tumor model systems studied. In the skin tumor studies tumor burden was reduced from 4.86 to 1.20 and 1.15 by the low and high dose treatment respectively. In stomach tumor studies tumor burden was 2.05 and 1.73 by the low and high doses of Spirulina treatment against 3.73 that of control.     

Genomic structures and population histories of linguistically distinct tribal groups of India

There are various conflicting hypotheses regarding the origins of the tribal groups of India, who belong to three major language groups--Austro-Asiatic, Dravidian and Tibeto-Burman. To test some of the major hypotheses we designed a genetic study in which we sampled tribal populations belonging to all the three language groups. We used a set of autosomal DNA markers, mtDNA restriction-site polymorphisms (RSPs) and mtDNA hypervariable segment-1 (HVS-1) sequence polymorphisms in this study. Using the unlinked autosomal markers we found that there is a fair correspondence between linguistic and genomic affinities among the Indian tribal groups. We reconstructed mtDNA RSP haplotypes and found that there is extensive haplotype sharing among all tribal populations. However, there is very little sharing of mtDNA HVS-1 sequences across populations, and none across language groups. Haplogroup M is ubiquitous, and the subcluster U2i of haplogroup U occurs in a high frequency. Our analyses of haplogroup and HVS-1 sequence data provides evidence in support of the hypothesis that the Austro-Asiatic speakers are the most ancient inhabitants of India. Our data also support the earlier finding that some of the western Eurasian haplogroups found in India may have been present in India prior to the entry of Aryan speakers. However, we do not find compelling evidence to support the theory that haplogroup M was brought into India on an "out of Africa" wave of migration through a southern exit route from Ethiopia. On the contrary, our data raise the possibility that this haplogroup arose in India and was later carried to East Africa from India.     

Structural and interactional homology of clinically potential trypsin inhibitors: molecular modelling of cucurbitaceae family peptides using the X-ray structure of MCTI-II

Several trypsin inhibitor peptides (with 28-32 amino acid residues) belonging to the Cucurbitaceae (LA-1, LA-2, MCTI-I, CMTI-I, CMTI-III, CMTI-IV), characterized by a distinctive tertiary fold with three conserved disulphide bonds and with mostly arginine at their active centre, were modelled using the high-resolution X-ray structure of a homologous inhibitor, MCTI-II, isolated from bitter gourd. All the inhibitors were modelled in both their native and complexed state with the trypsin molecule, keeping the active site the same as was observed in the trypsin-MCTI-II complex, by homology modelling using the InsightII program. The minimized energy profile supported the binding constants (binding behaviour) of the inhibitor-trypsin complexes in the solution state. A difference accessible surface area (DASA) study of the trypsin with and without inhibitors revealed the subsites of trypsin where the inhibitors bind. It revealed that the role of mutation of these peptides through evolution is to modulate their inhibitory function depending on the biological need rather than changing the overall structural folding characteristics which are highly conserved. The minor changes of amino acids in the non-conserved regions do not influence significantly the basic conformational and interactional sequences at the trypsin binding subsites during complex formation.     

5-Membered NH...N hydrogen bonded molecular scaffold in a model dipeptide containing 3-aminophenylacetic acid: Crystal and solution conformations

Crystallographic and spectroscopic studies of a model dipeptidecontaining unusual amino acid residues establish the presence ofan intramolecular, 5-membered NH...N hydrogen bond involvingan amide NH (from 3-amino phenyl acetic acid residue) and anamide N atom from an adjacent amino acid residue in solid stateand in solution. The dipeptide also forms an infinite -sheet ribbon structure in crystals.     

Thermodynamic and kinetic characterization of Co-C bond homolysis catalyzed by coenzyme B(12)-dependent methylmalonyl-CoA mutase

Methylmalonyl-CoA mutase is a member of the family of coenzyme B(12)-dependent isomerases and catalyzes the 1,2-rearrangement of methylmalonyl-CoA to succinyl-CoA. A common first step in the reactions catalyzed by coenzyme B(12)-dependent enzymes is cleavage of the cobalt-carbon bond of the cofactor, leading to radical-based rearrangement reactions. Comparison of the homolysis rate for the free and enzyme-bound cofactors reveals an enormous rate enhancement which is on the order of a trillion-fold. To address how this large rate acceleration is achieved, we have examined the kinetic and thermodynamic parameters associated with the homolysis reaction catalyzed by methylmalonyl-CoA mutase. Both the rate and the amount of cob(II)alamin formation have been analyzed as a function of temperature with the protiated substrate. These studies yield the following activation parameters for the homolytic reaction at 37 degrees C: DeltaH(f)() = 18.8 +/- 0.8 kcal/mol, DeltaS(f)() = 18.2 +/- 0.8 cal/(mol.K), and DeltaG(f)() = 13.1 +/- 0.6 kcal/mol. Our results reveal that the enzyme lowers the transition state barrier by 17 kcal/mol, corresponding to a rate acceleration of 0.9 x 10(12)-fold. Both entropic and enthalpic factors contribute to the observed rate acceleration, with the latter predominating. The substrate binding step is exothermic, with a DeltaG of -5.2 kcal/mol at 37 degrees C, and is favored by both entropic and enthalpic factors. We have employed the available kinetic and spectroscopic data to construct a qualitative free energy profile for the methylmalonyl-CoA mutase-catalyzed reaction.