Search PubMed⌕ Search

Biomedical subjects

D Bhattacharyya

Publications and source records attributed to D Bhattacharyya.

At least 73 records · Page 4Linked to original sources
PubMed1996

Positioning of positively charged residues in the V3 loop correlates with HIV type 1 syncytium-inducing phenotype.

Although the V3 loop of the envelope glycoprotein (gp120) plays a role in determining the phenotype, pathogenesis, and tropism of human immunodeficiency virus-1 (HIV-1), there has not been any consistent correlation between structure and phenotype. Theoretically determined structures of the V3 loop of gp120, from 20 different viral strains, 10 syncytium-inducing (SI) and 10 non-syncytium-inducing (NSI) phenotype, revealed that all V3 loops from SI phenotypic strains had at least two positively charged residues in close proximity, on the same face of the loop. All of the SI phenotypic V3 loop structures were capable of forming strong divalent electrostatic interactions with disulfated sugars. The ability to form this interaction may be a determinant of the phenotype, tropism, and pathogenicity of HIV-1 viral strains. This structural motif was absent in all V3 loops from viral strains with the NSI phenotype.

Amino Acid Sequence↗
PubMed1995

A 25-kDa beta-lactam-induced outer membrane protein of Vibrio cholerae. Purification and characterization.

A 25-kDa outer membrane protein, induced following treatment of Vibrio cholerae cells with beta-lactam antibiotics and constituting about 8-10% of the total outer membrane proteins of beta-lactam-resistant mutants, has been purified to homogeneity. It is a basic (pI 8.5) protein rich in beta-sheet structure and is a homodimer, the monomers being held together by hydrophobic interactions. The effective hydrophobicity of the protein is low, and a large part of the protein is exposed on the surface of the outer membrane. The protein does not have beta-lactamase or autolytic activity and is not a penicillin-binding protein. The Stoke's radius of the 25-kDa protein (26 A) is comparable to the pore size of the V. cholerae OmpF-like porin. Proteoliposome swelling assay showed that the 25-kDa protein might block the pores of OmpF through which beta-lactam antibiotics normally enter the cells. Twenty-two amino acid residues from the N-terminal end of the 25-kDa protein have been sequenced, and a 32-mer oligonucleotide probe was synthesized using the amino acid residues 2-12. This probe was used to identify the gene encoding the 25-kDa protein. The beta-lactam-resistant cells are insensitive to changes in the osmolarity of the growth medium in contrast to the wild type cells which exhibit osmoregulation of OmpF and OmpC synthesis. All beta-lactam-resistant mutants examined are resistant to novobiocin.

Anti-Bacterial Agents↗
PubMed1995

NUPARM and NUCGEN: software for analysis and generation of sequence dependent nucleic acid structures.

Software packages NUPARM and NUCGEN, are described, which can be used to understand sequence directed structural variations in nucleic acids, by analysis and generation of non-uniform structures. A set of local inter basepair parameters (viz. tilt, roll, twist, shift, slide and rise) have been defined, which use geometry and coordinates of two successive basepairs only and can be used to generate polymeric structures with varying geometries for each of the 16 possible dinucleotide steps. Intra basepair parameters, propeller, buckle, opening and the C6...C8 distance can also be varied, if required, while the sugar phosphate backbone atoms are fixed in some standard conformation in each of the nucleotides. NUPARM can be used to analyse both DNA and RNA structures, with single as well as double stranded helices. The NUCGEN software generates double helical models with the backbone fixed in B-form DNA, but with appropriate modifications in the input data, it can also generate A-form DNA and RNA duplex structures.

Base Composition↗
PubMed1995

NUVIEW: software for display and interactive manipulation of nucleic acid models.

The NUVIEW software package allows skeletal models of any double helical nucleic acid molecule to be displayed on a graphics monitor and to apply various rotations, translations and scaling transformations interactively, through the keyboard. The skeletal model is generated by connecting any pair of representative points, one from each of the bases in the basepair. In addition to the above mentioned manipulations, the base residues can be identified by using a locator and the distance between any pair of residues can be obtained. A sequence based color coded display allows easy identification of sequence repeats, such as runs of Adenines. The real time interactive manipulation of such skeletal models for large DNA/RNA double helices, can be used to trace the path of the nucleic acid chain in three dimensions and hence get a better idea of its topology, location of linear or curved regions, distances between far off regions in the sequence etc. A physical picture of these features will assist in understanding the relationship between base sequence, structure and biological function in nucleic acids.

Animals↗
PubMed1994

CA/TG sequence at the 5' end of oligo(A)-tracts strongly modulates DNA curvature.

An analysis of the base pair doublet geometries in available crystal structures indicates that the often reported intrinsic curvature of DNA containing oligo-(d(A).d(T)) tracts may also depend on the nature of the flanking sequences. The presence of CA/TG doublet in particular at the 5' end of these tracts is expected to enhance their intrinsic bending property. To test this proposition, three oligonucleotides, d(GAAAAAC-CCCCC), d(CCCCCCAAAAAG), d(GAAAAATTTTTC), and their complementary sequences were synthesized to study the effect of various flanking sequences, at the 5' and 3' ends of the A-tracts, on the curvature of DNA in solution. An analysis of the polyacrylamide gel electrophoretic mobilities of these sequences under different conditions of salts and temperatures (below their melting points) clearly showed that the oligomer with CA/TG sequence in the center was always more retarded than the oligomer with AC/GT sequence, as well as the oligomer with AT/AT sequence. Hydroxyl radical probing of the sequences with AC/GT and CA/TG doublet junctions gives a similar cutting pattern in the A-tracts, which is quite different from that in the C-tracts, indicating that the oligo(A)-tracts have similar structures in the two oligomers. KMnO4 probing shows that the oligomer with a CA/TG doublet junction forms a kink that is responsible for its inherent curvature and unusual electrophoretic mobility. UV melting shows a reduced thermal stability of the duplex with CA/TG doublet junction, and circular dichroism (CD) studies indicate that a premelting transition occurs in the oligomer with CA/TG doublet step before global melting but not in the oligomer with AC/GT doublet step, which may correspond to thermally induced unbending of the oligomer. These observations indicate that the CA/TG doublet junction at the 5' end of the oligo(A)-tract has a crucial role in modulating the overall curvature in DNA.

Base Sequence↗
PubMed1994

Analysis of sequence dependent variations in secondary and tertiary structure of tRNA molecules.

The double helical regions of the five tRNA(Phe) and two tRNA(Asp) crystal structures have been analyzed using the local basepair step parameters. The sequence dependent effects in the mini double helices of tRNA are very similar to those observed in the crystal structures of oligonucleotides in the A-form, the purine-pyrimidine and purine-purine steps have small roll angles when compared to the fiber models of A-DNA as well as A-RNA, while the pyrimidine-purine doublet steps have large roll angles. The orientation of the basepairs in the D-stem is unusual and invariant i.e. they are different from the other three stems but are very similar in all the five tRNA(Phe) crystal structures, presumably due to tertiary interaction of the Watson-Crick basepairs with other bases, with all bases being highly conserved. The origin of the differences between the tertiary structures of tRNA(Phe) and tRNA(Asp) from yeast has also been investigated. It is found that even though the angle between the acceptor arm and the D-stem is very similar in the two structures, the angle subtended by the acceptor arm and the anticodon arm is smaller in the tRNA(Phe) structure (by more than 10 degrees). This is due to differences in the orientation of the two mini helices constituting the anticodon arm, which are inclined to each other by approximately 25 degrees in tRNA(Phe) and 16 degrees in tRNA(Asp). In addition, the acceptor arm, the D-stem and the anticodon stem are nearly coplanar in tRNA(Phe), while in tRNA(Asp) the anticodon stem projects out of the plane defined by the acceptor arm and the anticodon stem. These two features together lead to a larger separation between the acceptor and anticodon ends in tRNA(Asp) and indicate that the junction between the D-stem and the anticodon stem is quite variable, with features characteristic of a ball-and-socket type joint and determined for each tRNA molecule by the base sequence at the junction.

Anticodon↗
PubMed1993

Reversible folding of UDP-galactose-4-epimerase from yeast Kluyveromyces fragilis.

UDP-galactose-4-epimerase from yeast Kluyveromyces fragilis is a dimeric molecule with one molecule of cofactor NAD per dimer. In presence of 8 M urea, the enzyme is inactivated with complete disorganization of its structure and dissociation of the subunits together with the cofactor. Dilution of the denaturant by sodium phosphate buffer (20 mM, pH 7.0) containing 1 mM NAD recovers the activity to the extent of 80-100%. At a monomer concentration of 0.8 microM, the reactivation follows second-order kinetics, k = 1.48 x 10(3) M-1 s-1, at 20 degrees C. The renatured enzyme resembles the native enzyme in terms of most of its physicochemical properties, e.g., circular dichroism spectrum, fluorescence spectrum, interaction with hydrophobic fluorophore 1-anilino-8-naphthalenesulfonic acid (ANS), hydrodynamic volume, Km for the substrate UDP-galactose, etc. The reactivation process has an energy of activation of 15.2 kcal/mol. The folding pathway could be divided into four major parts: in the first phase (within 10 min), the secondary and tertiary structures of the monomers are formed as found by circular dichroism and fluorescence spectroscopy; in the second phase (within 20 min), the folded monomers associate to form an inactive dimer as observed by size-exclusion HPLC; in the third phase, the NAD binding site is formed, which is possibly induced by the cofactor; and finally, NAD assembles over the cofactor binding site to yield an active holoenzyme (within 120 min). The third and fourth steps have been detected kinetically. They are slow and rate-limiting, depending upon the concentration of extraneous NAD.

Circular Dichroism↗
PubMed1993

Inhibition of intestinal peroxidase activity by nonsteroidal antiinflammatory drugs.

The peroxidase activity of the mitochondrial fraction of rat intestine is inhibited in vitro by non-steroidal antiinflammatory drugs (NSAIDs), such as indomethacin (IMN) and acetylsalicylic acid (ASA), the former being more potent than the latter. The peroxidase was solubilised by cetab-NH4Cl extraction and purified to apparent homogeneity by Sephadex G-150 gel filtration and affinity chromatography on Con-A Sepharose. The purified enzyme activity was 80% inhibited by 150 microM IMN and 50% by 2.67 mM ASA. IMN could also inhibit lactoperoxidase activity to the same extent but not the horseradish peroxidase activity. The inhibition of peroxidase-catalysed iodide oxidation by IMN and ASA was optimal at pH 5.5 and 4.5, respectively. Kinetic studies revealed that the inhibition by IMN was competitive with respect to iodide or guaiacol, while the inhibition by ASA was noncompetitive and reversible in nature. Studies of some structural analogues showed that indole-3-acetic acid was as effective as IMN, while salicylic acid was more potent than ASA. Spectral studies showed a small bathochromic shift of the Soret band of the enzyme by IMN, suggesting its possible interaction at or near the heme moiety. The competitive nature of IMN may be explained as due to its oxidation by the peroxidase to a product absorbing at 412 nm, the formation of which is inhibited by iodide. We suggest that IMN inhibits intestinal peroxidase activity by acting as a competitive substrate for the enzyme. As intestinal peroxidase is mainly contributed by the invading eosinophils, NSAIDs may affect the host defence mechanism by inhibiting the activity of the enzyme.

Animals↗
PubMed1993

Purification and partial characterization of a haemorrhagin (VRH-1) from Vipera russelli russelli venom.

A haemorrhagic toxin (VRH-1) has been purified to homogeneity from Vipera russelli russelli venom by subjecting it to chromatography twice successively on CM-Sephadex C-50. It is a protein of mol. wt 22,000 and contains one mole of Mg2+. Intradermal administration of this haemorrhagin in mice resulted in severe lung haemorrhage but produced little haemorrhage in skin. This apparent organ preference led us to develop a new haemorrhage assay method utilizing dye diffusion from lung in vitro. Proteolytic activity of VRH-1 was demonstrated using dimethylcasein as substrate following quantitation by reaction with trinitrobenzoyl sulfonic acid. Both haemorrhagic and proteolytic activities of VRH-1 were inhibited by serine protease inhibitors like phenylmethyl sulfonyl fluoride and chymostatin, but metal chelators had no effect. Lung haemorrhage is unlikely to be a direct reflection of a high local concentration of VRH-1. The administration of supernatant generated by incubation of chopped liver from untreated mouse and VRH-1 (in subhaemorrhagic dose) results in severe lung haemorrhage. This raises the possibility that VRH-1 leads to the formation of intermediate(s) which causes the haemorrhage.

Amino Acids↗
PubMed1992

Groove width and depth of B-DNA structures depend on local variation in slide.

The groove widths of DNA helix, especially minor groove width, are generally believed to be important for recognition of DNA by various types of ligands. It has been postulated earlier that large negative propeller twist, in the AT rich regions compresses the minor groove of duplex DNA. A systematic study has now been carried out by generating models with different values of local doublet and intra-basepair parameters and calculating their minor groove widths. It is found that several local doublet parameters affect the minor groove width but it depends most strongly on the local step parameters roll and slide when each parameter is considered individually. However, a detailed analysis of the various local parameters within the B-DNA family of crystal structures indicates that propeller twist and slide are most strongly correlated with the observed values of minor groove width. The groove depth is also strongly correlated with slide. Thus the local base sequence dependent variations in slide can modify both the groove width and depth and consequently determine the ligand binding properties of DNA.

Base Sequence↗
PubMed1990

Physicochemical studies of human O6-methylguanine-DNA methyltransferase.

O6-Methylguanine-DNA methyltransferase, present in most organisms, removes mutagenic and carcinogenic O6-alkylguanine from DNA by accepting the alkyl group in a stoichiometric reaction. The protein has been partially purified from human placenta. It reacts with second-order rate constants of 2.20 x 10(8) and 0.067 x 10(8) lmol-1 min-1 at 37 degrees C for duplex and single-stranded DNA substrates, respectively. The corresponding value for the alkylated base in synthetic poly(dC, dG, m6dG) is 0.02 x 10(8) l mol-1 min-1. The native protein is monomeric with a molecular mass of 22-24 kDa. Methylation of the protein does not lead to a gross change in its conformation but causes a slight reduction in its isoelectric point of 6.2. Although DNA protects the protein from heat inactivation, both duplex and single-stranded DNAs inhibit its activity in a concentration-dependent manner. The transferase reaction rate is also strongly inhibited by salt with about 20% of the maximum rate observed in physiological ionic strength. This inhibition is nonspecific with respect to the ions of univalent salts.

Animals↗
PubMed1990

Local variability and base sequence effects in DNA crystal structures.

The importance and usefulness of local doublet parameters in understanding sequence dependent effects has been described for A- and B-DNA oligonucleotide crystal structures. Each of the two sets of local parameters described by us in the NUPARM algorithm, namely the local doublet parameters, calculated with reference to the mean z-axis, and the local helical parameters, calculated with reference to the local helix axis, is sufficient to describe the oligonucleotide structures, with the local helical parameters giving a slightly magnified picture of the variations in the structures. The values of local doublet parameters calculated by NUPARM algorithm are similar to those calculated by NEWHELIX90 program, only if the oligonucleotide fragment is not too distorted. The mean values obtained using all the available data for B-DNA crystals are not significantly different from those obtained when a limited data set is used, consisting only of structures with a data resolution of better than 2.4 A and without any bound drug molecule. Thus the variation observed in the oligonucleotide crystals appears to be independent of the quality of their crystallinity. No strong correlation is seen between any pair of local doublet parameters but the local helical parameters are interrelated by geometric relationships. An interesting feature that emerges from this analysis is that the local rise along the z-axis is highly correlated with the difference in the buckle values of the two basepairs in the doublet, as suggested earlier for the dodecamer structures (Bansal and Bhattacharyya, in Structure & Methods: DNA & RNA, Vol. 3 (Eds., R.H. Sarma and M.H. Sarma), pp. 139-153 (1990)). In fact the local rise values become almost constant for both A- and B-forms, if a correction is applied for the buckling of the basepairs. In B-DNA the AA, AT, TA and GA basepair sequences generally have a smaller local rise (3.25 A) compared to the other sequences (3.4 A) and this seems to be an intrinsic feature of basepair stacking interaction and not related to any other local doublet parameter. The roll angles in B-DNA oligonucleotides have small values (less than +/- 8 degrees), while mean local twist varies from 24 degrees to 45 degrees. The CA/TG doublet sequences show two types of preferred geometries, one with positive roll, small positive slide and reduced twist and another with negative roll, large positive slide and increased twist.(ABSTRACT TRUNCATED AT 400 WORDS)

Algorithms↗
PubMed1989

A self-consistent formulation for analysis and generation of non-uniform DNA structures.

A fully self-consistent formulation is described here for the analysis and generation of base-pairs in non-uniform DNA structures, in terms of various local parameters. It is shown that the internal "wedge parameters" are mathematically related to the parameters describing the base-pair orientation with respect to an external helix axis. Hence any one set of three translation and three rotation parameters are necessary and sufficient to completely describe the relative orientation of the base-pairs comprising a step (or doublet). A general procedure is outlined for obtaining an average or global helix axis from the local helix axes for each step. A graphical representation of the local helix axes in the form of a polar plot is also shown and its application for estimating the curvature of oligonucleotide structures is illustrated, with examples of both A and B type structures.

Algorithms↗
PubMed1989

Site-directed mutation of the Escherichia coli ada gene: effects of substitution of methyl acceptor cysteine-321 by histidine in Ada protein.

Oligodeoxynucleotide-mediated mutagenesis of the ada gene of Escherichia coli was used to produce two mutant Ada proteins. In mutant I the methyl acceptor Cys-321 for O6-methylguanine was replaced by histidine; and in mutant II the positions of Cys-321 and His-322 of the wild-type protein were inverted. Neither mutant protein had O6-methylguanine-DNA methyltransferase activity, but both retained the phosphotriester-DNA methyltransferase activity involving methyl group transfer to Cys-69. Under the control of the endogenous promoter, synthesis of mutant I protein was undetectable before or after adaptation treatment with promoter, synthesis of mutant I protein was undetectable before or after adaptation treatment with N-methyl-N'-nitro-N-nitrosoguanidine. This appeared to be due to both inhibition of transcription of the mutant gene and degradation of the synthesized protein. On the other hand, mutant II protein was inducible by N-methyl-N'-nitro-N-nitrosoguanidine, although to a smaller extent than the wild-type protein was, and the phosphotriester-DNA methyltransferase activity appeared to reside in 24- to 30-kilodalton cleavage products. Mutant I protein could be produced under lac promoter control, and its cleavage products, unlike those of mutant II protein, tended to aggregate. These results indicate that (i) Cys-321 cannot be replaced or transposed with the nucleophilic amino acid histidine for O6-methylguanine-DNA methyltransferase function, (ii) single amino acid replacement or transposition at the O6-methylguanine methyl acceptor site can have a profound effect on the in vivo stability and regulatory function of the Ada protein, and (iii) the integrity of the protein may not be absolutely needed for its transcription-activation function.

Amino Acid Sequence↗
PubMed1988

Rapid, large-scale purification and characterization of 'Ada protein' (O6 methylguanine-DNA methyltransferase) of E. coli.

The E. coli Ada protein (O6-methylguanine-DNA methyltransferase) has been purified using a high-level expression vector with a yield of about 3 mg per liter of E. coli culture. The 39-kDa protein has an extinction coefficient (E280 nm (1%)) of 5.3. Its isoelectric point of 7.1 is lower than that predicted from the amino acid content. The homogeneous Ada protein is fully active as a methyl acceptor from O6-methylguanine in DNA. Its reaction with O6-methylguanine in a synthetic DNA has a second-order rate constant of 1.1 x 10(9) M-1 min-1 at O degree C. Both the native form and the protein methylated at Cys-69 are monomeric. The CD spectrum suggests a low alpha-helical content and the radius of gyration of 23 A indicates a compact, globular shape. The middle region of the protein is sensitive to a variety of proteases, including an endogenous activity in E. coli, suggesting that the protein is composed of N-terminal and C-terminal domains connected by a hinge region. E. coli B has a higher level of this protease than does K12.

Circular Dichroism↗
PubMed1988

Effect of polyvalent metal ions on the reactivity of human O6-methylguanine-DNA methyltransferase.

Polyvalent metal ions are highly effective in inhibiting human O6-methylguanine-DNA methyltransferase, the repair protein responsible for the removal of the promutagenic and presumably procarcinogenic adduct, O6-alkylguanine, in DNA. The sulfhydryl group-reacting metal ions (Cd2+, Zn2+, Hg2+, Pb2+) completely inhibited the reaction at concentrations of 100-500 microM while other metal ions (Al3+, Fe3+) required concentrations of 1 mM or greater for significant reduction of the reaction rate. Inhibition by the former group of metals could be reversed by dithiothreitol but not by EDTA, while the opposite was true for the second group. Under conditions of partial inhibition of the initial reaction rate by either Hg2+ or Al3+, the extent of reaction was not significantly affected, indicating reversible binding of these ions.

Cations↗
PubMed1987

Energetics of left and right handed models of DNA.

It has been shown by model building studies that various right handed and left handed models are compatible with X-ray data of B-DNA and C-DNA. These models are also found to be in good agreement with infrared dichroism data. Detailed potential energy calculations have now been carried out for these models, viz., right and left handed B-DNA and right and left handed C-DNA. It is found that base sugar stacking and interactions involving the phosphate groups are the dominant forces for stabilizing a particular structure. For some sequences, viz., A-A, T-A and C-A, left handed stacking is quite favourable in both B and C structures. But intranucleotide interactions make the left B-DNA unfavourable while the left C-DNA structure is more stable, for all the sequences, than the right C-DNA structure, proposed from fibre data. For the hexanucleoside pentaphosphate fragments the same trend is observed, with the right handed B-DNA being the most stable of the four models studied. However, the left C-DNA structure is only marginally higher in energy, particularly if the shielding effect of the counter ions, on the phosphate group is taken into consideration.

Chemical Phenomena↗