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Biomedical subjects

C G Suresh

Publications and source records attributed to C G Suresh.

At least 19 recordsLinked to original sources
PubMed2006

Structural and functional analysis of a conjugated bile salt hydrolase from Bifidobacterium longum reveals an evolutionary relationship with penicillin V acylase.

Bile salt hydrolase (BSH) is an enzyme produced by the intestinal microflora that catalyzes the deconjugation of glycine- or taurine-linked bile salts. The crystal structure of BSH reported here from Bifidobacterium longum reveals that it is a member of N-terminal nucleophil hydrolase structural superfamily possessing the characteristic alphabetabetaalpha tetra-lamellar tertiary structure arrangement. Site-directed mutagenesis of the catalytic nucleophil residue, however, shows that it has no role in zymogen processing into its corresponding active form. Substrate specificity was studied using Michaelis-Menten and inhibition kinetics and fluorescence spectroscopy. These data were compared with the specificity profile of BSH from Clostridium perfrigens and pencillin V acylase from Bacillus sphaericus, for both of which the three-dimensional structures are available. Comparative analysis shows a gradation in activity toward common substrates, throwing light on a possible common route toward the evolution of pencillin V acylase and BSH.

Amidohydrolases↗
PubMed2006

Crystallization and preliminary characterization of a highly thermostable lectin from Trichosanthes dioica and comparison with other Trichosanthes lectins.

A lectin from Trichosanthes dioica seeds has been purified and crystallized using 25%(w/v) PEG 2K MME, 0.2 M ammonium acetate, 0.1 M Tris-HCl pH 8.5 and 50 microl 0.5%(w/v) n-octyl beta-D-glucopyranoside as thick needles belonging to hexagonal space group P6(4). Unit-cell parameters were a = b = 167.54, c = 77.42 A. The crystals diffracted to a Bragg spacing of 2.8 A. Both the structures of abrin-a and T. kirilowii lectin could be used as a model in structure determination using the molecular-replacement method; however, T. kirilowii lectin coordinates gave better values of reliability and correlation parameters. The thermal, chemical and pH stability of this lectin have also been studied. When heated, its haemagglutination activity remained unaffected up to 363 K. Other stability studies show that 4 M guanidinium hydrochloride (Gdn-HCl) initiates unfolding and that the protein is completely unfolded at 6 M Gdn-HCl. Treatment with urea resulted in a total loss of activity at higher concentrations of denaturant with no major structural changes. The protein remained stable over a wide pH range, from pH 6 to pH 12, except for partial unfolding at extremely alkaline pH. The role of disulfide bonds in the protein stability was found to be insignificant. Rayleigh light-scattering studies showed no molecular aggregation in any of the extreme treated conditions. The unusual stability of this lectin resembles that of type II ribosome-inactivating proteins (type II RIPs), which is also supported by structure determination. The structural features observed in a preliminary electron-density map were compared with the other two available Trichosanthes lectin structures.

Crystallization↗
PubMed2006

High-resolution mtDNA studies of the Indian population: implications for palaeolithic settlement of the Indian subcontinent.

The population of the Indian subcontinent represents a very complex social and cultural structure. Occupying a geographically central position for the early modern human migrations, indications are that the founder group that migrated out of East Africa also reached India. In the present study we used the twin strategy of mapping the whole mitochondrial DNA (mtDNA) using the standard 14 restriction enzymes, and sequencing the non-transcribed HVSI region, to derive maximum maternal lineages from a sample of non-tribal Indians. The essential features of the reduced median network of the two datasets were the same. Both showed two demographic expansions of two major haplogroups, 'M' and 'N'. The reduced median network was drawn with inputs from other studies on the Indian population, and correlated with data from other ethnic populations. The coalescence time of expansions and genetic diversity were estimated. A reduced median network was also drawn combining data from studies on Africans, Southeast Asians and West-Eurasians, tracing the migration of 'M' from East Africa to India. A time estimate of the migration of major mtDNA haplogroups from Africa was attempted. The comparison of a set of Indian maternal lineages belonging to different geographical regions of the country, with other populations revealed the in-situ differentiation and antiquity of the Indian population. Our analysis places the 'southern route' migration as the source of haplogroup 'M'. Multiple migrations might have brought the other major haplogroups, 'N' and 'R', found in our sample to India. Archaeological evidence of modern humans in the subcontinent supports this mtDNA study.

Cluster Analysis↗
PubMed2005

X-ray crystallographic studies on C-phycocyanins from cyanobacteria from different habitats: marine and freshwater.

C-phycocyanins from three cyanobacterial cultures of freshwater and marine habitat, Spirulina, Phormidium and Lyngbya spp., were purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method. Blue-coloured crystals in different crystal forms, monoclinic and hexagonal, were obtained for the three species. The crystals took 1-12 weeks to grow to full size using polyethylene glycols of different molecular weights as precipitants. The amino-acid sequences of these proteins show high similarity to other known C-phycocyanins from related organisms; however, the C-phycocyanins reported here showed different biochemical and biophysical properties, i.e. molecular weight, stability etc. The X-ray diffraction data were collected at resolutions of 3.0 A for the monoclinic and 3.2 and 3.6 A for the hexagonal forms. The unit-cell parameters corresponding to the monoclinic space group P2(1) are a = 107.33, b = 115.64, c = 183.26 A, beta = 90.03 degrees for Spirulina sp. C-phycocyanin and are similar for crystals of Phormidium and Lyngbya spp. C-phycocyanins. Crystals belonging to the hexagonal space group P6(3), with unit-cell parameters a = b = 154.97, c = 40.35 A and a = b = 151.96, c = 39.06 A, were also obtained for the C-phycocyanins from Spirulina and Lyngbya spp., respectively. The estimated solvent content is around 50% for the monoclinic crystals of all three species assuming the presence of two hexamers per asymmetric unit. The solvent content is 66.5 and 64.1% for the hexagonal crystals of C-phycocyanin from Spirulina and Lyngbya spp. assuming the presence of one alphabeta monomer per asymmetric unit.

Crystallography, X-Ray↗
PubMed2005

Cloning, purification, crystallization and preliminary structural studies of penicillin V acylase from Bacillus subtilis.

Penicillin acylase proteins are amidohydrolase enzymes that cleave penicillins at the amide bond connecting the side chain to their beta-lactam nucleus. An unannotated protein from Bacillus subtilis has been expressed in Escherichia coli, purified and confirmed to possess penicillin V acylase activity. The protein was crystallized using the hanging-drop vapour-diffusion method from a solution containing 4 M sodium formate in 100 mM Tris-HCl buffer pH 8.2. Diffraction data were collected under cryogenic conditions to a spacing of 2.5 A. The crystals belonged to the orthorhombic space group C222(1), with unit-cell parameters a = 111.0, b = 308.0, c = 56.0 A. The estimated Matthews coefficient was 3.23 A3 Da(-1), corresponding to 62% solvent content. The structure has been solved using molecular-replacement methods with B. sphaericus penicillin V acylase (PDB code 2pva) as the search model.

Amino Acid Sequence↗
PubMed2004

Cloning, preparation and preliminary crystallographic studies of penicillin V acylase autoproteolytic processing mutants.

The crystallization of three catalytically inactive mutants of penicillin V acylase (PVA) from Bacillus sphaericus in precursor and processed forms is reported. The mutant proteins crystallize in different primitive monoclinic space groups that are distinct from the crystal forms for the native enzyme. Directed mutants and clone constructs were designed to study the post-translational autoproteolytic processing of PVA. The catalytically inactive mutants will provide three-dimensional structures of precursor PVA forms, plus open a route to the study of enzyme-substrate complexes for this industrially important enzyme.

Amino Acid Substitution↗
PubMed2004

Crystallization and preliminary X-ray characterization of a lectin from Cicer arietinum (chickpea).

The lectin isolated from mature seeds of Cicer arietinum (CAL) agglutinates pronase-treated rabbit and human erythrocytes and its haemagglutination activity is inhibited by fetuin and desialated fetuin but not by simple monosaccharides or oligosaccharides. The purified lectin is a dimer of molecular weight 43,000 Da composed of two identical subunits (MW 21,500), as confirmed by SDS-PAGE. The lectin has been crystallized using the hanging-drop vapour-diffusion method at 295 K over a well solution containing 0.2 M sodium acetate, 0.1 M sodium phosphate buffer pH 6.5 and 14%(w/v) polyethylene glycol 8000. The triangular prism-shaped crystals belong to space group R3 and have unit-cell parameters a = b = 81.2, c = 69.4 A. The diffraction data are 93.8% complete to 2.3 A Bragg spacing with an Rmerge of 0.103.

Amino Acid Sequence↗
PubMed2004

Functional form of Caveolin-1 is necessary for the assembly of alpha-hemolysin.

The assembly of alpha-HL was shown to rapidly progress upon its interaction with Caveolin-1. Treatment of A431 cells with alpha-HL has resulted in clustering of Caveolin-1 at cell-cell contacts. Consistent with this observation, alpha-HL mutants devoid of assembly property have not induced the clustering of Caveolin-1. While cholesterol depletion of A431 cells completely arrests the assembly of alpha-HL, chelation of membrane cholesterol results in its retarded assembly. Interestingly, HT29 cells, with low Caveolin-1 levels, are resistant to alpha-HL attack. Clustering of Caveolin-1, as seen in case of A431 cells, was readily observed in case of HT29 cells transfected with Caveolin-1 construct, thus overexpressing the full length Caveolin-1, upon alpha-HL treatment. A model was constructed to visualize the interactions between alpha-HL and Caveolin-1 which suggests that facile penetration of alpha-HL's beta-barrel might occur through protein-protein interactions with the surrounding 7 alpha-helices of Caveolin-1.

Amino Acid Motifs↗
PubMed2004

Expression, purification, crystallization and preliminary X-ray diffraction analysis of conjugated bile salt hydrolase from Bifidobacterium longum.

Conjugated bile salt hydrolase (BSH) catalyses the hydrolysis of the amide bond that conjugates bile acids to glycine and to taurine. The BSH enzyme from Bifidobacterium longum was overexpressed in Escherichia coli BL21(DE3), purified and crystallized. Crystallization conditions were screened using the hanging-drop vapour-diffusion method. Crystal growth, with two distinct morphologies, was optimal in experiments carried out at 303 K. The crystals belong to the hexagonal system, space group P622 with unit-cell parameters a = b = 124.86, c = 219.03 A, and the trigonal space group P321, with unit-cell parameters a = b = 125.24, c = 117.03 A. The crystals diffracted X-rays to 2.5 A spacing. Structure determination using the multiple isomorphous replacement method is in progress.

Amidohydrolases↗
PubMed2004

Two orthorhombic crystal structures of a galactose-specific lectin from Artocarpus hirsuta in complex with methyl-alpha-D-galactose.

Based on their carbohydrate specificity, the jacalin family of lectins can be divided into two groups: galactose-specific and mannose-specific. The former are cytoplasmic proteins, whereas the latter are localized in the storage vacuoles of cells. It has been proposed that the post-translational modification in some of the lectins that splits their polypeptide chains into two may be crucial for galactose specificity. The mannose-specific members of the family are single-chain proteins that lack the above modification. Although the galactose-specific and the mannose-specific jacalin-type lectins differ in their sequences, they share a common fold: the beta-prism I fold, which is characteristic of Moraceae plant lectins. Here, two crystal structures of a jacalin-related lectin from Artocarpus hirsuta, which is specific for galactose, in complex with methyl-alpha-D-galactose are reported. The lectin crystallized in two orthorhombic forms and one hexagonal form under similar conditions. The crystals had an unusually high solvent content. The structure was solved using the molecular-replacement method using the jacalin structure as a search model. The two orthorhombic forms were refined using data to 2.5 and 3.0 A resolution, respectively. The structures of the A. hirsuta lectin and jacalin are identical. In orthorhombic form I the crystal packing provides three different micro-environments for sugar binding in the same crystal. The observed difference in the specificity for oligosaccharides between the A. hirsuta lectin and jacalin could only be explained based on differences in the molecular associations in the packing and variation of the C-terminal length of the beta-chain. The observed insecticidal activity of A. hirsuta lectin may arise from its similar fold to domain II of the unrelated delta-endotoxin from Bacillus thuringiensis.

Amino Acid Sequence↗
PubMed2004

Evidence for the involvement of arginyl residue at the active site of penicillin G acylase from Kluyvera citrophila.

Penicillin G acylase (PGA) is used for the commercial production of semi-synthetic penicillins. It hydrolyses the amide bond in penicillin producing 6-aminopenicillanic acid and phenylacetate. 6-Aminopenicillanic acid, having the beta-lactam nucleus, is the parent compound for all semi-synthetic penicillins. Penicillin G acylase from Kluyvera citrophila was purified and chemically modified to identify the role of arginine in catalysis. Modification with 20 mM phenylglyoxal and 50 mM 2,3-butanedione resulted in 82% and 78% inactivation, respectively. Inactivation was prevented by protection with benzylpenicillin or phenylacetate at 50 mM. The reaction followed psuedo-first order kinetics and the inactivation kinetics (V(max), K(m), and k(cat)) of native and modified enzyme indicates the essentiality of arginyl residue in catalysis.

Amino Acid Substitution↗
PubMed2001

Differences in thrombolytic treatment and in-hospital mortality between women and men after acute myocardial infarction.

There is evidence for gender differences in the treatment and outcome of acute myocardial infarction (AMI). However, little data exist about these differences in patients from the Arab Middle East. Therefore, we studied the influence of patient gender on the presentation, the use of thrombolytic therapy, and in-hospital mortality after AMI in Kuwaiti nationals. This is a retrospective study of all consecutive Kuwaiti patients admitted to the coronary care unit of a university hospital with the diagnosis of AMI between June 1994 and May 1997. A total of 89 women and 267 men were included. Women were older than men and had significantly higher rates of diabetes (72% vs 46%), hypertension (58% vs 33%) and hypercholesterolemia (80% vs 53%). Women were less likely to receive thrombolytic therapy (40% vs 62%, p=0.001). Fewer women were eligible for thrombolytic therapy (50% vs 66%, p<0.05). Of those who were eligible for thrombolysis there was no sex difference in receiving such treatment. The in-hospital mortality among women younger than 70 years was 2.5 times higher than among men in the same age group, while there was no difference in mortality between women and men aged 70 years and older. We conclude that women and men with AMI have different clinical characteristics and outcomes following AMI. There was no gender bias for the use of thrombolytic therapy. The higher in-hospital mortality in younger women, i.e. less than 70 years, compared to younger men, indicates that younger women with AMI should be considered as a high-risk group.

Aged↗
PubMed2000

Indian-Asian relationship: mtDNA reveals more.

Recent studies on human mtDNA have identified continent-specific restriction enzyme sites and resultant haplo-groups among populations from different regions of the world. Such studies have helped in elaborating the models for human migrations. We have studied Indian mtDNAs to identify the recognized world ethnic elements present in it. The results presented here are based on the study of DdeI 10394 site along with the associated Asian-specific AluI 10397 site in the mtDNA sequences of the Indian samples. On examining all the related haplo-groups, this study suggests that the apparent affinities of Indians and East Asians (comprising Chinese, Japanese, Southeast Asians etc.) could be due to a proto-Asiatic element present in Indians.

Animals↗
PubMed1999

Crystallization and preliminary X-ray studies of the basic lectin from the seeds of Artocarpus hirsuta.

The basic lectin from Artocarpus hirsuta specific towards methyl alpha-galactose has been purified and crystallized using the hanging-drop vapour-diffusion method with ammonium sulfate as precipitant. Three different crystal forms, orthorhombic I, orthorhombic II and hexagonal, were grown under the same crystallization conditions. The orthorhombic forms belonged to space group P212121 with unit-cell dimensions a = 92.9, b = 99.8, c = 166. 2 A and a = 89.9, b = 121.9, c = 131.6 A, respectively. The unit-cell dimensions of the hexagonal form were a = b = 84.1 and c = 271.7 A and the space group was P6122.

Crystallization↗
PubMed1999

Crystallization and preliminary x-ray diffraction studies of a Bowman-Birk inhibitor from Vigna unguiculata seeds.

A Bowman-Birk type trypsin/chymotrypsin inhibitor isolated from Vigna unguiculata seeds has been crystallized. Crystals were grown using the vapour-diffusion method at pH 4.0 using citrate/phosphate as a buffer and 30% saturated ammonium sulfate as precipitant. The crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 32.4, b = 61.8, c = 32.9 A, beta = 114.5 degrees. The Matthews coefficient calculated assuming two molecules in the asymmetric unit was 1.95 A(3) Da(-1), which corresponds to a 37% solvent content. X-ray data were collected to 2.5 A resolution from a flash-frozen crystal. The structure was solved using the molecular-replacement method using tracy soybean inhibitor structure (PDB entry 1pi2) as a model.

Chymotrypsin↗
PubMed1996

Ancestry and interrelationships of the Indians and their relationship with other world populations: a study based on mitochondrial DNA polymorphisms.

Mitochondrial DNA variation was studied in 100 Indians using the same set of six restriction enzymes used in the study of other world ethnic groups in order to compare and discern possible relationships of the Indian populations. Twenty nine mtDNA types were found including the ones from an earlier study (Semino et al. 1991) and unweighted pair-group method (UPGMA) and maximum parsimony trees were constructed using the mtDNA types. The nucleotide diversity values were calculated using the maximum likelihood method. From a study of the shared mitochondrial DNA types and the parsimony tree (Fig. 2) we came to the conclusion that the Indian population is closer to Caucasians and has an admixture with Asians. The North Indian population appears to have a recent admixture of the Caucasian mtDNA types which is absent in the south.

DNA, Mitochondrial↗