Biomedical subjects

V B Singh

Publications and source records attributed to V B Singh.

At least 37 records · Page 2Linked to original sources

PubMed1991

Coagglutination test for rapid detection of Salmonella enterotoxin.

The Staphylococcal Coagglutination (CoA) test using Staphylococcus aureus (Cowan type-1) cells coated with antiserum against purified Salmonella enterotoxin was standardized and carried out to screen Salmonella serotypes for their enterotoxigenicity. Of the 101 cell-free culture supernatants from an equal number of Salmonella strains belonging to 15 different serotypes tested, 76 were found to be enterotoxigenic. All of the 24 strains which gave positive reactions in the rabbit ligated ileal loop test were also positive with this test. As little as 7.5 ng of purified Salmonella enterotoxin could be detected by the CoA test. The presence of enterotoxin in polymyxin B extract of cells and cell-free preparation of a S. typhimurium strain was detected as early as after 1.5 and 3.0 h of incubation, respectively. Besides the test standardized with the antiserum to purified V. cholerae toxin (CT) could detect purified toxin in a quantity as little as 5.0 ng and also enterotoxigenicity in an E. coli strain (LT+). The usefulness of the test as a routine, rapid and economic one for the detection of enterotoxin of Salmonella, Vibrio cholerae and E. coli is discussed.

Agglutination Tests↗

PubMed1991

Evaluation of ELISA and GM1-ELISA for detection of Salmonella enterotoxin.

The ELISA and GM1-ELISA, by using antiserum to purified Salmonella enterotoxin (SE), were standardized and carried out to screen salmonellae isolated from foods of animal origin for enterotoxigenicity. Of the 101 strains of Salmonella belonging to 15 different serogroups tested, 76 (75.24%) strains from 13 serogroups were found enterotoxigenic. ELISA correlated well with rabbit ligated ileal loop (RLIL) test for the detection of enterotoxin producing salmonellae with 24 test strains. ELISA also yielded positive reaction with 7 of 13 RLIL negative strains. GM1-ELISA could not be carried out as none of the 101 cell free culture supernatants (CFCS) were able to bind with GM1-ganglioside. ELISA and GM1-ELISA were also standardized with antiserum to cholera toxin for the detection of salmonellae producing cholera related enterotoxin. None of the 101 strains was found to produce cholera related enterotoxin. ELISA could detect as low as 15 ng/100 microliters of purified SE and 10 ng/100 microliters of cholera toxin when tested with their homologous antisera.

Cholera Toxin↗

PubMed1991

Comparative evaluation of some bioassay models & serological tests for the detection of Salmonella enterotoxin.

Two bioassay models viz., rabbit ligated ileal loop (RLIL) and the Chinese hamster ovary (CHO) cell culture and three serological methods viz., enzyme-linked immunosorbent assay (ELISA), Biken and staphylococcal coagglutination (CoA) tests were standardized and used for the detection of Salmonella enterotoxin. A total of 37 strains belonging to 14 different serotypes of Salmonella were tested simultaneously by all the five assay models. Serological tests were standardized with antiserum against the purified enterotoxin of S. typhimurium (P/536). The CHO test was found to be the most sensitive (86.37%) followed by ELISA and CoA test (83.78%) each and Biken test (78.37%). The RLIL test was found to be least sensitive (64.86%) for detection of Salmonella enterotoxin.

Agglutination Tests↗

PubMed1990

The increases in rat cortical and midbrain tryptophan hydroxylase activity in response to acute or repeated sound stress are blocked by bilateral lesions to the central nucleus of the amygdala.

Sound stress (SS) (120-dB pulses of 100 ms duration, every min for 1 h) produces an elevation of in vitro cortical or midbrain tryptophan hydroxylase activity from male Sprague-Dawley rats that is abolished, in vitro, by incubation of the enzyme preparation with alkaline phosphatase. SS, when repeated on 3 different occasions, the first 2 sessions 24 h apart and the 2nd and 3rd session separated by 48 h, produces a stable increase in the in vitro enzyme activity that is unaffected by alkaline phosphatase. Bilateral lesions to the central nucleus of the amygdala block both increases in enzyme activity obtained in response to acute and repeated SS, but leave enzyme activity from sham-stressed rats unaffected.

Amygdala↗

PubMed1990

Increases in the activity of tryptophan hydroxylase from rat cortex and midbrain in response to acute or repeated sound stress are blocked by adrenalectomy and restored by dexamethasone treatment.

Exposure of male Sprague-Dawley rats to acute sound stress (2 s, 110 dB sound pulses presented randomly every minute for 1 h) increases the in vitro activity of cortical and midbrain tryptophan hydroxylase by an alkaline phosphatase-reversible mechanism. Repeated exposure to sound stress on three separate days produces a stable increase in enzyme activity that persists 24 h after the termination of the stress and is insensitive to alkaline phosphatase. Adrenalectomy abolishes both increases in enzyme activity to acute or repeated sound stress but does not change baseline levels of enzyme activity. The synthetic glucocorticoid, dexamethasone, (500 micrograms/day i.p.) given for 3 days or 5 out of 6 days, starting day 3 after adrenalectomy, restores the increases in enzyme activity in adrenalectomized rats exposed, respectively, to acute or repeated sound stress. The mineralocorticoid, aldosterone (5 micrograms/day s.c.), does not substitute for dexamethasone in acutely sound-stressed, adrenalectomized rats. Dexamethasone does not alter control levels of enzyme activity in either adrenalectomized rats or rats with intact adrenals (sham-adrenalectomized), but is required to allow the increase in enzyme activity in response to acute or repeated sound stress to be expressed. The effect of the glucocorticoid, thus, appears to be a permissive one.

Acoustic Stimulation↗

PubMed1986

Transformation and phosphorylation of purified molybdate-stabilized chicken oviduct progesterone receptor.

The non-transformed, molybdate-stabilized chick oviduct cytosol progesterone receptor was purified approx. 7000-fold using biospecific affinity resin (NADAC-Sepharose), DEAE-Sephacel chromatography and gel filtration on Bio-Gel A-0.5m agarose. The purified preparation contained progesterone receptor which sedimented as a 7.9S molecule, had a Stokes' radius of 7.5 nm, was composed of three major peptides corresponding to Mr 108,000, 90,000 and 79,000. Upon removal of molybdate, the purified [3H]progesterone-receptor complex could be transformed from the 8S form to a 4S form by exposure to 23 degrees C or by an incubation with 10 mM ATP at 0 degrees C. The purified thermally transformed receptor could be adsorbed to columns of ATP-Sepharose. No cytosol factor(s) appeared to be required for the 8S to 4S transformation of purified receptor or for its subsequent binding to ATP-Sepharose. Incubation of purified non-transformed receptor preparation with [gamma-32P]ATP and cAMP-dependent protein kinase led to incorporation of radioactivity in all the three major peptides at serine residues. The results of this study show for the first time that purified 8S progesterone receptor can be phosphorylated in vitro by a cAMP-dependent protein kinase, and that it can be transformed to a 4S form by 0 degrees C incubation with 10 mM ATP.

Adenosine Triphosphate↗

PubMed1985

Phosphorylation of rat liver glucocorticoid receptor.

Rat liver glucocorticoid-receptor complex (GRc) was purified 2000-fold by a combination of methods including (NH4)2SO4-fractionation and phosphocellulose and DNA-cellulose chromatography. The purified glucocorticoid receptor preparation contained a major peptide of Mr = 90,000 and the GRc sedimented as 4 S in 5-20% sucrose gradients. An additional peptide of Mr = 45,000 (45K) was also observed. Some preparations yielded only the Mr = 90,000 (90K) peptide suggesting that the 45K peptide may be a proteolyzed portion of the 90K protein. The purified GRc was incubated with [gamma-32P]ATP in the presence of cAMP-dependent kinase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the above preparation revealed the presence of two 32P-containing bands with apparent Mr = 90,000 and 45,000. The 32P incorporation was dependent on the availability of divalent cation (Mg2+). GRc in cytosol labeled with [3H]dexamethasone mesylate and purified as above co-migrated with 32P-containing bands. GRc was also purified from cytosol obtained from livers of rats injected with [32P]orthophosphate. Both 32P and 3H bands were associated with 90K and 45K peptides. Our results indicate that rat liver glucocorticoid receptor is a phosphoprotein and that both the phosphorylated peptides 90K and 45K also contain the steroid and the DNA binding regions of the glucocorticoid receptor.

Adrenalectomy↗

PubMed1985

Interaction of chick oviduct progesterone receptor with immobilized aurintricarboxylic acid.

Aurintricarboxylic acid (ATA) was immobilized on Sepharose 4B via a carbodiimide coupling mechanism. A majority of the chick oviduct progesterone receptor was retained on the affinity resin and could be recovered upon washing the column with buffer containing free ligand or 3 M guanidine-HCl. The [3H]progesterone-receptor complex retained its integrity following the chromatography on ATA-Sepharose as judged by sedimentation analysis. The procedure allowed significant purification of progesterone receptor: SDS-polyacrylamide gel electrophoresis of the purified preparation revealed elimination of many peptide bands present in the cytosol prior to ATA-Sepharose chromatography. The technique thus has a clear potential in characterization and purification of progesterone receptor.

Animals↗

PubMed1985

Chromatographic separation of nontransformed and transformed glucocorticoid-receptor complexes from rat liver cytosol. Use of tungstate in the purification, inactivation, and resolution of receptor forms.

Aliquots of rat liver cytosol glucocorticoid-receptor complexes (GRc) were transformed by an incubation with 8-10 mM ATP at 0 degrees C and were compared with those transformed by an exposure to 23 degrees C. The extent of receptor transformation was measured by chromatography of the samples over columns of DEAE-Sephacel. The ATP-transformed complexes, like those which were heat-transformed, exhibited lower affinity for the positively charged ion-exchange resin and were eluted with 0.12 M KCl (peak-I): the nontransformed complexes appeared to possess higher affinity and required 0.21 M KCl (peak II) for their elution. As expected, the receptor in the peak-I exhibited the DNA-cellulose binding capacity and sedimented as 4S in sucrose gradients. Peak II contained an 8-9S glucocorticoid receptor (GR) form that showed reduced affinity for DNA-cellulose. Presence of sodium tungstate (5 mM) prevented both heat and ATP transformation of the GRc resulting in the elution of the complexes in the region of nontransformed receptors. When parallel experiments were performed, binding of the cytosol GRc to rat liver nuclei or DNA-cellulose was seen to increase 10-15 fold upon transformation by heat or ATP: tungstate treatment blocked this process completely. The transformed and nontransformed GRc were also differentially fractionated by (NH4)2SO4: tungstate-treated (nontransformed) receptor required higher salt concentration and was precipitated at 55% saturation. In addition, the GRc could be extracted from DNA-cellulose by an incubation of the affinity resin with sodium tungstate resulting in approximately 500-fold purification of the receptor with a 30% yield. These studies show that the nontransformed, and the heat-, salt-, and ATP-transformed GRc from the rat liver cytosol can be separated chromatographically, and that the use of tungstate facilitates the resolution of these different receptor forms. In addition, extraction of the receptor from DNA-cellulose by tungstate provides another new and efficient method of partial receptor purification.

Adenosine Triphosphate↗

PubMed1984

Protein kinase activity of purified rat liver glucocorticoid receptor.

Molybdate-stabilized nonactivated rat liver glucocorticoid receptor (GR) was purified to near homogeneity using a biospecific affinity adsorbent, Bio Gel A 0.5 m and DEAE-Sephacel. The purified GR sedimented in the 9-10S region in 5-20% sucrose gradients containing 0.10M KCl and 20mM Na2MoO4. SDS-polyacrylamide gel electrophoresis revealed a major single band with an apparent molecular weight of 90,000 +/- 2,000. Affinity labeling of GR with [3H]-dexamethasone mesylate showed association of the radioactivity with a peptide of 90,000 molecular weight. Purified receptor preparation was dialyzed to remove molybdate and was incubated with different protein substrates in the presence of 50 microM [gamma-32P]-ATP and divalent cations. Radioactive phosphate from [gamma-32P]-ATP was seen to be incorporated into calf thymus histones, turkey gizzard myosin light chain kinase and rabbit skeletal muscle kinase in the presence of Mg2+ and Ca2+ ions. Addition of steroid ligand exogenously to the reaction mixture appeared to increase the extent of protein phosphorylation. No autophosphorylation of GR was evident under the above conditions. The data suggest that purified rat liver GR displays protein kinase activity.

Adrenalectomy↗

PubMed1984