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Biomedical subjects

P Ghosh

Publications and source records attributed to P Ghosh.

At least 235 records · Page 13Linked to original sources
PubMed1992

Cloning, expression, and nucleotide sequence of glgC gene from an allosteric mutant of Escherichia coli B.

The Escherichia coli B mutant strain CL1136 accumulates glycogen at a 3.4- to 4-fold greater rate than the parent E. coli B strain and contains an ADPglucose synthetase with altered kinetic and allosteric properties. The enzyme from CL1136 is less dependent on the allosteric activator, fructose 1,6-bisphosphate, for activity and less sensitive to inhibition by AMP than the parent strain enzyme. The structural gene, glgC, for the allosteric mutant enzyme was selected by colony hybridization and cloned into the bacterial plasmid pBR322 by insertion of the chromosomal DNA at the PstI site. One recombinant plasmid, designated pKG3, was isolated from the genomic library of CL1136 containing glgC. The cloned ADPglucose synthetase from the mutant CL1136 was expressed and characterized with respect to kinetic and allosteric properties and found to be identical to the enzyme purified from the CL1136 strain. The mutant glgC was then subcloned into pUC118/119 for dideoxy sequencing of both strands. The mutant glgC sequence was found to differ from the wild-type at the deduced amino acid residue 67 where a single point mutation resulted in a change from arginine to cysteine.

Allosteric Regulation↗
PubMed1992

A comparison of the high buoyant density proteoglycans isolated from the intervertebral discs of chondrodystrophoid and non-chondrodystrophoid dogs.

Groups of purebred beagles and greyhounds of similar ages (1.5-2.5 years) were used for the study. Intervertebral disc proteoglycans (PGs) were radiolabelled in vivo (with [35SO4(2-)], 24 hours and 60 days prior to euthanasia, when lumbar discs were dissected into nucleus pulposus (NP) and annulus fibrosus (AF). Aliquots of each disc region were separately analysed for total PG content as hexuronate. The remaining tissue was subjected to extraction with 4.0 M GuHCl. High buoyant density PGs were isolated from these extracts by CsCl density gradient ultracentrifugation. The hydrodynamic size and aggregatability of the 24-hour, 60-day-old, and resident PG populations were determined by Sepharose CL2B chromatography in the presence or absence of excess hyaluronic acid. While the hydrodynamic sizes of the newly synthesized (24-hour) disc PG preparations appeared to be similar, the 60-day-old greyhound disc PGs were found to be larger than the corresponding beagle disc PG populations. However, the keratan sulphate-core protein complexes prepared by chondroitinase ABC digestion of the newly synthesized (24 hour) disc PGs showed that the greyhound disc preparations were also larger than those from beagle discs. Approximately 80% of the newly synthesized PGs from beagle and greyhound discs were capable of aggregating with hyaluronic acid, however, this was reduced to 55% for the 60 day-old PGs in NP and AF and even less for the resident PG populations (as determined by hexuronate analysis). Significantly, PG aggregation was lower in the greyhound NP and AF preparations than in the corresponding PGs isolated from the beagle disc.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

The serine proteinase inhibitory proteins of the human intervertebral disc: their isolation, characterization and variation with ageing and degeneration.

Serine proteinase inhibitory proteins (SPIs) were extracted from human disc tissues using 2 M GuHCl and subjected to CsCl density gradient ultracentrifugation. The SPIs recovered in the low buoyant density fractions (rho < or = 1.35 g/ml) were purified by a combination of gel-permeation, ion-exchange, trypsin affinity, and reverse-phase high performance chromatographies. Characterisation of the major disc SPI by polyacrylamide gel electrophoresis, isoelectric focussing, enzyme inhibition and pH stability studies indicated that this small molecular weight (12-14 kDa), highly basic (pI > 9.5), acid-stable but alkaline-labile protein possessed potent inhibitory activity against bovine pancreatic trypsin and chymotrypsin, and human leukocyte elastase and cathepsin G. Two-major and two-minor low molecular weight cationic SPI species were identified by reverse-phase HPLC. The predominant species was identical to a human articular cartilage SPI sharing amino terminal sequence homology with the mucus proteinase inhibitors (MPIs). It also cross-reacted with an antiserum to the MPIs and behaved identically to secretory leucocyte proteinase inhibitor (SLPI) when examined by reverse phase HPLC, and SDS PAGE. A higher molecular weight (54 kDa), anionic (pI approximately 4.6) SPI was also purified and identified as alpha 1-proteinase inhibitor (alpha 1-PI). Quantification of alpha 1-PI and the small molecular weight cationic disc inhibitors indicated that the latter were depleted in morphologically degenerate disc tissues while levels of alpha 1-PI were somewhat higher although a large proportion of the alpha 1-PI was inactive. A depletion of total SPI levels was evident overall in degenerate discs suggesting a functional role for these inhibitory proteins in the maintenance of IVD matrix homeostasis.

Adolescent↗
PubMed1992

Kappa B site-dependent activation of the interleukin-2 receptor alpha-chain gene promoter by human c-Rel.

The cis-acting control elements of the interleukin-2 receptor alpha-chain (IL-2R alpha) gene contain a potent kappa B-like enhancer whose activity can be induced by various mitogenic stimuli. Recent cloning of the p50 and p65 subunits of the kappa B-binding protein NF-kappa B complex revealed a striking sequence homology of these proteins with the c-rel proto-oncogene product (c-Rel). On the basis of this homology, we examined the potential role of c-Rel in controlling IL-2R alpha transcription. We now demonstrate that the recombinant human c-Rel protein binds to the kappa B element in the IL-2R alpha promoter and results in alteration of the DNA structure in the adjacent downstream regulatory elements containing the CArG box and the GC box. We found that human c-Rel can activate transcription from the IL-2R alpha promoter, but not the kappa B-containing human immunodeficiency virus type 1 promoter, upon cotransfection into Jurkat T cells. Furthermore, truncation of the carboxyl terminus of c-Rel results in a c-Rel mutant (RelNA) that (i) localizes exclusively in the nucleus and (ii) acts in synergy with wild-type c-Rel in activating transcription from the kappa B site of the IL-2R alpha promoter. Finally, induction of surface IL-2R alpha expression coincides with the induced levels of endogenous c-Rel and induced c-Rel binding to the IL-2R alpha kappa B site. Our study identified c-Rel as one component of the Rel/NF-kappa B-family proteins involved in the kappa B-dependent activation of IL-2R alpha gene expression. Furthermore, our results suggest that a Re1NA-like cellular factor (e.g., NF-kappa B p50 or p49 subunit) acts in synergy with c-Re1 during T-cell activation.

Base Sequence↗
PubMed1992

Effects of tiaprofenic acid (Surgam) on cartilage proteoglycans in the rabbit joint immobilisation model.

A well established model of arthritis induced in rabbit knee joints by immobilisation in full extension for 30 days was used to evaluate the in vivo effects of 2.5, 5.0, and 10.0 mg/kg body weight of tiaprofenic acid on articular cartilage proteoglycans. The drug was given subcutaneously every 24 hours during the entire immobilisation period. Immobilised animals not treated with drugs and normal animals were used as controls. In the non-drug treated immobilised animals articular cartilage showed evidence of surface damage accompanied by synovial hypertrophy and effusion. Proteoglycan concentrations were reduced in cartilages of these joints and the incorporation of 35SO2-4 into macromolecular proteoglycans was higher than in cartilages of non-immobilised controls. Gel filtration chromatographic studies of both resident and 35S labelled proteoglycans isolated from immobilised joint cartilage showed reduced aggregation and the presence of degraded proteoglycan subunit species. Whereas the administration of 10.0 mg/kg tiaprofenic acid every 24 hours to immobilised animals exacerbated the degradation and loss of proteoglycans from joint cartilages, 5.0 mg/kg tiaprofenic acid provided some protection of these macromolecules, as shown by the concentrations and extractability of proteoglycans from cartilages, which were comparable with those from non-immobilised controls. A high incorporation of 35S into proteoglycans was demonstrated, together with reduced catabolism of proteoglycans, indicating preservation of chondrocyte anabolic activity. At a tiaprofenic acid dose of 2.5 mg/kg, however, no beneficial effects on cartilage proteoglycans could be shown.

Animals↗
PubMed1992

Suppression of Helicobacter pylori reduces gastrin releasing peptide stimulated gastrin release in duodenal ulcer patients.

Helicobacter pylori increases gastrin release in duodenal ulcer patients. This may be through disruption or changes in the mucus layer affecting the access of luminal stimulants to gastrin releasing cells. The effect of suppressing H pylori on gastrin release stimulated by a non-luminal stimulus, gastrin releasing peptide (GRP), was examined. Eleven patients with active duodenal ulcer disease and colonised with H pylori received an intravenous infusion of GRP (2.9 pmol/kg/minute for 30 minutes) and the plasma gastrin response was measured. Basal and peak pentagastrin stimulated acid output were also determined. Patients were treated with tripotassium dicitratobismuthate (De-Nol) and metronidazole to suppress H pylori and the tests were repeated. Suppression of H pylori decreased plasma gastrin concentrations during GRP infusion, but acid output was not affected. Chromatographic analysis of the forms of gastrin in plasma showed a significant fall in gastrin 17, the predominant form found in the gastric antrum. Gastrin 34 did not fall significantly. This study shows that suppression of H pylori decreases the hypergastrinaemia caused by the nonluminal stimulant, GRP, mainly via decreasing gastrin 17.

Adult↗
PubMed1992

A comparative study of the low-molecular mass serine proteinase inhibitors of human connective tissues.

Low molecular mass serine proteinase inhibitors isolated from human articular cartilage, intervertebral disc, meniscus, and costal cartilage were compared chromatographically. Similar charge and size properties were exhibited when these inhibitors were examined by gel permeation and cation exchange chromatography. The individual proteinase inhibitory species separated by these procedures all cross-reacted with a polyclonal antibody raised against the mucous proteinase inhibitors (MPIs) obtained from human bronchial secretions, however the distribution of these MPI-like species varied with the origin of the connective tissue. The major inhibitory species present in human articular cartilage and intervertebral disc were purified to homogeneity using gel filtration, cation exchange, trypsin affinity and high performance reverse phase chromatography. The amino-terminal sequences of the purified cartilage intervertebral disc inhibitors was found to be identical to the published sequence of MPIs isolated from parotid and seminal secretions. These findings indicate that the endogenous small molecular mass cationic serine proteinase inhibitory proteins present in human cartilaginous connective tissues are members of the MPI family of proteinase inhibitors.

Amino Acid Sequence↗
PubMed1992

Time dependent tissue distribution of 203Hg in the white rat and Anabas testudineus, a freshwater teleost.

The distribution of mercury, an environmentally important toxicant, has been evaluated in a time dependent manner in different tissues of white rat and a freshwater teleost, Anabas testudineus. Sampling was performed at 15 min, 2, 6 and 48 h post injection (im) of 203Hg mercuric nitrate. Radioactivity of the 5% tissue homogenate, serum and bile was measured in a Gamma Counter. The rate of 203Hg accumulation is higher in fish immediately after administration which, however, is more or less of equal rate in the later period of observation in both the experimental animals. Partitioning of 203Hg occurs in a species specific manner with higher levels recorded in the brain and gonad of white rat. Spleen, liver and kidney, however, are the major tissues to accumulate mercury in both the species. The present study highlights that kidney is the target site of mercury retention with a higher kidney/liver ratio of mercury.

Animals↗
PubMed1992

In vivo and in vitro acetylcholinesterase inhibition by metacid-50 and carbaryl in Channa punctatus under natural field condition.

Channa punctatus (Bloch), cultivated under paddy-cum-fish culture programs, was exposed to non-lethal levels of 0.239 ppb of Metacid-50 and 3.73 ppm of Carbaryl for 7 days in the field. These insecticides effected significant inhibition of brain acetylcholinesterase (AChE) activity accompanied by a concurrent increase in acetylcholine (ACh). In vitro inhibition of brain acetylcholinesterase was also compared between unexposed and pesticide exposed fish. Data revealed that the time taken for 50% inhibition (t0.5) of the enzyme was less in the pesticide exposed fish as compared to that of the unexposed fish. This indicates a preformed effect of the test pesticides on fish brain AChE during the in vivo exposure while the in vitro effect is an additive inhibition of the enzyme caused by the circulating inhibitors reducing the t0.5. Acetylcholinesterase inhibition may thus be utilized to monitor pesticide pollution in agricultural fields.

Acetylcholine↗
PubMed1992

Acetylcholinesterase characteristics of termite queen exposed to anticholinesterase compounds.

A regional profile of AChE activity was noted in the Indian termite queen Odontotermes redemanni with the head recording the higher and body the lower range of activity. The enzyme characteristics such as substrate and temperature optima were more or less similar while pH requirement for optimum AChE activity varied from 7.0 to 7.6 In vitro inhibition of head and body AChE was studied using pure and commercial anticholinesterase compounds. Interestingly, the commercial formulations like Metacid-50 and Carbaryl are potent enough at 1 x 10(-8) M to produce 50% in vitro inhibition of AChE of head and body regions within 15 min of preincubation. A 20 min of preincubation (t0.5) was necessary to record 50% in vitro inhibition of AChE with known and pure anticholinesterase compounds such as DFP (3.5 x 10(-10) M) and physostigmine (3.6 x 10(-10) M). It is surmised that (a) the response of the head and body AChE to the commercial formulations of the insecticides may be used as a reliable and sensitive bioindicator of pesticidal contamination of the terrestrial environment and (b) termite control may be successfully done with the application of organophosphate or carbamate compounds.

Acetylcholinesterase↗
PubMed1991

Hypergastrinaemia: a new mechanism.

The mechanism by which lack of gastric acid causes hypergastrinaemia was investigated in seven patients with pernicious anaemia. Perfusion of gastric juice from the fasting patients (adjusted to pH 7.0) into the stomachs of conscious rats caused a significant rise in plasma gastrin (median 14 [range 2-20] pmol/l to 27 [12-65] pmol/l; p less than 0.01), whereas perfusion of bicarbonate buffer (18 [13-23] pmol/l to 20 [9-25] pmol/l) or gastric juice from a duodenal ulcer patient (also pH 7.0; 13 [3-30] pmol/l to 17 [9-27] pmol/l) had no significant effect. During gastric lavage at pH 7.0 the median plasma gastrin concentration of the pernicious anaemia patients fell from 320 (63-1760) pmol/l to 125 (46-760) pmol/l (p less than 0.03).

Aged↗
PubMed1991

Mechanism of action of 2,3,7,8-tetrachlorodibenzo-p-dioxin on intermediary metabolism in the rat.

A single 20-microgram/kg dose of TCDD caused, within 3 days, a significant mobilization of depot fat into the plasma compartment resulting in 1.44- to 2.8-fold increase in plasma free-fatty acid concentrations. With respect to the fate of mobilized fatty acids, the same treatment of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) caused a 32% inhibition (P less than .008) of the rate of hepatic oleate oxidation without significantly affecting the rate of fatty acid esterification. In contrast, hepatic ketogenic rate from oleate and octanoate was stimulated markedly by 85% (P less than .001) and 69% (P less than .001), respectively. These results support the concept that although the beta-oxidation pathway of the fatty acids must be operating normally, their complete oxidation to CO2 via the Tricarboxylic acid (TCA) cycle is impaired. At the same time, TCDD seems to preferentially divert the acetyl CoA generated from the beta-oxidation of fatty acids to the ketogenic pathway. Surprisingly, TCDD treatment inhibited the hepatic ketogenic rate from glycerol by 21% (P less than .001) and its oxidation to CO2 by 31% (P less than .025) without affecting its esterification to triglycerides. These results imply that the other possible major site which is sensitive to TCDD inhibition may be the generation of acetyl CoA from glycerol via the pyruvate dehydrogenase complex.

Animals↗
PubMed1991

Comparison of the effects of non-steroidal anti-inflammatory drugs (NSAIDs) on proteoglycan synthesis by articular cartilage explant and chondrocyte monolayer cultures.

Experiments were conducted to study proteoglycan biosynthesis by rabbit articular chondrocytes cultured in the presence of NSAIDs and 35SO4(2-) for up to 8 days. Both articular cartilage explants and confluent chondrocyte monolayer culture models were used. Medium was changed every 2 days and the [35SO4]proteoglycans which had accumulated in the medium and the extracellular matrix during the culture intervals were assayed separately. In long-term experiments, drugs were removed on day 8, and proteoglycan production during a 10-12 day culture interval also was assayed. The drugs studied were diclofenac, indomethacin, ketoprofen, piroxicam and tiaprofenic acid, at concentrations of 0, 0.1, 1, 10, 50 and 100 micrograms/mL. Whereas proteoglycan production by cell cultures was maximal early in the culture period, explants produced more proteoglycans as time progressed. The highest concentrations of all of the drugs, especially diclofenac and indomethacin, inhibited proteoglycan secretion by both cell and explant cultures. However, after removal of the drugs from the cultures, suppressed proteoglycan production reversed to levels equivalent to, or higher than controls in the cell cultures, but largely persisted in explant cultures. About 70-80% of proteoglycans produced by explants were retained in the matrix, whereas about 80-90% of proteoglycans produced by cell cultures were secreted into the medium. Where drugs inhibited proteoglycan production, the levels were reduced by approximately the same proportions in both extracellular matrix and culture medium fractions. Of the NSAIDs examined only ketoprofen demonstrated a stimulatory effect on PG synthesis in explant cultures at a physiological concentration (0.1 microgram/mL).

Animals↗
PubMed1991

Ion channels formed by a highly charged peptide.

A peptide (MA-beta) corresponding to a segment of the nicotinic acetylcholine receptor (AChR) that has amphipathic alpha-helical periodicity forms ion channels in artificial phospholipid bilayers. The MA-beta ion channels are very stable, comprise two discrete conductance states, and undergo rapid, flickering-type closings. The discrete-conductance ion channels formed by MA-beta contrast with the continuous-conductance ion channels formed by a peptide (M2-delta) identical in sequence with M2 [Oiki, S., Danho, W., Madison, V., & Montal, M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8703-8707], a putative transmembrane segment of the AChR. Neither MA-beta nor M2-delta sufficiently mimics the electrophysiological properties of the native AChR. We suggest that peptide ion channels can be classified into at least three general groups: discrete-conductance channels, such as MA-beta; continuous-conductance channels, such as M2-delta; and membrane disruptors, such as those formed by short, amphipathic alpha-helical peptides.

Amino Acid Sequence↗