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Biomedical subjects

M Ghosh

Publications and source records attributed to M Ghosh.

At least 91 records · Page 5Linked to original sources
PubMed1996

Restoration of tRNA3Lys-primed(-)-strand DNA synthesis to an HIV-1 reverse transcriptase mutant with extended tRNAs. Implications for retroviral replication.

The mechanism for the initiation of reverse transcription in human immunodeficiency virus type 1 (HIV-1) was studied utilizing a unique reverse transcriptase (RT) mutant altered in its noncatalytic p51 subunit. This mutant (p66/p51Delta13) retains full DNA- and RNA-dependent DNA polymerase activity but has reduced affinity for tRNA3Lys, the cognate HIV primer. When the ability to support(-)-strand DNA synthesis on a viral RNA template was evaluated, this mutant initiated from an 18-nucleotide (nt) oligoribo- or oligodeoxyribonucleotide primer complementary to the primer binding site (pbs). However, it failed to do so from natural and synthetic versions of tRNA3Lys. tRNA-primed(-)-strand synthesis could, however, be rescued by substituting the 76-nt tRNA3Lys with 81- and 107-nt tRNA-DNA chimeras, i.e. tRNA3Lys extended by 5 and 31 deoxyribonucleotides complementary to the viral genome upstream of the pbs. These findings imply that through interactions involving its p51 subunit, RT may be required to disrupt additional tRNA-viral RNA duplexes outside the pbs to proceed into productive(-)-strand DNA synthesis. Alternatively, specific interactions between tRNA3Lys and HIV-1 RT may be necessary for efficient initiation of(-)-strand DNA synthesis.

Base Sequence↗
PubMed1996

Synthesis and in vitro antibacterial activity of spermidine-based mixed catechol- and hydroxamate-containing siderophore--vancomycin conjugates.

The first antibiotic conjugates of vancomycin (1) and siderophore analogues containing spermidine-based catechol ligands (conjugate 11) as well as mixed catechol and hydroxamate ligands (conjugate 13) are described. The design of the conjugates was based on the earlier observation that conjugation of siderophore components to beta-lactam antibiotics induced active iron transport-mediated drug delivery. The novel conjugates (11 and 13) were synthesized by selective acylation of the primary amino group of 1. Preliminary biological studies indicated that siderophore modified vancomycins lost some activity (4- to 16-fold) against Gram-positive bacteria relative to vancomycin itself, and were generally similar to vancomycin in activity against Gram-negative bacteria under iron-sufficient conditions. However, under iron-depleted conditions which mimic human serum, conjugate 11 displayed enhanced antibacterial activity against an antibiotic hypersensitive strain of Pseudomonas aeruginosa.

Anti-Bacterial Agents↗
PubMed1996

Iron transport-mediated drug delivery using mixed-ligand siderophore-beta-lactam conjugates.

BACKGROUND: Assimilation of iron is essential for microbial growth. Most microbes synthesize and excrete low molecular weight iron chelators called siderophores to sequester and deliver iron by active transport processes. Specific outer membrane proteins recognize, bind and initiate transport of species-selective ferric siderophore complexes. Organisms most often have specific receptors for multiple types of siderophores, presumably to ensure adequate acquisition of the iron that is essential for their growth. Conjugation of drugs to synthetic hydroxamate or catechol siderophore components can facilitate active iron-transport-mediated drug delivery. While resistance to the siderophore-drug conjugates frequently occurs by selection of mutants deficient in the corresponding siderophore-selective outer membrane receptor, the mutants are less able to survive under iron-deficient conditions and in vivo. We anticipated that synthesis of mixed ligand siderophore-drug conjugates would allow active drug delivery by multiple iron receptor recognition and transport processes, further reducing the likelihood that resistant mutants would be viable. RESULTS: Mixed ligand siderophore-drug conjugates were synthesized by combining hydroxamate and catechol components in a single compound that could chelate iron, and that also contained a covalent linkage to carbacephalosporins, as representative drugs. The new conjugates appear to be assimilated by multiple active iron-transport processes both in wild type microbes and in selected mutants that are deficient in some outer membrane iron-transport receptors. CONCLUSIONS: The concept of active iron-transport-mediated drug delivery can now be extended to drug conjugates that can enter the cell through multiple outer membrane receptors. Mutants that are resistant to such conjugates should be severely impaired in iron uptake, and therefore particularly prone to iron starvation.

Anti-Bacterial Agents↗
PubMed1996

Species selectivity of new siderophore-drug conjugates that use specific iron uptake for entry into bacteria.

Siderophores selectively bind ferric iron and are involved in receptor-specific iron transport into bacteria. Several types of siderophores were synthesized, and growth-promoting or inhibitory activities when they were conjugated to carbacephalosporin, erythromycylamine, or nalidixic acid were investigated. Overall, 11 types of siderophores and 21 drug conjugates were tested against seven different bacterial species: Escherichia coli, Bordetella bronchiseptica, Pasteurella multocida, Pasteurella haemolytica, Streptococcus suis, Staphylococcus aureus, and Staphylococcus epidermidis. In some species, the inhibitory activities of the drug conjugates were associated with the ability of the bacteria to use the siderophore portion of the molecules for growth promotion in disc diffusion tests (0.04 mumol of conjugate or siderophore per disc). E. coli used catechol-based siderophore portions as well as hydroxamate-based tri-delta-OH-N-OH-delta-N-acetyl-L-ornithine ferric iron ligands for growth under iron-restricted conditions achieved by supplemental ethylenediamine di (O-hydroxyphenylacetic acid) (100 micrograms/ml) and was sensitive to carbacephalosporin conjugated to these siderophore types (up to a 34-mm-diameter inhibition zone). B. bronchiseptica used desferrioxamine B and an isocyanurate-based or trihydroxamate in addition to catechol-based siderophore portions for promotion but was not inhibited by beta-lactam conjugates partly because of the presence of beta-lactamase. P. multocida and P. haemolytica did not use any of the synthetic siderophores for growth promotion, and the inhibitory activities of some conjugates seemed partly linked to their ability to withhold iron from these bacteria, since individual siderophore portions showed some antibacterial effects. Individual siderophores did not promote S. suis growth in restrictive conditions, but the type of ferric iron ligands attached to beta-lactams affected inhibitory activities. The antibacterial activities of the intracellular-acting agents erythromycylamine and nalidixic acid were reduced or lost, even against S. aureus and S. epidermidis, when the agents were conjugated to siderophores. Conjugate-resistant E. coli mutants showed the absence of some iron-regulated outer membrane proteins in gel electrophoresis profiles and in specific phage or colicin sensitivity tests, implying that the drugs used outer membrane receptors of ferric complexes to get into cells.

Animals↗
PubMed1996

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

Volume 60, no. 12, p 4620-4623: errors appear to have been made in the original identification of the microorganism used in this study and in the cultures submitted in 1993 to both the American Type Culture Collection (ATCC) and the Indian Institute of Microbial Technology. A resubmitted culture has now been assigned to the species Aspergillus citrinum by Dr. Frank Dugan of the ATCC. The authors have confirmed that the resubmitted culture produces xylanase, so that the major findings of the original publication remain valid. [This corrects the article on p. 4620 in vol. 60.].

Journal Article↗
PubMed1996

Evaluation of phthalmustine, a new anticancer compound. II. Tumor inhibitory effect in murine Ehrlich ascites carcinoma and sarcoma-180.

The antitumor activity of phthalmustine (Neoplasma, 41, 1994, 35) has been evaluated in murine Ehrlich ascites carcinoma (EAC) and sarcoma-180 (S-180). The hematological changes associated with the application of phthalmustine were also evaluated in tumor bearing as well as in normal male Swiss mice. The results indicate that phthalmustine induces marked inhibition of tumor growth and substantially prolongs host survival having curative effect while it does not adversely affect hematopoiesis at the optimum dose tested. No toxic symptoms were noted with respect to external appearance, body weight changes and behavioral pattern.

Animals↗
PubMed1996

Delta F 508 molecular mutation in Indian children with cystic fibrosis.

A preliminary report of 13 Indian children with cystic fibrosis who were screened for the commonest mutation (delta F 508) is presented. Six (46%) patients were homozygous for delta F 508, while two patients were compound heterozygotes. Thus 14 (53.8%) of 26 mutant chromosomes had delta F 508 mutation. These findings confirm that cystic fibrosis occurs in India and all children with persistent respiratory problems and/or malabsorption should be screened for this disease.

Child↗
PubMed1996

Regulation of pineal melatonin secretion: comparison between mammals and birds.

The melatonin secretory pattern of the perifused rat and chicken pineal was compared in response to different lighting conditions and norepinephrine administration. The following main differences were observed. 1. Explanted (perifused) rat pineal showed, after a rapid initial surge, a steady continuous basal secretion of melatonin. This was independent from the day-night (light-dark) periods. Chicken pineal, however, showed a characteristic daily rhythm of melatonin production with peak in the dark and nadir in the light phase. 2. This daily rhythm could not be extinguished by keeping the pineal donor birds in constant light- or constant dark environment for at least 2 weeks immediately before sacrifice. 3. Short light impulse (5 min), applied in the middle of the dark phase, was ineffective in birds. Keeping the perifusion chambers in continuous light or darkness, however, depressed the amplitude of the circadian rhythm even in the next cycle. 4. Rat pineal responds to norepinephrine stimulation with a dose-related increase of melatonin release, independently from the phase of the day, while in the chicken, norepinephrine slightly inhibits both the diurnal and the nocturnal level of melatonin secretion. 5. It can be inferred that melatonin secretion of the mammalian pineal gland is primarily regulated by a peripheral (sympathetic) innervation. This is modulated, under in vivo circumstances, by different environmental factors, mainly by light conditions transmitted by neural mechanisms. In contrast, the primary secretory process of the avian pineal is based on an intrinsic circadian rhythm. This might be genetically coded or maintained by yet unknown neurohormonal mechanisms and/or external factors (e.g. magnetic fields). This fairly stable circadian rhythm is only modulated by environmental lighting conditions.

Animals↗
PubMed1995

Truncating alpha-helix E' of p66 human immunodeficiency virus reverse transcriptase modulates RNase H function and impairs DNA strand transfer.

The properties of recombinant p66/p51 human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) containing C-terminal truncations in its p66 polypeptide were evaluated. Deletion end points partly or completely removed alpha-helix E' of the RNase H domain (p66 delta 8/p51 and p66 delta 16/p51, respectively), while mutant p66 delta 23/p51 lacked alpha E' and the beta 5'-alpha E' connecting loop. Although dimerization and DNA polymerase properties of all mutants were not significantly different from those of the parental enzyme, p66 delta 16/p51 and p66 delta 23/p51 RT lacked ribonuclease H (RNase H) activity. In contrast, RT mutant p66 delta 8/p51 retained endonuclease activity but lacked the directional processing feature of the parental enzyme. Despite retaining full endoribonuclease function, p66 delta 8/p51 RT barely supported transfer of nascent (-)-strand DNA between RNA templates representing the 5' and 3' ends of retroviral genome, shedding light on the requirement for the endonuclease and directional processing functions of the RNase H domain during replication.

Amino Acid Sequence↗
PubMed1995

The refined structure of the quinoprotein methanol dehydrogenase from Methylobacterium extorquens at 1.94 A.

BACKGROUND: Methanol dehydrogenase (MDH) is a bacterial periplasmic quinoprotein; it has pyrrolo-quinoline quinone (PQQ) as its prosthetic group, requires Ca2+ for activity and uses cytochrome cL as its electron acceptor. Low-resolution structures of MDH have already been determined. RESULTS: The structure of the alpha 2 beta 2 tetramer of MDH from Methylobacterium extorquens has now been determined at 1.94 A with an R-factor of 19.85%. CONCLUSIONS: The alpha-subunit of MDH has an eight-fold radial symmetry, with its eight beta-sheets stabilized by a novel tryptophan docking motif. The PQQ in the active site is held in place by a coplanar tryptophan and by a novel disulphide ring formed between adjacent cysteines which are bonded by an unusual non-planar trans peptide bond. One of the carbonyl oxygens of PQQ is bonded to the Ca2+, probably facilitating attack on the substrate, and the other carbonyl oxygen is out of the plane of the ring, confirming the presence of the predicted free-radical semiquinone form of the prosthetic group.

Alcohol Oxidoreductases↗
PubMed1995

Design, synthesis, and biological evaluation of isocyanurate-based antifungal and macrolide antibiotic conjugates: iron transport-mediated drug delivery.

The syntheses and preliminary biological evaluation of conjugates of a synthetic isocyanurate-based trihydroxamate siderophore with two antifungal agents, 5-FU (conjugate 9) and norneoenactin (conjugate 12), and a macrolide antibiotic, erythromycylamine (conjugate 18), are described. A 19F NMR study was used to determine the hydrolytic stability of conjugate 9 under assay conditions. Preliminary biological studies with ferric complexes of conjugates 9 and 12 indicated that these antifungal agents are recognized by Candida and perhaps are actively transported into the cell by the siderophore-transport mechanisms. While conjugate 18 did not show any significant antibacterial activity, presumably due to size restriction, the 5-FU conjugate 9 appeared to be moderately active against a variety of Gram-positive strains, and was more active than the 5-FC control against some strains of Staphylococcus.

Anti-Bacterial Agents↗
PubMed1995

Identification of fat-cell enhancer activity in Drosophila melanogaster using P-element enhancer traps.

To identify genes important in fat-cell metabolism and development, we have screened Drosophila stocks carrying an engineered transposable element that can reveal the presence of nearby enhancer elements. We have identified those "enhancer-trap lines" that contain transposable P elements integrated near fat-cell specific enhancer elements. We anticipate that the genes associated with these enhancers will provide information concerning fat-cell function and serve as target genes for studying fat-cell specific gene expression. Furthermore, the identification of enhancer-trap lines active in the developing fat cell should provide an entry point into the molecular and genetic analysis of early fat-cell development. Analysis of two lines has revealed that the transcription factors svp, a steroid-hormone receptor, and Kr, a zinc-finger protein, are present in the fat body; these factors are likely to be involved in fat-cell gene expression. In two other lines, beta-galactosidase was detected in a subset of adepithelial cells that may be the precursors to the adult fat cell. And finally, in a single line transgene activity is present in the progenitor cells of the embryonic fat body. The genes associated with these enhancer-trap lines may be involved in fat-cell development.

Animals↗
PubMed1994

Evaluation of toxicity of cypenhymustine, a new anticancer compound, in mice.

The toxicity of cypenhymustine, a potential anticancer compound 1 (Cancer Letters, 70 (1993) 1-6), was assessed in normal as well as in Ehrlich ascites carcinoma (EAC), Sarcoma-180 (S-180) and Dalton's lymphoma (DL)-bearing Swiss male mice by measuring drug-induced changes in (1) hematological parameters and (2) femoral bone marrow cellularity on day 9 following drug treatment at the optimum dose of 3.0 mg/kg body weight from days 1 to 7. Detailed studies were also made by noting sequential changes in the above parameters in normal and EAC-bearing mice on days 12, 15, 18 and 21, respectively. The results indicate that the compound did not adversely affect hematopoiesis. From the sequential studies, it was observed that after a mild initial decrease in hematological counts, particularly in EAC-bearing treated mice, normalcy was reached within 11-14 days after termination of drug therapy. Drug induced hepatotoxicity and nephrotoxicity were also sequentially evaluated in normal and EAC-bearing mice on days 9, 12 and 15 but no such toxicities were detected. Also, body weight, skin and hair texture, and behavioural pattern (food and water intake and activity) did not reflect any toxic reaction in the host mice at this optimum dose.

Alanine Transaminase↗
PubMed1994

Probing the function(s) of active-site arginine residue in Leishmania donovani adenosine kinase.

The presence of arginine at the active site of Leishmania donovani adenosine kinase was studied by chemical modification, followed by the characterization of the modified enzyme. The arginine-specific reagents phenylglyoxal (PGO), butane-2,3-dione and cyclohexane-1,2-dione all irreversibly inactivated the enzyme. In contrast, adenosine kinase from hamster liver was insensitive to these reagents. The inactivation of the enzyme by PGO followed pseudo-first-order kinetics, with a second-order rate constant of 39.2 min-1.M-1. Correlation between the stoichiometry of PGO modification and extent of inactivation indicated that modification of a single residue per molecule suffices for the loss of activity. Reactivity of the essential arginine residue towards PGO was affected by the presence of adenosine (Ado) and other competing alternative substrates, consistent with an arginine residue located proximal to the Ado-binding site. The enzyme showed an intrinsic fluorescence with an emission maximum at 340 nm when excited at 295 nm. The protein fluorescence was partially quenched on addition of Ado. PGO modification also led to significant quenching of the fluorescence. However, the fluorescence of the Ado-protected enzyme, which displayed 82% of the original activity after PGO treatment, was retained. The kinetic analyses of the partially modified enzyme showed an increase in the Km for Ado from 14 to 55 microM. Furthermore, the inability of the modified enzyme to bind to 5'-AMP-Sepharose 4B affinity column provided additional evidence that modification is attended by decrease in affinity of the enzyme for Ado. The results are consistent with the interpretation that modification of the active-site arginine residue affects activity by interfering with the binding of the substrate to the active site.

Adenosine↗
PubMed1994

X-ray structure of PQQ-dependent methanol dehydrogenase.

The three-dimensional structure of the PQQ-dependent quinoprotein, methanol dehydrogenase from Methylobacterium extorquens AM1, has been determined at 3A resolution. The a2b2 tetrameric enzyme has a large a-chain of almost spherical form with a chain fold in which eight 4-stranded antiparallel b-sheets segments are arranged radially around a pseudo 8-fold molecular symmetry axis. The much smaller b-chain is surprisingly not globular, but has an extended conformation running across the surface of the alpha-subunit. The PQQ prosthetic group is buried within the large a-subunit located on the pseudo 8-fold molecular symmetry axis. It is surrounded by protein side-chains but not covalently bound. Associated with the PQQ are two unexpected features: a vicinal disulphide bridge formed between Cys103 and Cys104, and a calcium ion bound between the protein and the PQQ. Vicinal disulphide bridges forming highly distorted structures containing a cis peptide bond, have been proposed to be present in one or two enzymes but have not previously been available for detailed structural investigation. Activity studies have indicated that the ability of the enzyme to transfer electrons derived from the reduction of the alcohols to the specific cytochrome CL receptor is lost when the vicinal disulphide bridge is reduced. The roles of the calcium ions and the b-chain in the enzyme's activity remain to be determined.

Alcohol Oxidoreductases↗
PubMed1994

Rearranged p53 gene with loss of normal allele in a low-grade nonrecurrent glioma.

We are studying the p53 gene profile in primary glial tumors by seeking alterations in the hybridization pattern of the tumor DNA probed with a p53 gene probe. This report documents a rearranged p53 gene with loss of the normal allele in a low-grade mixed glioma which has not recurred during 4-year follow-up. The tumor had a low 5-bromodeoxyuridine (BrdU)-labeling index and low AgNOR count. The p53 protein was not detected on immunochemical staining. To our knowledge, this is the first report of an altered p53 gene in a low-grade nonrecurrent glial tumor and highlights the presence of further checks and balances on the control of cell proliferation and other malignancy-associated phenotypes, even in an already-established tumor.

Alleles↗
PubMed1994

The active site of methanol dehydrogenase contains a disulphide bridge between adjacent cysteine residues.

Adjacent cysteine residues can only form disulphide bridges in a distorted structure containing a cis-peptide link. Such bridges are extremely uncommon, identified so far in the acetyl choline receptor alone where the structure of the bridge is undetermined. Here we present the first molecular description of a disulphide bridge of this type in the quinoprotein methanol dehydrogenase from Methylobacterium extorquens. We show that this structure occurs in close proximity to the pyrrolo-quinoline quinone prosthetic group and a calcium ion in the active site of the enzyme. This unusual disulphide bridge appears to play a role in the electron transfer reaction mediated by methanol dehydrogenase.

Alcohol Oxidoreductases↗