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H Singh

Publications and source records attributed to H Singh.

At least 235 records · Page 13Linked to original sources
PubMed1993

Targeted disruption of the Oct-2 locus in a B cell provides genetic evidence for two distinct cell type-specific pathways of octamer element-mediated gene activation.

The Oct-2 protein is a tissue-specific POU-homeodomain transcription factor. It has been considered to represent a developmental regulator of immunoglobulin gene expression by virtue of its interaction with a functionally essential octamer element found in immunoglobulin gene promoters. This proposal has been most strongly challenged by several in vitro transcription analyses which have shown that the related ubiquitous factor Oct-1 can activate transcription from immunoglobulin gene promoters as efficiently as Oct-2. We have genetically analyzed Oct-2 function by using gene targeting to disrupt both alleles of the locus in the murine B cell line WEHI-231. This cell line expresses productively rearranged immunoglobulin genes as well as the Oct-2 gene at high levels which are comparable to those observed in activated murine splenic B cells. In spite of a drastic reduction in Oct-2 levels (20-fold), no effect was observed on the expression of endogenous immunoglobulin genes or on the activity of a transfected immunoglobulin promoter or a heterologous promoter with a single octamer element. In contrast, expression of a reporter construct containing multiple octamer motifs upstream of a heterologous promoter was severely reduced in the double-disruptant cells. The differential responses of the single- and multiple-octamer motif reporter constructs in the mutant B cells are unlikely to be a consequence of differing concentration requirements for activation by Oct-2. The two constructs are activated equivalently over the same range of Oct-2 concentration in a non-B cell. These results provide genetic support for the existence of an Oct-2-independent, but octamer element-dependent, B cell-specific pathway for immunoglobulin gene transcription. They also genetically reveal a distinct Oct-2-dependent pathway of octamer-mediated gene activation. This study demonstrates the feasibility of targeting a diploid locus in a somatic mammalian cell line. Extension of this approach to genes encoding other transcription factors will allow a genetic dissection of their functions within the context of cell lines representing various differentiation states.

Alleles↗
PubMed1993

Mechanism of stimulatory action of growth hormone on ovarian steroidogenesis in spotted seatrout, Cynoscion nebulosus.

The mechanism of growth hormone's (bovine growth hormone, bGH) stimulatory action on steroidogenesis was examined using ovarian tissue of spotted seatrout (Cynoscion nebulosus) in an in vitro incubation system and compared to that of a gonadotropin (human chorionic gonadotropin, hCG). Testosterone and estradiol production by seatrout ovaries in vitro were stimulated even with low concentrations of bGH (50 ng/ml), and increasing concentrations demonstrated a concentration-dependent relationship similar to that observed with hCG. The steroidogenic responses to combined treatments with bGH and hCG approximately equaled the sum of the stimulatory effects of the two hormones, possibly indicating that bGH does not potentiate the action of gonadotropin. Cyanoketone treatment of vitellogenic follicles inhibited bGH-induced estradiol production, but the stimulatory effect of bGH on the conversion of exogenous testosterone to estradiol was not altered by cyanoketone, which suggests that GH stimulates aromatase activity in addition to an earlier step in the biosynthetic pathway for estradiol production. The stimulatory effects of bGH and hCG on aromatase activity were mimicked by forskolin and dibutyryl cAMP, both of which are known to raise the intracellular level of cAMP. These results suggest that bGH- as well as hCG-induced increases in aromatase activity are mediated through adenylate cyclase-cAMP-dependent mechanism(s). The cAMP content of ovarian follicles was markedly increased within 15 min of bGH stimulation and was maximal at 1 hr, whereas steroid production did not increase significantly until 30 min and was maximal after 3 to 9 hr of incubation. The cAMP response to bGH, like that of steroid production, was concentration-dependent. The time-course and concentration-response effects of bGH on cAMP accumulation and steroid production were similar to those of hCG and forskolin, although the magnitude of the responses was less. The addition of cycloheximide or actinomycin D to the media did not affect the basal production of estradiol but completely blocked the bGH and hCG stimulation of the aromatization of exogenous testosterone to estradiol. These results suggest that the stimulatory effects of bGH and hCG on aromatase activity depend upon the synthesis of new RNA and regulatory protein(s).

Adenylyl Cyclases↗
PubMed1993

Characterization of the Schistosoma mansoni gene encoding the glycolytic enzyme, triosephosphate isomerase.

The complete gene encoding Schistosoma mansoni triosephosphate isomerase (TPI) was isolated from a lambda phage genomic library on 2 overlapping clones. These genomic clones have been characterized by restriction mapping and DNA sequencing of the 5' flanking region, the exons, the intron boundaries and the polyadenylation addition site. S. mansoni TPI is encoded by 6 exons spanning a region of about 12 kb. The 5 introns are located at positions precisely analogous to those of mammalian TPI genes but one of the 6 mammalian TPI introns is missing in S. mansoni. We find no evidence of spliced leader involvement in TPI gene expression. The gene is preceded by at least 4 tandem copies of a 2.5-kb repetitive sequence. While the 12-kb size for the S. mansoni TPI gene is much larger than the 3-4 kb typical of mammalian TPI genes, the 42-bp first intron is unusually short. The transcription initiation site for the S. mansoni TPI gene is heterogeneous. Genomic Southern blot analysis suggests that TPI is expressed from a single copy gene.

Amino Acid Sequence↗
PubMed1993

Dipeptidyl peptidase I from goat brain: purification, characterization and its action on Leu-enkephalin.

Brain dipeptidyl peptidase (DPP) I has been purified 2990-fold to apparent homogeneity shown by a single protein band in electrophoreses at pH 4.5, 8.4 and in SDS-PAGE at pH 7.2. The purification techniques included homogenization of brain acetone powder, autolysis at pH 4.2, 30-80% (NH4)2SO4 fractionation. Sephadex G-100 column chromatography, heat treatment at 65 C. organomercurial affinity chromatography. CM-Sephadex cation-exchange chromatographies at pH 5.6 and 5.0 and anion-exchange chromatography on DEAE-Sephadex at pH 6.8. The enzyme hydrolysed synthetic substrate Gly-Arg-4-methoxy-beta-naphthyl-amide maximally at pH 6.0. The Km values for Gly-Arg-beta-naphthylamide and Gly-Arg-4-methoxy-beta-naphthylamide substrates were 0.10 mM and 0.14 mM respectively. The enzyme was inhibited by thiol inhibitors like p-chloromercuribenzoic acid, iodoacetic acid, iodoacetamide and microbial inhibitors leupeptin and antipain. Molecular weight estimations on a calibrated Sephadex G-200 column afforded a value of 180,000 Da while in denaturing conditions on sodium dodecyl sulphate polyacrylamide gel electrophoresis, the subunit molecular weight was 22,000 Da. The subunit structure of the native enzyme was unfolded in presence of different concentrations of urea. In 8 M urea, the enzyme dissociated completely into monomers of 25,000 Da but 6, 5 and 4 M urea concentrations revealed the existence of dimers, tetramers and hexamers. Leu-enkephalin. Tyr-Gly-Gly-Phe-Leu was degraded by DPPI into Tyr-Gly and Gly-Phe-Leu with no further degradation of the newly generated tripeptide.

Amino Acid Sequence↗
PubMed1993

Study on the stability of 17D-204 yellow fever vaccine before and after stabilization.

To monitor the parameters controlling yellow fever vaccine production, eight different lots produced without stabilizers were studied. It was found that the freeze-thaw cycle did not have any adverse effect on virus infectivity and the mean loss in virus titre during lyophilization was 0.51 log10 ml-1. A thermodegradation study after storage of vaccine at different temperatures showed that the vaccine did not pass the accelerated stability test. To stabilize the 17D-204 substrain vaccine, four stabilizers with different sugars and amino acids were examined. The optimum time for addition of the stabilizers was found to be during homogenization of infected embryos. An accelerated stability test at 37 degrees C indicated that stabilizers S1, S2 and S3 kept the vaccine stable for up to 4 weeks, whereas the vaccine was only stable for up to 3 weeks in stabilizer S4. Stabilizer S3 was found statistically to be the best. Reproducibility in production methodology was established by preparing and testing more batches of the vaccine using stabilizer S3.

Animals↗
PubMed1993

A selective colorimetric assay for cathepsin L using Z-Phe-Arg-4-methoxy-beta-naphthylamide.

Among the intracellular proteinases, the thiol proteinases such as cathepsin B (EC 3.4.22.1), cathepsin H (EC 3.4.22.16) and cathepsin L (EC 3.4.22.15) which act at slightly acidic pHs are more likely to play an important role in lysosomal protein catabolism. Out of these, cathepsin L plays a major role primarily because it has high degradative activity on cellular and matrix proteins. However, the studies on cathepsin L in crude homogenates and subcellular fractions have always been hampered by the lack of a specific substrate to exclusively measure the activity of this proteinase. The only synthetic substrate alpha-N-benzyloxycarbonyl-L-Phe-L-Arg-4-methoxy-beta-naphthylamide (Z-Phe-Arg-NNapOMe) which is hydrolysed by cathepsin L is hydrolysed equally well by cathepsin B. This substrate was manipulated to act as a selective substrate for cathepsin L. In presence of 4 M urea at pH 5.0, cathepsin B (the only other cathepsin which also hydrolyses Z-Phe-Arg-NNapOMe) was inactivated and, therefore, under these conditions, the enzyme activity quantitated by using this substrate is only due to cathepsin L. Using this newly-developed colorimetric assay method specific for cathepsin L, the subcellular and regional distribution of this proteinase were established in goat brain tissue. About 80% cathepsin L activity was recovered in the lysosomal fraction thus establishing its lysosomal nature. Among the various brain parts, highest activity was found in cerebrum followed by cerebellum, pituitary body, pons-varolli, thalamus, medulla-oblongata and hypothalamus.

Amino Acid Sequence↗
PubMed1993

Anesthetic potency (MAC) is independent of forebrain structures in the rat.

BACKGROUND: The ability of general anesthetics to suppress somatomotor responses to surgical incision and other noxious stimuli is of particular clinical relevance. When the blockade is due to inhaled agents, this effect can be quantified as the minimum alveolar concentration (MAC), i.e., that concentration that blocks movement evoked by a noxious stimulus (ED50). METHODS: To identify the neural structures that subtend this somatomotor response, we anesthetized 14 rats with isoflurane in oxygen and performed bilateral parietal-temporal craniotomies. In each rat, MAC was repeatedly tested using tail-clamping and Dixon's up-down concentration technique. After determination of baseline MAC, seven rats underwent aspiration decerebration, after which MAC was repeatedly measured. RESULTS: In the control group (N = 7), MAC (mean +/- SD) remained constant at 1.30 +/- 0.25% for more than 6 h. In the seven rats that underwent aspiration decerebration, baseline MAC was 1.26 +/- 0.14%. These seven rats with histologically validated precollicular decerebration demonstrated no change in MAC relative to control rats, as much as 11 h after decerebration (P = 0.14). CONCLUSIONS: These findings suggest that the anesthetic-induced unresponsiveness to noxious stimuli measured by MAC testing does not depend on cortical or forebrain structures in the rat.

Administration, Inhalation↗
PubMed1993

PU.1 is a component of a multiprotein complex which binds an essential site in the murine immunoglobulin lambda 2-4 enhancer.

B-cell-specific enhancers have been identified in the immunoglobulin lambda locus 3' of each constant-region cluster. These enhancers contain two distinct domains, lambda A and lambda B, which are essential for enhancer function. lambda B contains a near-consensus binding site for the Ets family of transcription factors. In this study, we have identified a B-cell-specific protein complex which binds the lambda B motif of the lambda 2-4 enhancer in vitro and appears necessary for the activity of the enhancer in vivo, since mutations in lambda B which prevent this interaction also eliminate enhancer function. This complex contains PU.1, a member of the Ets family, and a transcriptional activator whose expression is restricted to cells of the hematopoietic system with the exception of T lymphocytes. In addition, it contains a factor which binds specifically to a region adjacent to the PU.1 binding site. This factor cannot bind lambda B autonomously but appears to require interaction with the PU.1 protein to stabilize its association with the DNA. This complex may be identical or related to the PU.1/NF-EM5 complex which interacts with a homologous DNA element in the immunoglobulin kappa 3' enhancer.

Animals↗
PubMed1993

Spontaneous coronary artery dissection: a report of three cases and review of the literature.

We describe the clinical course of three patients who developed spontaneous coronary artery dissection. All patients were young women, one 9 weeks pregnant. All presented with chest pain; one died suddenly proving refractory to resuscitation, another developed unstable angina culminating in myocardial infarction, cardiogenic shock and death, and the third patient underwent coronary artery bypass grafting following diagnosis of a spontaneous coronary dissection of the left anterior descending artery at angiography. Pathological findings in the two fatal cases are reported. This condition, although rare, is a prominent cause of ischaemic coronary events in young women, when it is frequently associated with pregnancy or the puerperium. Most patients die suddenly, but a clinical spectrum is seen including stable and unstable angina, myocardial infarction and cardiogenic shock. The left anterior descending artery is most frequently affected. The classical histological finding is that of a large haematoma occupying the outer third of the media resulting in complete compression of the true lumen. The cause of spontaneous dissection remains unclear but theories of aetiology include a medial eosinophilic angiitis, pregnancy-induced degeneration of collagen in conjunction with the stresses of parturition, and rupture of the vasal vasorum. The diagnosis must be considered when a patient presents with a suggestive clinical profile. Urgent angiography should be undertaken to establish the diagnosis and consideration given to the need for coronary artery bypass grafting, which has been successfully employed in a number of patients. The uneventful long-term survival of cases treated conservatively has been reported.

Adult↗
PubMed1993

Effect of halothane exposure on motor skills & memory in anaesthetists.

In a prospective study involving 24 anaesthesia residents, each resident was assessed three times i.e., first as control group, not exposed to anaesthesia at all; the second group in which the resident was exposed to anaesthesia while using controlled respiration with closed circuit without halothane (non-halothane group) and the third group in which they were exposed to anaesthesia using spontaneous respiration with semiclosed circuit using halothane (halothane group). After each exposure of 3-4 h duration in each group, the resident was subjected to specific psychological tests to assess motor skills and memory. In the non-halothane group, a depression of motor skills by 5.51 per cent and of memory by 17.14 per cent was observed. In the halothane group, the reduction of motor skills was up to 27 per cent and of memory by 45 per cent as compared to the control values. It is concluded that 3-4 h exposure to halothane (1.5-4%) obtunds the motor skills and memory in anaesthetists.

Adult↗
PubMed1993

Midazolam as an induction agent in mitral stenosis patients for closed mitral commissurotomy.

In 30 patients of rheumatic heart disease with mitral stenosis (MS) belonging to NYHA class II and III scheduled for closed mitral commissurotomy anaesthesia was induced with morphine 0.15 mg/kg followed by either thiopentone (group A, n = 15) or midazolam (group B, n = 15) titrated to produce sleep. Patients were intubated with pancuronium bromide in a dose of 0.12 mg/kg. Minimum mean arterial blood pressure following induction was significantly lower in thiopentone group (77 +/- 7 mm Hg) than midazolam group (85 +/- 6 mm Hg; P < 0.05). After intubation blood pressure was significantly higher in thiopentone group (99 +/- 8 mm Hg) than midazolam group patients (89 +/- 7 mm Hg). Heart rate was significantly higher in thiopentone treated patients both before and after endotracheal intubation. During surgery, three patients in group A had hypotensive episodes (mean arterial blood pressure 20% below basal at two successive readings 5 min apart) while one in group B had a hypotensive episode. Average duration of surgery was comparable between the two groups (102 +/- 15 and 95 +/- 18 min) and postoperatively there was no significant difference in sedation score and incidence of nausea and vomiting between the two groups.

Adult↗
PubMed1993

Effect of some steroidal & non-steroidal anti-inflammatory drugs on purified goat brain cathepsin L.

The lysosomal cysteine proteinases have been found to be involved in various inflammatory conditions. Inhibitory effects of certain commonly used anti-inflammatory drugs were observed on lysosomal thiol proteinase cathepsin L. Of the non-steroidal anti-inflammatory drugs tested, phenylbutazone was found to be most potent inhibitor of cathepsin L activity. The half maximal inhibition was achieved at 0.6 mM concentration. The inhibition by phenylbutazone was of non-competitive type, with a ki of 1.3 x 10(-3) M. Flufenamic acid and indomethacin were also inhibitory to cathepsin L activity, giving half maximal inhibitions at 3.5 mM and 4.5 mM concentrations respectively. In contrast, cathepsin L activity was not affected at all by steroidal anti-inflammatory agents. Aspirin was also found to have no effect on cathepsin L activity whereas salicylic acid, its m- and p-analogs exhibited inhibitory effects but to a lesser degree.

Animals↗