Understanding iron acquisition by pathogenic leptospires: a review.
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Bacterial Proteins↗
Biomedical subjects
V Sridhar
Publications and source records attributed to V Sridhar.
Sublethal effects of monocrotophos on locomotor behavior and gill architecture of the mosquito fish, Gambusia affinis.
Subacute studies of monocrotophos [Dimethyl (E)-1-methyl-2-(methyl-carbamoyl) vinyl phosphate] on mosquito fish, Gambusia affinis, were carried out in vivo for 24 days to assess the locomotor behavior, structural integrity of gill, and targeted enzyme acetylcholinesterase (AChE, EC: 3.1.1.7) interactions. Monocrotophos (MCP) can be rated as moderately toxic to G. affinis, with a median lethal concentration (LC(50)) of 20.49 +/- 2.45 mgL(-1). The fish exposed to sublethal concentration of LC(10) (7.74 mgL(-1)) were under stress and altered their locomotor behavior, such as distance traveled per unit time (m min(-1)) and swimming speed (cm sec(-1)) with respect to the length of exposure. Inhibition in the activity of brain AChE and deformities in the primary and secondary lamellae of gill may have resulted in failure of exchange of gases. The maximum inhibition of 95% of AChE activity was observed on days 20 and 24. Morphological aberrations in the gills were also studied during exposure to the sublethal concentration of monocrotophos for a period ranging from 8 to 24 days. The extent of damage in gill was dependent on the duration of exposure. The findings revealed that inhibition in brain AChE activity and structural alteration in gill were responsible for altering the locomotor behavior of exposed fish.
Acetylcholinesterase↗
Comparative proteomics of the Mycobacterium leprae binding protein myelin P0: its implication in leprosy and other neurodegenerative diseases.
Mycobacterium leprae, the causative agent of leprosy invades Schwann cells of the peripheral nerves leading to nerve damage and disfigurement, which is the hallmark of the disease. Wet experiments have shown that M. leprae binds to a major peripheral nerve protein, the myelin P zero (P0). This protein is specific to peripheral nerve and may be important in the initial step of M. leprae binding and invasion of Schwann cells which is the feature of leprosy. Though the receptors on Schawann cells, cytokines, chemokines and antibodies to M. leprae have been identified the molecular mechanism of nerve damage and neurodegeneration is not clearly defined. Recently pathogen and host protein/nucleotide sequence similarities (molecular mimicry) have been implicated in neurodegenerative diseases. The approach of the present study is to utilise bioinformatic tools to understand leprosy nerve damage by carrying out sequence and structural similarity searches of myelin P0 with leproma and other genomic database. Since myelin P0 is unique to peripheral nerve, its sequence and structural similarities in other neuropathogens have also been noted. Comparison of myelin P0 with the M. leprae proteins revealed two characterised proteins, Ferrodoxin NADP reductase and a conserved membrane protein, which showed similarity to the query sequence. Comparison with the entire genomic database (www.ncbi.nlm.nih.gov) by basic local alignment search tool for proteins (BLASTP) and fold classification of structure-structure alignment of proteins (FSSP) searches revealed that myelin P0 had sequence/structural similarities to the poliovirus receptor, coxsackie-adenovirus receptor, anthrax protective antigen, diphtheria toxin, herpes simplex virus, HIV gag-1 peptide, and gp120 among others. These proteins are known to be associated directly or indirectly with neruodegeneration. Sequence and structural similarities to the immunoglobin regions of myelin P0 could have implications in host-pathogen interactions, as it has homophilic adhesive properties. Although these observed similarities are not highly significant in their percentage identity, they could be functionally important in molecular mimicry, receptor binding and cell signaling events involved in neurodegeneration.
Amino Acid Sequence↗
Receiver operating characteristics curve analysis of body fat & body mass index in dyslipidaemic Asian Indians.
BACKGROUND & OBJECTIVES: Optimal limit of body mass index (BMI) for Asian Indians remains to be defined. In this study, we describe the anthropometric and lipid profiles and determine the appropriate cut-offs of BMI for defining obesity in dyslipidaemic patients. METHODS: Correlations were carried out between lipid profile and anthropometric variables in 217 dyslipidaemic Asian Indians and the data were compared to those of 123 healthy historical controls. Receiver operating characteristics (ROC) curve analysis was carried out to determine the appropriate cut-offs of BMI for defining obesity taking the percentage of body fat (% BF) as the standard. RESULTS: Dyslipidaemic patients had high waist-hip ratio (W-HR) and percentage of BF. The prevalence of obesity as measured by percentage of BF was significantly (P < 0.05) higher as compared to obesity defined by the BMI cut-off. W-HR was the most important independent predictor (odds ratio: 2.8; 95% CI: 1.02-7.83) of atherogenic dyslipidaemia on multivariate logistic regression analysis. On ROC curve analysis the suggested appropriate cut-offs of BMI were; males 24.0 kg/m2 (sensitivity, 74.7%, and specificity, 79.7%), and females 23.0 kg/m2 (sensitivity, 85.7% and specificity, 62.5%). According to the suggested lower limits of BMI, an additional 15 per cent dyslipidaemic patients will be diagnosed as obese. INTERPRETATION & CONCLUSION: The observations in dyslipidaemic Asian Indians suggest high prevalence rates of generalized and abdominal obesity, and that high values of W-HR alone predisposes to atherogenic dyslipidaemia. Further, obesity may be optimally defined by a lower cut-off of BMI. The revised criteria for the BMI-based diagnosis of obesity will lead to a more rational management of dyslipidaemia in Asian Indians.
Adolescent↗
Disseminated cryptococcal infection in immune competent patients.
Cryptococcal meningeal or cerebral infection has become an increasing global problem.(1) In this respect there are many anacedotal Indian case reports.(2) More than 50% of CNS infections occur in immunosuppressed patients and other debilitating conditions. Neurological form of cryptococcosis in immunocompetant patients needs to be considered in situations with intractable headache, papilloedema, hydrocephalus and prior to decisions on shunt placements.(3) We report on two such immunocompetent patients who presented with CNS involvement. Their clinical features and outcome is discussed.
Adolescent↗
Recombinant cytochrome rC557 obtained from Escherichia coli cells expressing a truncated Thermus thermophilus cycA gene. Heme inversion in an improperly matured protein.
Cytochrome rC(557) is an improperly matured, dimeric cytochrome c obtained from expression of the "signal peptide-lacking" Thermus thermophilus cycA gene in the cytoplasm of Escherichia coli. It is characterized by its Q(00) (or alpha-) optical absorption band at 557 nm in the reduced form (Keightley, J. A., Sanders, D., Todaro, T. R., Pastuszyn, A., and Fee, J. A. (1998) J. Biol. Chem. 273, 12006-12016). We report results of a broad ranging, biochemical and spectral characterization of this protein that reveals the presence of a free vinyl group on the porphyrin and a disulfide bond between the protomers and supports His-Met ligation in both valence states of the iron. A 3-A resolution x-ray structure shows that, in comparison with the native protein, the heme moiety is rotated 180 degrees about its alpha,gamma-axis; cysteine 14 has formed a thioether bond with the 2-vinyl of pyrrole ring I instead of the 4-vinyl of pyrrole ring II, as occurs in the native protein; and a cysteine 11 from each protomer has formed an intermolecular disulfide bond. Numerous, minor perturbations exist within the structure of rC(557) in comparison with that of native protein, which result from heme inversion and protein-protein interactions across the dimer interface. The unusual spectral properties of rC(557) are rationalized in terms of this structure.
Crystallography↗
Microsomal cytochrome P450 2C5: comparison to microbial P450s and unique features.
Although microsomal P450s represent the majority of P450s, only microbial P450s have been amenable to crystal structure solution. We have recently solved the first crystal structure of a microsomal P450, 2C5, a progesterone hydroxylase from rabbit. We discuss the features of the protein in common with existing structures of microbial P450s and limitations of homology modeling mammalian P450s based on the microbial structures. Unique features involving membrane, substrate and cytochrome P450 reductase interactions are also discussed.
Animals↗
Mammalian microsomal cytochrome P450 monooxygenase: structural adaptations for membrane binding and functional diversity.
Microsomal cytochrome P450s participate in xenobiotic detoxification, procarcinogen activation, and steroid hormone synthesis. The first structure of a mammalian microsomal P450 suggests that the association of P450s with the endoplasmic reticulum involves a hydrophobic surface of the protein formed by noncontiguous portions of the polypeptide chain. This interaction places the entrance of the putative substrate access channel in or near the membrane and orients the face of the protein proximal to the heme cofactor perpendicular to the plane of the membrane for interaction with the P450 reductase. This structure offers a template for modeling other mammalian P450s and should aid drug discovery and the prediction of drug-drug interactions.
Amino Acid Sequence↗
Crystallization and preliminary crystallographic characterization of recombinant L-methionine-alpha-deamino-gamma-mercaptomethane lyase (methioninase).
L-Methionine-alpha-deamino-gamma-mercaptomethane lyase (rMETase) is involved in the alpha,gamma-elimination of methionine to alpha-ketobutyrate, methanethiol and ammonia. The reaction catalyzed by rMETase reduces the methionine concentration of methionine-dependent tumor cells, arresting their growth. Towards the goal of developing rMETase into an effective antitumor therapeutic and also to understand the catalytic mechanism of this enzyme, rMETase from Pseudomonas putida has been expressed, purified and crystallized. The crystals belong to space group P2(1)2(1)2 and diffract X-rays to at least 2.68 A resolution at 100 K using synchrotron radiation. The unit cell has parameters a = 152.8, b = 154.6, c = 80.8 A and contains four molecules in the asymmetric unit.
Carbon-Sulfur Lyases↗
Integrity of thermus thermophilus cytochrome c552 synthesized by Escherichia coli cells expressing the host-specific cytochrome c maturation genes, ccmABCDEFGH: biochemical, spectral, and structural characterization of the recombinant protein.
We describe the design of Escherichia coli cells that synthesize a structurally perfect, recombinant cytochrome c from the Thermus thermophilus cytochrome c552 gene. Key features are (1) construction of a plasmid-borne, chimeric cycA gene encoding an Escherichia coli-compatible, N-terminal signal sequence (MetLysIleSerIleTyrAlaThrLeu AlaAlaLeuSerLeuAlaLeuProAlaGlyAla) followed by the amino acid sequence of mature Thermus cytochrome c552; and (2) coexpression of the chimeric cycA gene with plasmid-borne, host-specific cytochrome c maturation genes (ccmABCDEFGH). Approximately 1 mg of purified protein is obtained from 1 L of culture medium. The recombinant protein, cytochrome rsC552, and native cytochrome c552 have identical redox potentials and are equally active as electron transfer substrates toward cytochrome ba3, a Thermus heme-copper oxidase. Native and recombinant cytochromes c were compared and found to be identical using circular dichroism, optical absorption, resonance Raman, and 500 MHz 1H-NMR spectroscopies. The 1.7 A resolution X-ray crystallographic structure of the recombinant protein was determined and is indistinguishable from that reported for the native protein (Than, ME, Hof P, Huber R, Bourenkov GP, Bartunik HD, Buse G, Soulimane T, 1997, J Mol Biol 271:629-644). This approach may be generally useful for expression of alien cytochrome c genes in E. coli.
Amino Acid Sequence↗
Alteration of the reduction potential of the [4Fe-4S](2+/+) cluster of Azotobacter vinelandii ferredoxin I.
The [4Fe-4S](2+/+) cluster of Azotobacter vinelandii ferredoxin I (FdI) has an unusually low reduction potential (E(0')) relative to other structurally similar ferredoxins. Previous attempts to raise that E(0') by modification of surface charged residues were unsuccessful. In this study mutants were designed to alter the E(0') by substitution of polar residues for nonpolar residues near the cluster and by modification of backbone amides. Three FdI variants, P21G, I40N, and I40Q, were purified and characterized, and electrochemical E(0') measurements show that all had altered E(0') relative to native FdI. For P21G FdI and I40Q FdI, the E(0') increased by +42 and +53 mV, respectively validating the importance of dipole orientation in control of E(0'). Protein Dipole Langevin Dipole calculations based on models for those variants accurately predicted the direction of the change in E(0') while overestimating the magnitude. For I40N FdI, initial calculations based on the model predicted a +168 mV change in E(0') while a -33 mV change was observed. The x-ray structure of that variant, which was determined to 2.8 A, revealed a number of changes in backbone and side chain dipole orientation and in solvent accessibility, that were not predicted by the model and that were likely to influence E(0'). Subsequent Protein Dipole Langevin Dipole calculations (using the actual I40N x-ray structures) did quite accurately predict the observed change in E(0').
Azotobacter vinelandii↗
Crystal structure of transhydrogenase domain III at 1.2 A resolution.
The nicotinamide nucleotide transhydrogenases (TH) of mitochondria and bacteria are membrane-intercalated proton pumps that transduce substrate binding energy and protonmotive force via protein conformational changes. In mitochondria, TH utilizes protonmotive force to promote direct hydride ion transfer from NADH to NADP, which are bound at the distinct extramembranous domains I and III, respectively. Domain II is the membrane-intercalated domain and contains the enzyme's proton channel. This paper describes the crystal structure of the NADP(H) binding domain III of bovine TH at 1.2 A resolution. The structure reveals that NADP is bound in a manner inverted from that previously observed for nucleotide binding folds. The non-classical binding mode exposes the NADP(H) nicotinamide ring for direct contact with NAD(H) in domain I, in accord with biochemical data. The surface of domain III surrounding the exposed nicotinamide is comprised of conserved residues presumed to form the interface with domain I during hydride ion transfer. Further, an adjacent region contains a number of acidic residues, forming a surface with negative electrostatic potential which may interact with extramembranous loops of domain II. Together, the distinctive surface features allow mechanistic considerations regarding the NADP(H)-promoted conformation changes that are involved in the interactions of domain III with domains I and II for hydride ion transfer and proton translocation.
Amino Acid Motifs↗
A T14C variant of Azotobacter vinelandii ferredoxin I undergoes facile [3Fe-4S]0 to [4Fe-4S]2+ conversion in vitro but not in vivo.
[4Fe-4S]2+/+ clusters that are ligated by Cys-X-X-Cys-X-X-Cys sequence motifs share the general feature of being hard to convert to [3Fe-4S]+/0 clusters, whereas those that contain a Cys-X-X-Asp-X-X-Cys motif undergo facile and reversible cluster interconversion. Little is known about the factors that control the in vivo assembly and conversion of these clusters. In this study we have designed and constructed a 3Fe to 4Fe cluster conversion variant of Azotobacter vinelandii ferredoxin I (FdI) in which the sequence that ligates the [3Fe-4S] cluster in native FdI was altered by converting a nearby residue, Thr-14, to Cys. Spectroscopic and electrochemical characterization shows that when purified in the presence of dithionite, T14C FdI is an O2-sensitive 8Fe protein. Both the new and the indigenous clusters have reduction potentials that are significantly shifted compared with those in native FdI, strongly suggesting a significantly altered environment around the clusters. Interestingly, whole cell EPR have revealed that T14C FdI exists as a 7Fe protein in vivo. This 7Fe form of T14C FdI is extremely similar to native FdI in its spectroscopic, electrochemical, and structural features. However, unlike native FdI which does not undergo facile cluster conversion, the 7Fe form T14C FdI quickly converts to the 8Fe form with a high efficiency under reducing conditions.
Amino Acid Sequence↗
A motif in human histidyl-tRNA synthetase which is shared among several aminoacyl-tRNA synthetases is a coiled-coil that is essential for enzymatic activity and contains the major autoantigenic epitope.
In myositis, disease-specific autoantibodies may be directed against an aminoacyl-tRNA synthetase, usually histidyl-tRNA synthetase. To explore the basis for this phenomenon, we have made recombinant histidyl-tRNA synthetase in the baculovirus system. It was enzymatically active and recognized by human autoantibodies. A truncated protein lacking the first 60 amino acids was inactive as an antigen and as an enzyme. This region is within the first two exons, is predicted to have a coiled-coil configuration, and is found in some other synthetases but not in Escherichia coli or yeast histidyl-tRNA synthetase. Circular dichroism showed that the peptides from this region (amino acids 1-60 and 1-47) have the predicted high alpha-helical content, but smaller fragments (1-30, 14-45, and 31-60) do not. The peptides with a high alpha-helical content could inhibit autoantibodies almost completely, whereas the smaller peptides were unable to do so. The amino acid sequence of this coiled-coil domain in human histidyl-tRNA synthetase resembles the sequence of the extended this coiled-coil arm near the NH2 terminus of bacterial seryl-tRNA synthetase as well as similar regions in some eukaryotic aminoacyl-tRNA synthetases, raising the possibility that this domain serves a similar tRNA-stabilizing role and has been preserved from a common ancestor.
Amino Acid Sequence↗
Substructure-based support vector machine classifiers for prediction of adverse effects in diverse classes of drugs.
Unforeseen adverse effects exhibited by drugs contribute heavily to late-phase failure and even withdrawal of marketed drugs. Torsade de pointes (TdP) is one such important adverse effect, which causes cardiac arrhythmia and, in some cases, sudden death, making it crucial for potential drugs to be screened for torsadogenicity. The need to tap the power of computational approaches for the prediction of adverse effects such as TdP is increasingly becoming evident. The availability of screening data including those in organized databases greatly facilitates exploration of newer computational approaches. In this paper, we report the development of a prediction method based on a support machine vector algorithm. The method uses a combination of descriptors, encoding both the type of toxicophore as well as the position of the toxicophore in the drug molecule, thus considering both the pharmacophore and the three-dimensional shape information of the molecule. For delineating toxicophores, a novel pattern-recognition method that utilizes substructures within a molecule has been developed. The results obtained using the hybrid approach have been compared with those available in the literature for the same data set. An improvement in prediction accuracy is clearly seen, with the accuracy reaching up to 97% in predicting compounds that can cause TdP and 90% for predicting compounds that do not cause TdP. The generic nature of the method has been demonstrated with four data sets available for carcinogenicity, where prediction accuracies were significantly higher, with a best receiver operating characteristics (ROC) value of 0.81 as against a best ROC value of 0.7 reported in the literature for the same data set. Thus, the method holds promise for wide applicability in toxicity prediction.
Algorithms↗