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J Das

Publications and source records attributed to J Das.

At least 55 records · Page 3Linked to original sources
PubMed1996

Crystallographic determination of the structures of human alpha-thrombin complexed with BMS-186282 and BMS-189090.

The crystallographic structures of the ternary complexes of human alpha-thrombin with hirugen (a sulfated hirudin fragment) and the small-molecule active site thrombin inhibitors BMS-186282 and BMS-189090 have been determined at 2.6 and 2.8 A. In both cases, the inhibitors, which adopt very similar bound conformations, bind in an antiparallel beta-strand arrangement relative to the thrombin main chain in a manner like that reported for PPACK, D-Phe-Pro-Arg-CH2Cl. They do, however, exhibit differences in the binding of the alkyl guanidine moiety in the specificity pocket. Numerous hydrophilic and hydrophobic interactions serve to stabilize the inhibitors in the binding pocket. Although PPACK forms covalent bonds to both serine and the histidine of the catalytic triad of thrombin, neither BMS-186282 nor BMS-189090 bind covalently and only BMS-186282 forms a hydrogen bond to the serine of the catalytic triad. Both inhibitors bind with high affinity (Ki = 79 nM and 3.6 nM, respectively) and are highly selective for thrombin over trypsin and other serine proteases.

Amino Acid Chloromethyl Ketones↗
PubMed1996

Porins of Vibrio cholerae: purification and characterization of OmpU.

Three outer membrane proteins with molecular masses of 40, 38, and 27 kDa of the hypertoxinogenic strain 569B of Vibrio cholerae have been purified to homogeneity. The synthesis of all the three proteins is regulated by the osmolarity of the growth medium. The pore-forming ability of the 40-kDa protein, OmpT, and the 38-kDa protein, OmpU, has been demonstrated by using liposomes, in which these proteins were embedded. The 27-kDa protein, OmpX, though osmoregulated, is not a porin. OmpU constitutes 30% of the total outer membrane protein when grown in the presence of 1.0% NaCl in the growth medium and 60% in the absence of NaCl. OmpU is an acidic protein and is a homotrimer of 38-kDa monomeric units. Its secondary structure contains predominantly a beta-sheet, and three to four Ca2+ ions are associated with each monomeric unit. Removal of Ca2+ irreversibly disrupts the structure and pore-forming ability of the protein. The pore size of OmpU is 1.6 nm, and the specific activity of the OmpU channel is two- to threefold higher than that of Escherichia coli porin OmpF, synthesis of which resembles that of OmpU with respect to the osmolarity of the growth medium. The pore size of OmpT, which is analogous to OmpC of E. coli, is smaller than that of OmpU. Southern blot hybridization of V. cholerae genomic DNA digested with several restriction endonucleases with nick-translated E. coli ompF as the probe revealed no nucleotide sequence homology between the ompU and ompF genes. OmpU is also not antigenically related to OmpF. Anti-OmpF antiserum, however, cross-reacted with the 45-kDa V. cholerae outer membrane protein, OmpS, the synthesis of which is regulated by the presence of maltose in the growth medium. OmpU hemagglutinated with rabbit and human blood. This toxR-regulated protein is one of the possible virulence determinants in V. cholerae (V. L. Miller and J. J. Mekalanos, J. Bacteriol. 170:2575-2583, 1988).

Adhesins, Bacterial↗
PubMed1996

Physical map of the genome of Vibrio cholerae 569B and localization of genetic markers.

A combined physical and genetic map of the genome of the classical O1 hypertoxinogenic strain 569B of Vibrio cholerae has been constructed. The enzymes NotI, SfiI and CeuI generated DNA fragments of suitable size distribution that could be resolved by pulsed-field gel electrophoresis. The digests produced 37, 22, and 7 fragments, respectively. The CeuI maps of the genomes of strains 569B and O395, constructed by partial restriction digestion, were identical, and the data are consistent with the concept of circular chromosomes. The genome size of each of the strains was estimated to be about 3.2 Mb. The NotI and SfiI digestion profiles of the genomic DNAs of strains 569B and O395 exhibited distinct restriction fragment length polymorphism. The linkages between the 37 NotI fragments of the genome of strain 569B were determined by combining three approaches: isolation of linking clones, analysis of partial digestion fragments, and identification of NotI fragments in isolated CeuI and SfiI fragments. To align linked fragments precisely, NotI-digested genomic DNA was end labeled and separated in the same gel with the NotI-digested DNA to be probed with linking clones. This also allowed the identification of smaller restriction fragments that are not visible in ethidium bromide-stained gels. The presence of repetitive DNA sequences in the V. cholerae 569B genome has been demonstrated. Twenty cloned homologous and heterologous genes and seven rrn operons have been positioned on the physical map. The two copies of the Ctx genetic element in the genome of strain 569B are located about 1,000 kb apart.

Base Sequence↗
PubMed1996

Antipyretic activity of Nelumbo nucifera rhizome extract.

Antipyretic activity of methanolic extract of rhizome of N. nucifera was studied on normal body temperature and yeast induced pyrexia in rats. Yeast suspension (10 ml/kg, s.c.) increased rectal temperature after 19 hr of administration. The extract, in doses of 200, 300 or 400 mg/kg (po) produced significant dose dependent lowering of normal body temperature and yeast provoked elevation of body temperature in rats. The effect produced was comparable with the standard antipyretic drug, paracetamol (150 mg/kg, i.p.).

Analgesics, Non-Narcotic↗
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

Thrombin active site inhibitors.

Development of small molecule thrombin active site inhibitors has been an area of intense research. A brief review on recent progress and challenges is outlined.

Anticoagulants↗
PubMed1994

The DNA adenine methyltransferase-encoding gene (dam) of Vibrio cholerae.

The DNA adenine methyltransferase (MTase)-encoding gene (dam) of Vibrio cholerae, an organism belonging to the family Vibrionaceae, has been cloned and the complete nucleotide (nt) sequence determined. V. cholerae dam encodes a 21.5-kDa protein and is directly involved in methyl-directed DNA mismatch repair. It can substitute for the Escherichia coli enzyme and can suppress the phenotypic traits associated with E. coli dam mutants. Overproduction of V. cholerae Dam MTase does not result in hypermutability in either V. cholerae or E. coli cells. Overproduction of V. cholerae Dam in a pUC plasmid, however, fails to suppress the 2-aminopurine (2-AP)-sensitive phenotype of E. coli dam mutants. Homology between the nt and deduced amino acid (aa) sequences of the E. coli and V. cholerae dam genes is only 30-35%.

Amino Acid Sequence↗
PubMed1994

Genome size and restriction fragment length polymorphism analysis of Vibrio cholerae strains belonging to different serovars and biotypes.

The genome size of Vibrio cholerae has been determined by pulsed field gel electrophoresis following digestion of chromosomal DNA with endonucleases. The genome size of all the classical strains examined was about 3000 kb and that of El Tor biotype was 2500 kb. The NotI and SfiI digestion patterns of the genomes of several V. cholerae strains belonging to different serovars and biotypes showed distinct restriction fragment length polymorphism (RFLP). RFLP analysis together with the genome size can be used to differentiate strains of different serovars and biotypes of V. cholerae.

Bacterial Typing Techniques↗
PubMed1994

Recombinant derivative of a naturally occurring non-toxinogenic Vibrio cholerae 01 expressing the B subunit of cholera toxin: a potential oral vaccine strain.

A clinical isolate of Vibrio cholerae 01 was identified which did not possess the heat-labile (CT), the heat-stable (ST) or the zonula occludens (Zot) toxin genes. Rabbit ileal loop assays showed that no other CT-like toxin was produced by this strain. The partly deleted cholera toxin gene which carries the intact gene for the B subunit was cloned and the recombinant plasmid, pURD110, was introduced into this non-toxinogenic natural human isolate. The transformed cells (strain URD2) secreted the B subunit gene product which competed with the holotoxin secreted by the hypertoxinogenic strain 569B of V. cholerae for the GM1 ganglioside binding sites in vivo. This strain can colonize the rabbit intestine as detected by the removable intestinal tie adult rabbit diarrhoea (RITARD) model. This construct has an advantage over other live oral attenuated V. cholerae strains used as vaccines in that the latter strains were made non-toxinogenic by only deleting part of the gene coding for the A subunit of cholera toxin while the strain described here is naturally non-toxinogenic.

Administration, Oral↗
PubMed1994

Heat shock response and heat shock protein antigens of Vibrio cholerae.

Sixteen heat shock proteins (Hsps) have been identified in the hypertoxinogenic strain 569B of Vibrio cholerae which are synthesized in response to small and large elevations of temperature. The induction of the Hsps is necessary for the cells to survive the deleterious effects of heat. There is no difference in the pattern of induction of the Hsps in V. cholerae strains varying in levels of toxinogenicity. One of the major low-molecular-mass Hsps, a 16-kDa protein, is preferentially degraded following shift down of temperature. This protein is induced at a much lower level at high temperatures in cells maintained in the laboratory for a prolonged period. The only Hsp located in the outer membrane of V. cholerae cells is a 23-kDa protein. Western immunoblot analysis with human immune sera collected from convalescent cholera patients revealed that this protein is markedly immunogenic. The human immune serum also reacted with the 69- and 16-kDa major Hsps and the 88-, 66-, and 46-kDa Hsps but not with the 61-kDa major Hsp identified as the groEL gene product. All major Hsps reacted with rabbit anti-V. cholerae sera. Ethanol stress leads to the induction of four of the major Hsps and three additional proteins.

Adaptation, Biological↗
PubMed1994

Cholera vaccine: developmental strategies and problems.

Over a hundred years have elapsed since Vibrio cholerae, the etiological agent for the disease cholera, was discovered by Robert Koch. Ever since then serious efforts have been made to develop prophylactic measures to combat the disease without much success. Seven pandemics have so far been reported and cholera still remains a public health problem in developing countries. Several strategies have been adopted to develop vaccines against the disease and many of these vaccines have undergone field trials. During the last two decades, an enormous amount of information has accumulated regarding the organism V. cholerae, its virulence factors, including cholera toxin, and the molecular basis of its pathogenicity. In recent years, with the advent of recombinant DNA technology and major breakthroughs in molecular biology and immunology, a new dimension has been given to the design of vaccine strains. The second generation live oral vaccines will perhaps soon replace the long-used first generation parenterally administered killed whole cell vaccines which offered protection for not more than three months. All the recombinant vaccines tested so far produced adverse reactions in volunteers, although they provided varying degrees of protection upto about one year of surveillance. Parallel to the trials of live oral vaccines, combination vaccines comprising killed whole cells and purified B subunit of cholera toxin was also tried. These vaccines had minimal side-effects but the efficacy was not upto expectations. From the failure of each vaccine strain, new information had emerged and improved strategies were adopted.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗
PubMed1993

Depressed patients' parental representations: stability across changes in depressed mood and specificity across diagnoses.

Parental representations of a Dutch sample of psychiatric patients with diagnoses of dysthymia and unipolar depression were compared with those of a matched sample of non-depressed patients and a matched sample of healthy controls. No differences in recalled parental rearing styles were found between depressives with a diagnosis of dysthymia and those with a diagnosis of unipolar depression. Depressive did not differ from the mixed (but non-depressed) sample of psychiatric patients, whereas both the depressed and the mixed group of patients reported more adverse parenting than the healthy controls. Analysis of repeated measurements of parental representations showed that memories of parental behavior were highly stable across clinically significant changes in depressed mood, so that it seems unlikely that patients' relatively negative recollections of their parents' behavior were due to mood state dependent recall. Results are discussed within the framework of depression theories and with regard to the validity and utility of self-report instruments for parental rearing styles.

Adolescent↗
PubMed1993

1-Benzazepin-2-one calcium channel blockers--VI. Receptor-binding model and possible relationship to desmethoxyverapamil.

We have prepared a series of potent antihypertensive 1-benzazepin-2-one calcium channel blockers (CCBs) 1 that are structurally related to diltiazem 2. Structural studies and the preparation of conformationally constrained analogs of 1-benzazepin-2-ones have led us to postulate a receptor-bound conformation for both 1 and 2. We believe that these compounds bind to the calcium channel protein in an MI ("inboard") binding conformation in which the amine of the side chain is placed over the heptagonal benzazepione ring and in close proximity to the phenyl methyl ether pharmacophore. This receptor-bound conformation places the side chain amine and methyl ether pharmacophores in the same spatial relationship as 3-methoxyphenylethalamine. Combined with our SAR, this binding model rationalizes literature findings that desmethoxyverapamil can demonstrate pharmacology typical of both phenylalkylamine (PA) and benzothiazepinone (DTZ) calcium channel blockers. Simple experiments are proposed to test the hypothesis that desmethoxyverapamil can bind at the benzothiazepinone site on the calcium channel.

Animals↗