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M Ghosh

Publications and source records attributed to M Ghosh.

At least 73 records · Page 4Linked to original sources
PubMed1998

The structure and function of the PQQ-containing quinoprotein dehydrogenases.

Bacterial methanol and glucose dehydrogenases containing a novel type of prosthetic group, subsequently identified as pyrrolo-quinoline quinone (PQQ), were first described about 30 years ago. Quinoproteins were originally defined as proteins containing PQQ but this definition has since been broadened to include those proteins containing other types of quinone-containing prosthetic groups, and the X-ray structures of representatives of each type of quinoprotein have recently been published. This review is mainly concerned with the structure and function of the PQQ-containing methanol dehydrogenase, whose structure has been determined at high resolution, and related proteins. Their basic structure consists of a 'propeller' fold superbarrel made up of 8-sheet 'propeller blades' which are held together by novel tryptophan-docking motifs. In methanol dehydrogenase the PQQ in the active site is coordinated to a Ca2+ ion and is maintained in position by a stacked tryptophan and a novel 8-membered ring structure made up of a disulphide bridge between adjacent cysteine residues. This review describes these features and discusses them in relation to previously proposed mechanisms for this enzyme.

Alcohol Oxidoreductases↗
PubMed1998

Skin banking in the UK: the need for proper organization.

Skin banking was set up in Sheffield in 1991 to provide a readily available source of allograft material to be used both for research purposes and also as a means of providing immediate wound cover for major burns patients. Once skin was available, however, clinical demand for it both within and outside Sheffield, outstripped the resources to run the bank. Logistical difficulties were encountered in the day to day running of the bank. These revolved around shortage of staff available for harvesting, the relative lack of public awareness of skin donation, shortage of banked skin as the bank became more widely known and lack of space and finance to expand. The decision was made to transfer the now established skin bank to the National Blood Service where it now operates with staff and resources dedicated specifically to this purpose. Experience leads to the suggestion that there is a clinical need for allograft skin in the UK which is not being met at the present time. There is a need for dedicated properly resourced skin banks and for the Department of Health to introduce regulation, monitoring and inspection of skin bank facilities in order to safeguard standards.

Blood Banks↗
PubMed1998

Selective growth promotion and growth inhibition of gram-negative and gram-positive bacteria by synthetic siderophore-beta-lactam conjugates.

Conjugates of a carbacephalosporin with hydroxamate, spermexatol, N alpha,N epsilon-bis(2,3-dihydroxybenzoyl)-L-lysine, mixed catecholate/hydroxamate and cyanuric acid-based siderophores were investigated for their potential to promote growth of siderophore indicator strains of Gram-negative and Gram-positive bacteria under iron depleted conditions, for their antibacterial activity and for their ability to use iron transport pathways to penetrate the Gram-negative bacterial outer membrane. The selective growth promotion of enterobacterial and pseudomonas strains by hydroxamate, spermexatol and mixed catecholate-hydroxamate siderophore-based conjugates bearing a L- or D-amino acid spacer was correlated with TonB dependent uptake routes. The preferred outer membrane siderophore receptor used in Escherichia coli was found to be Fiu, followed by Cir. Antagonistic effects of siderophores administered with the conjugates to determine antibacterial activity confirmed the active transport of conjugates via siderophore receptors. All of the conjugates were still able to diffuse through the porin proteins OmpC and OmpF. Nevertheless, strong inhibition of E. coli and Pseudomones aeruginosa outer membrane mutants DC2 and K799/61 compared to the parent strains indicated inefficient penetrability of all types of conjugates tested. Mycobacterium smegmatis SG 987 was able to use all of the siderophore-cephalosporin conjugates as growth promotors. Consequently there was no growth inhibition of this strain.

Bacterial Outer Membrane Proteins↗
PubMed1998

Characterization of native and recombinant forms of an unusual cobalt-dependent proline dipeptidase (prolidase) from the hyperthermophilic archaeon Pyrococcus furiosus.

Proline dipeptidase (prolidase) was purified from cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus by multistep chromatography. The enzyme is a homodimer (39.4 kDa per subunit) and as purified contains one cobalt atom per subunit. Its catalytic activity also required the addition of Co2+ ions (Kd, 0.24 mM), indicating that the enzyme has a second metal ion binding site. Co2+ could be replaced by Mn2+ (resulting in a 25% decrease in activity) but not by Mg2+, Ca2+, Fe2+, Zn2+, Cu2+, or Ni2+. The prolidase exhibited a narrow substrate specificity and hydrolyzed only dipeptides with proline at the C terminus and a nonpolar amino acid (Met, Leu, Val, Phe, or Ala) at the N terminus. Optimal prolidase activity with Met-Pro as the substrate occurred at a pH of 7.0 and a temperature of 100 degrees C. The N-terminal amino acid sequence of the purified prolidase was used to identify in the P. furiosus genome database a putative prolidase-encoding gene with a product corresponding to 349 amino acids. This gene was expressed in Escherichia coli and the recombinant protein was purified. Its properties, including molecular mass, metal ion dependence, pH and temperature optima, substrate specificity, and thermostability, were indistinguishable from those of the native prolidase from P. furiosus. Furthermore, the Km values for the substrate Met-Pro were comparable for the native and recombinant forms, although the recombinant enzyme exhibited a twofold greater Vmax value than the native protein. The amino acid sequence of P. furiosus prolidase has significant similarity with those of prolidases from mesophilic organisms, but the enzyme differs from them in its substrate specificity, thermostability, metal dependency, and response to inhibitors. The P. furiosus enzyme appears to be the second Co-containing member (after methionine aminopeptidase) of the binuclear N-terminal exopeptidase family.

Amino Acid Sequence↗
PubMed1998

Sequences of intestinal and hepatic cytochrome P450 3A4 cDNAs are identical.

Cytochrome P450 (CYP or P450) 3A4 is known to be the major P450 expressed in the liver. More recently, CYP3A4 was also shown to be the major P450 in the intestine, where it plays an important role in the metabolism of some orally administered drugs. However, studies examining the catalytic properties of CYP3A4 have been largely based on the use of CYP3A4 enzyme obtained from liver or recombinant protein expressed from hepatic cDNA. To investigate whether intestinal and hepatic CYP3A4 enzymes are identical, we determined the sequences of intestinal CYP3A4 cDNAs. Compared with the published sequence for hepatic CYP3A4, we found a single base pair difference in the 3' untranslated region of intestinal cDNA from three individuals. We found this same base pair difference in cDNA obtained from the livers of three additional subjects. We conclude that hepatic and intestinal CYP3A4 cDNAs are identical and that the proteins expressed in these two tissues are therefore likely to be the same.

Cloning, Molecular↗
PubMed1997

An atomic model of the outer layer of the bluetongue virus core derived from X-ray crystallography and electron cryomicroscopy.

BACKGROUND: Bluetongue virus (BTV), which belongs to the Reoviridae family and orbivirus genus, is a non-enveloped, icosahedral, double-stranded RNA virus. Several protein layers enclose its genome; upon cell entry the outer layer is stripped away leaving a core, the surface of which is composed of VP7. The structure of the trimeric VP7 molecule has previously been determined using X-ray crystallography. The articulated VP7 subunit consists of two domains, one which is largely alpha-helical and the other, smaller domain, is a beta barrel with jelly-roll topology. The relative orientations of these two domains vary in different crystal forms. The structure of VP7 and the organizations of 780 subunits of this molecule in the core of virus is central to the assembly and function of BTV. RESULTS: A 23 A resolution map of the core, determined using electron cryomicroscopy (cryoEM) data, reveals that the 260 trimers of VP7 are organized on a rather precise T = 13 laevo icosahedral lattice, in accordance with the theory of quasi-equivalence. The VP7 layer occupies a shell that is between 260 A and 345 A from the centre of the core. Below this radius (230-260 A) lies the T = 1 layer of 120 molecules of VP3. By fitting the X-ray structure of an individual VP7 trimer onto the cryoEM BTV core structure, we have generated an atomic model of the VP7 layer of BTV. This demonstrates that one of the molecular structures seen in crystals of the isolated VP7 corresponds to the in vivo conformation of the molecule in the core. CONCLUSIONS: The beta-barrel domains of VP7 are external to the core and interact with protein in the outer layer of the mature virion. The lower, alpha-helical domains of VP7 interact with VP3 molecules which form the inner layer of the BTV core. Adjacent VP7 trimer-trimer interactions in the T = 13 layer are mediated principally through well-defined regions in the broader lower domains, to form a structure that conforms well with that expected from the theory of quasi-equivalence with no significant conformational changes within the individual trimers. The VP3 layer determines the particle size and forms a rather smooth surface upon which the two-dimensional lattice of VP7 trimers is laid down.

Bluetongue virus↗
PubMed1997

Mutations in HIV reverse transcriptase which alter RNase H activity and decrease strand transfer efficiency are suppressed by HIV nucleocapsid protein.

Structural studies of authentic HIV reverse transcriptase (RT) suggest a role for the p51 carboxyl terminus in forming an active RNase H conformation [Rodgers, D. W., Gamblin, S. J., Harris, B. A., Ray, S., Culp, J. S., Hellmig, B., Woolf, D. J., Debouck, C. & Harrison, S. C. (1995) Proc. Natl. Acad. Sci. USA 92, 1222-1226]. We have purified mutant RT heterodimers containing deletion of 5, 9, or 13 amino acids from the p51 carboxyl terminus. These "selectively deleted" heterodimers have been analyzed for changes in RNA-dependent DNA polymerase activity, RNase H activity, and the ability to catalyze DNA strand transfer. As deletions extended into the p51 subunit, a decrease in the stability of the RT-DNA complex was apparent. The largest effect was observed for p66/p51Delta13 RT, which showed a 3-fold decrease relative to wild-type RT. RNase H activity was measured by digestion of the RNA in a 5' 32P-labeled RNA/DNA hybrid. Deletion of 5 or 9 amino acids from p51 had little effect on synthesis-dependent and synthesis-independent RNase H activities. In contrast, deletion of 13 amino acids from p51 increased the length of the hydrolysis products of both RNase H activities by 8-10 bp, thus changing the spatial relationship between the polymerase and RNase H active sites from a distance of 17-18 bp to 26-27 bp. The Delta13 derivative was also incapable of efficient DNA strand transfer. This defect in strand transfer could be suppressed by the 71-amino acid form of HIV nucleocapsid protein (NC) but not by the 55-amino acid form (NC55) or by equine infectious anemia virus NC. These results provide evidence for the existence of a specific complex between RT and NC and are discussed in terms of the role of this complex in proviral DNA synthesis.

Base Sequence↗
PubMed1997

Alanine-scanning mutations in the "primer grip" of p66 HIV-1 reverse transcriptase result in selective loss of RNA priming activity.

Alanine-scanning mutants of the primer grip region of human immunodeficiency virus type 1 reverse transcriptase were tested for their ability to extend RNA and DNA versions of the polypurine tract primer, and an oligonucleotide representing the 18-nucleotide sequence at the 3' end of tRNALys3. A majority of the mutant enzymes were either completely or severely deficient in RNA priming activity, but, with only one exception, were able to efficiently extend DNA versions of the same primers. The mutant enzymes were able to bind to RNA primers, indicating that the defect in RNA priming was not simply a loss of binding activity. Mutations at positions 229, 233, and 235 dramatically reduced the amount of specific RNase H cleavage at the 3' terminus of the polypurine tract, which is required for primer removal. An alanine substitution at position 232 led to loss of cleavage specificity, although total activity was close to the wild-type level. Taken together, these results demonstrate for the first time that there are residues in human immunodeficiency virus type 1 reverse transcriptase which are specifically involved in protein-nucleic acid interactions with RNA primers.

Alanine↗
PubMed1997

Mutating a conserved motif of the HIV-1 reverse transcriptase palm subdomain alters primer utilization.

In order to investigate how primer grip residues of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) contribute toward the architecture of its palm subdomain and neighboring structural elements, the DNA polymerase and ribonuclease H (RNase H) activities of enzymes bearing aromatic substitutions at Trp229 and Tyr232 of the catalytically-competent p66 subunit were evaluated. Although all mutants retained RNase H function, the manner in which different RNA-DNA hybrids were hydrolyzed was affected. Depending on the nature of the substitution, DNA-dependent DNA synthesis was (i) unaffected, (ii) interrupted shortly after initiation, or (iii) stalled when the replication machinery encountered an intramolecular duplex on the single-stranded template. Evaluating (-) strand strong-stop DNA synthesis on an RNA template derived from the viral genome raises the additional possibility that DNA and RNA primers might be differentially recognized by the retroviral polymerase. In support of this, all mutants were unable to extend the HIV-1 polypurine tract (PPT) RNA primer into (+) strand DNA, despite supporting the equivalent event from an oligodeoxynucleotide primer. Collectively, our data illustrate that subtle alterations to primer grip architecture may manifest themselves in discrimination between oligoribo- and oligodeoxyribonucleic acid primers.

Conserved Sequence↗
PubMed1997

Transgenic elite indica rice plants expressing CryIAc delta-endotoxin of Bacillus thuringiensis are resistant against yellow stem borer (Scirpophaga incertulas).

Generation of insect-resistant, transgenic crop plants by expression of the insecticidal crystal protein (ICP) gene of Bacillus thuringiensis (Bt) is a standard crop improvement approach. In such cases, adequate expression of the most appropriate ICP against the target insect pest of the crop species is desirable. It is also considered advantageous to generate Bt-transgenics with multiple toxin systems to control rapid development of pest resistance to the ICP. Larvae of yellow stem borer (YSB), Scirpophaga incertulas, a major lepidopteran insect pest of rice, cause massive losses of rice yield. Studies on insect feeding and on the binding properties of ICP to brush border membrane receptors in the midgut of YSB larvae revealed that cryIAb and cryIAc are two individually suitable candidate genes for developing YSB-resistant rice. Programs were undertaken to develop Bt-transgenic rice with these ICP genes independently in a single cultivar. A cryIAc gene was reconstructed and placed under control of the maize ubiquitin 1 promoter, along with the first intron of the maize ubiquitin 1 gene, and the nos terminator. The gene construct was delivered to embryogenic calli of IR64, an elite indica rice cultivar, using the particle bombardment method. Six highly expressive independent transgenic ICP lines were identified. Molecular analyses and insect-feeding assays of two such lines revealed that the transferred synthetic cryIAc gene was expressed stably in the T2 generation of these lines and that the transgenic rice plants were highly toxic to YSB larvae and lessened the damage caused by their feeding.

Animals↗
PubMed1997

Identification and expression of the Saccharomyces cerevisiae cytoplasmic tryptophanyl-tRNA synthetase gene.

The enzymes that aminoacylate tRNAs have been studied extensively and can be organized into two distinct classes based on signature sequences and the position of aminoacylation. The class I enzymes have canonical HIGH and KMSKS sequences as part of a Rossman fold nucleotide-binding site. The tryptophan-specific enzymes have been placed in class I based on analysis of the cognate genes from Escherichia coli, B. stearothermophilus, B. taurus, and Homo sapiens. An unidentified open reading frame (ORF) on Saccharomyces cerevisiae chromosome XV, HRE342, has 46% identity with the bovine tryptophanyl-tRNA synthetase and possesses the appropriate signature sequences. The predicted molecular weight of the putative HRE342 protein also closely matched the expected monomer size of the S. cerevisiae enzyme. The HRE342 ORF plus about 250 bp of 5' and 3' flanking sequence was amplified by polymerase chain reaction, cloned into a 2 mu based vector, and transformed into a host strain, S. cerevisiae JG369.3B. Nucleotide sequence analysis of the clone confirmed the presence of HRE342. Extracts from transformed yeast have a 30- to 100-fold increase in specific activity of the tryptophanyl-tRNA synthetase. An HRE342 locus in a diploid strain, PTY33XPTY44, was disrupted with a LEU2 insert. Sporulation and tetrad analysis of the HRE342::LEU2 strain demonstrated that HRE342 is an essential gene. We conclude that HRE342 is the S. cerevisiae gene encoding the cytoplasmic tryptophanyl-tRNA synthetase, WRS1. A search of the Saccharomyces Genome Database using amino acid sequences from other eukaryotic aminoacyl-tRNA synthetase suggests there is sufficient similarity to identify both class I and class II genes.

Amino Acid Sequence↗
PubMed1997

Nickel-dependent oxidative cross-linking of a protein.

A model protein, ribonuclease A (bovine pancreas), was examined for its ability to coordinate Ni2+ and promote selective oxidation. In the presence of a peracid such as monopersulfate, HSO5-, nickel induced the monomeric RNase A to form dimers, trimers, tetramers, and higher oligomers without producing fragmentation of the polypeptide backbone. Co2+ and to a lesser extent Cu2+ exhibited similar activity. The nickel-dependent reaction appeared to result from a specific association between the protein and Ni2+ that allowed for transient and in situ oxidation of the bound nickel to yield intermolecular tyrosine-tyrosine cross-links. Macrocylic nickel complexes that had previously been shown to promote guanine oxidation were unable to mimic the activity of the free metal salt. Amino acid analysis of the protein dimer confirmed the expected consumption of one tyrosine per polypeptide and formation of dityrosine. The presence of excess tyrosine efficiently inhibited formation of the protein dimer and produced instead a ribonuclease-tyrosine cross-link. In contrast, high concentrations of the hydroxyl radical quenching agent mannitol only partially inhibited ribonuclease dimerization. The polypeptide-mediated activation of nickel and its subsequent reactivity mimic a process that could contribute to the adverse effects of nickel in vivo.

Amino Acids↗
PubMed1997

Purification and Some Properties of a Xylanase from Aspergillus sydowii MG49.

Volume 62, no. 10, p. 3915, Author's Correction (Ghosh and Nanda). Errors were made in the original identification of the microorganism used in this study and in the reporting of the corrected species identification. The culture resubmitted to the American Type Culture Collection was identified by Dr. Frank Dugan as Aspergillus flavus, not Aspergillus citrinum, and is available as ATCC 96916. [This corrects the article on p. 3915b in vol. 62.].

Journal Article↗
PubMed1997

Melatonin secretion of the rat pineal gland in response to norepinephrine in different types of the anovulatory syndrome.

Earlier experimental results show the role of altered pineal function in the development of the constant estrous-anovulatory (CEA) state induced by neonatal androgen sterilization (NA) or in the spontaneously developed anovulatory syndrome in the aging rat (AG-CEA state). Since norepinephrinergic (NE) innervation of pineal gland represents the main stimulus for melatonin secretion in mammals, in the present experimental series, the reactivity of pineal tissue to NE was investigated in in vitro perifusion system in rats suffering from NA-CEA or AG-CEA state, using the model of pineal body deprived from neural inputs. The data indicate that the 'melatonin deficiency' observed in the 2 types of anovulatory syndrome (NA-CEA and AG-CEA states) is due to disturbance of norepinephrinergic innervation of the pineal gland rather than to deficient secretory capacity of pineal tissue.

Animals↗
PubMed1997

Mediator substances of the pineal neuronal network of mammals.

In addition to receptor-type pinealocytes, the mammalian pineal organ contains small and large neurons and ependymal/glial cells as well. Axons of pinealocytes form synaptic ribbon-containing axo-dendritic synapses on large secondary pineal neurons and/or terminate as neurohormonal endings on the basal lamina of the vascular surface of the organ. The small pineal neurons were found to be gamma-aminobutyric acid (GABA)-immunoreactive, while large secondary neurons and pinealocytes contained immunoreactive amino acids (glutamate and aspartate). Glutamate accumulated presynaptically in pinealocytic axon terminals on large secondary neurons and in the axons of these neurons. Glutamate immunoreactive axons of pineal neurons were traced via the pineal tract to the habenular nucleus. Axons containing granular vesicles and coming from extrapineal perikarya are glutamate immunoreactive as well. Aspartate and GABA are also present in some of the myelinated axons, supposedly pinealopetal in the pineal tract.

Animals↗
PubMed1996

Alterations to the primer grip of p66 HIV-1 reverse transcriptase and their consequences for template-primer utilization.

Alanine scanning mutagenesis was undertaken to evaluate the structural significance of Met230-His235 of the 66 kDa subunit of p66/p51 human immunodeficiency virus reverse transcriptase (HIV-1 RT). Together with Glu224-Trp229, these residues provide the framework of the p66 "primer grip", whose proposed role is maintaining the primer terminus in an orientation appropriate for nucleophilic attack on an incoming dNTP. Of these residues, altering Leu234 results in a p66 subunit incapable of associating into heterodimer. The remaining selectively mutated enzymes were successfully reconstituted and purified to homogeneity for evaluation of RT-associated activities. We show here that alterations to any residue within the p66-Trp229-Met230-Gly231-Tyr232-quartet alter functions associated with both the DNA polymerase and ribonuclease H (RNase H) domains. Detailed analysis of mutant p66Y232A/p51 with an intact or a model "precleaved" RNA-DNA hybrid suggests an altered RNase H phenotype could result from relocation of template-primer in the nucleic acid binding cleft. As a consequence, template nucleotide-8 is positioned in the immediate vicinity of the RNase H catalytic center rather than nucleotide-17.

Amino Acid Sequence↗
PubMed1996

Two conformationally vicinal thiols at the active site of Leishmania donovani adenosine kinase.

Inactivation of adenosine kinase (Adk) from Leishmania donovani correlates with the modification of two conformationally vicinal cysteine residues. In contrast, Adk from hamster liver, despite being sensitive to monothiol-blocking reagents, was insensitive to dithiol modifiers. Inactivation kinetics and substrate-protection studies along with double-modification experiments successively with N-ethylmaleimide in the presence of Ado and sodium m-arsenite-2,3-dimercaptopropanol or diazenedicarboxylic acid bis-N,N'-dimethylamide supported assignment of the two thiols at the Ado-binding site. Cystine bridge formation impaired the ability of the modified enzyme to bind to the substrate. Tryptophan fluorescence of the enzyme was quenched after modification by dithiol-blocking reagents with concomitant loss of activity. However, treatment of the enzyme with methylmethanethiosulphonate (MMTS) led to complete inactivation without a marked change in protein fluorescence. Ado protected both fluorescence and catalytic activity against inactivation by both MMTS and dithiol-blocking reagents. Stern-Volmer quenching analysis of the native and Ado-complexed enzyme suggested that, of the four tryptophan residues, at least one is located at or near the active site. Furthermore quenching constants of native, Ado-complexed and dithiol-modified enzyme in the presence of either acrylamide or KI indicated spatial proximity of tryptophan and two cysteine residues within the hydrophobic domain of the Ado-binding site. Taken together the results suggest important function(s) for the cysteine residue(s). A schematic model is proposed to explain the inactivation of the enzyme by both monothiol- and dithiol-blocking reagents.

Adenosine↗