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Biomedical subjects

R Bhat

Publications and source records attributed to R Bhat.

At least 37 records · Page 2Linked to original sources
PubMed1997

Bile flow and composition in preterm, term and infant baboons.

UNLABELLED: We studied the maturational changes in bile composition, bile flow and choleretic effects of sodium taurocholate and secretin in preterm (160 +/- 2 days, n = 4, group I), term (184 +/- 2 days, n = 4, group II), 7-day postnatal age (n = 5, group III) and 60-day-old (n = 5, group IV) baboons. The canalicular bile flow was determined by 14C-erythritol clearance. RESULTS: Gall bladder volume increased from group I to group III (0.08 +/- 0.06 to 1.06 +/- 0.93 ml). Bile flow increased significantly from group I to IV (0.13 +/- 0.05 to 0.34 +/- 0.07 microliter/min/g liver weight, p < 0.05). This was associated with significant increases in total bile acid excretion (16 +/- 3.6 to 31 +/- 2.5 mEq/l, p < 0.05). The composition of bile also showed maturational changes with increasing postnatal age. Sodium taurocholate and secretin increased the bile flow significantly in all groups. CONCLUSION: Data from these studies clearly demonstrate that the bile flow and bile acid excretion is immature in preterm and term baboons when compared to 7- and 60-day-old baboons. The present studies also suggest that baboons can be used as a model to study the postnatal maturation of hepatic excretory function.

Animals↗
PubMed1996

ATP depletion affects the phosphorylation state, ligand binding, and nuclear transport of the 4 S polycyclic aromatic hydrocarbon-binding protein in rat hepatoma cells.

In the rat, cytochrome P4501A1 gene expression is thought to be regulated by several trans-acting factors including the 4 S polycyclic aromatic hydrocarbon (PAH)-binding protein. Phosphorylation and dephosphorylation have been suggested to influence the function of many cytosolic receptors and transcription factors. The ATP level within H4IIE rat hepatoma cells could be depleted by treatment with sodium azide or 2,4-dinitrophenol; restoration of the original ATP levels occurred with addition of glucose to the cell culture. ATP depletion reduced the phosphate content of the 4 S protein by approximately 25-30%, which lowered the binding of benzo[a]pyrene (B[a]P) to the 4 S protein by >60%. This effect could not be reversed by the addition of ATP to the binding reaction mixtures. Alkaline phosphatase treatment of the purified 4 S protein in a cell-free system also reduced the B[a]P binding to the protein. Cells treated with a protein phosphatase inhibitor, okadaic acid, and a protein kinase inhibitor, staurosporin, affected the B[a]P binding of the 4 S protein positively and negatively, respectively. These data suggested that phosphorylation is involved in the interaction of the 4 S protein with the PAH. The nuclear translocation of the predominantly cytosolic binding protein has been investigated after ligand binding. Western blots with the immunopurified 4 S PAH-binding protein from cytosolic and nuclear lysates showed significant differences in the distribution of the 4 S receptor between cytosolic and nuclear compartments in control and ATP-depleted cells. Ligand binding stimulated the movement of the receptor into the nucleus, which was completely blocked by reducing the intracellular ATP concentration. These findings provide new information on the role of ATP and phosphorylation on the interaction of B[a]P with 4 S PAH-binding protein and its nuclear translocation.

2,4-Dinitrophenol↗
PubMed1996

Thermostabilization of protective antigen--the binding component of anthrax lethal toxin.

Protective antigen (PA) is the binding component of anthrax lethal toxin produced by Bacillus anthracis, and constitutes a major ingredient of the vaccine against anthrax. PA and lethal factor when added together are cytolytic to mouse macrophages and J774G8 macrophage cell line. This in vitro lethal toxicity assay is very useful in understanding the molecular mechanism of action of lethal toxin. Effective utilization of PA is, however, hampered due to its thermolability. On prolonged storage at 37 degrees C, PA was found to lose its activity almost completely. The effect of solvent additives like trehalose, sorbitol, xylitol, sodium citrate and magnesium sulphate on the thermal stabilization of PA was examined. The results indicated an increase in the stability of PA when the incubation at 37 degrees C was carried out in the presence of solvent additives used in the 1-3 M range. Magnesium sulphate helped retain the activity up to 82.7% against the control in which no additive was used, as judged by cytolytic assay using J774G8 macrophage cell line. Trehalose or sodium citrate also showed an appreciable protection of PA activity, while sorbitol or xylitol were not very effective. Competitive binding assay using radiolabeled PA showed that PA had lost capacity of binding to macrophage cells on prolonged incubation at 37 degrees C. Circular dichroism results at 4, 18, and 37 degrees C indicated an increase in secondary structure at 37 degrees C relative to that at 4 or 18 degrees C, supporting the activity data.

Animals↗
PubMed1996

Nuclear transcription factor Oct-1 binds to the 5'-upstream region of CYP1A1 and negatively regulates its expression.

The cytochrome P450-dependent monooxygenases, which represent an extended superfamily, catalyze the biotransformation of many endogenous and exogenous substances. One of these hemoproteins, cytochrome P4501A1, is most closely associated with the bioactivation of polycyclic aromatic hydrocarbons such as benzo[a]pyrene, which may play a role in environmental carcinogenesis. A negative regulatory element (NRE) has been localized in the 5'-upstream region of the cytochrome P4501A1 gene (CYP1A1) at -843 to -746 base pairs from the site of transcription. The purpose of this research was to define any interactions of trans-acting proteins with this cis element. Rat liver nuclei were used as the source of trans-acting proteins and a biotinylated NRE-bearing fragment (-782 to -843 bp) from a plasmid which contained the CYP1A1 was prepared by the polymerase chain reaction technique. Gel mobility shift assays were used to demonstrate interactions between this NRE fragment and nuclear proteins. The specific binding to an octamer-containing motif in the 5'-upstream region of CYP1A1 was demonstrated; this was used as a step in the partial purification from rat liver of the transcription factor, Oct-1. Conventional chromatographic procedures and DNA recognition site affinity chromatography were also used. HepG2 human hepatoma cells were transfected with both pMCoLUC+ which contains the luciferase gene as a reporter gene driven by the CYP1A1 promoter (including the NRE), and an Oct-1 expression vector. Luciferase activity/mg protein in the doubly-transfected cells was significantly lower than in cells containing only pMCoLUC+. A nuclear transcription factor Oct-1 interacts with a portion of the NRE of the rat CYP1A1, suppressing the expression of this gene. These findings may help to explain the low level of basal expression of CYP1A1 in mammalian systems.

Animals↗
PubMed1996

Subunit structure of Ca++ dependent soluble goat hepatic lectin: evidence that it has antigenic structural relationship with soluble 14K lectin family.

The subunit structure of soluble goat hepatic lectin was studied by determining molecular weight under nondenaturing conditions by gel filtration and denaturing conditions by SDS PAGE. Affinity purified lectin was subjected to HPLC on asahipack column equilibrated with 10 mM Tris-HCl pH 7.5, containing 1 mM CaCl2, 1mM 2-mercaptoethanol and 0.1M NaCl. The lectin was eluted under single peak at retention time of 12 min. corresponding to molecular weight of 38Kd. On SDS-PAGE in the presence and absence of 2-mercaptoethanol protein moved as single band with Rm 0.45, which corresponds to molecular weight of 20 Kd. The results suggest that soluble goat hepatic lectin is a dimmer of identical subunits which are linked together by noncovalent interactions. The interaction of monoclonal antibodies raised against soluble goat hepatic lectin (MGHL/20) with hemagglutinin from different species as sheep, human, rat, bovine and chicken was studied in PBS by solid phase binding assay. MGHL/20 showed 29.89% binding with these lectin. However no binding was found with Ca++ dependent membrane bound lectin. These results indicate that soluble goat hepatic lectin possesses antigenic structural relationship with soluble 14 K lectin family.

Animals↗
PubMed1996

Regulating the private health care sector: the case of the Indian Consumer Protection Act.

Private medical provision is an important constituent of health care delivery services in India. The quality of care provided by this sector is a critical issue. Professional organizations such as the Medical Council of India and local medical associations have remained ineffective in influencing the behaviour of private providers. The recent decision to bring private medical practice under the Consumer Protection Act (COPRA) 1986 is considered an important step towards regulating the private medical sector. This study surveyed the views of private providers on this legislation. They believe the COPRA will be effective in minimizing malpractice and negligent behaviour, but it does have adverse consequences such as an increase in fees charged by doctors, an increase in the prescription of medicines and diagnostics, an adverse impact on emergency care, etc. The medical associations have also argued that the introduction of COPRA is a step towards expensive, daunting and needless litigation. A number of other concerns have been raised by consumer forums which focus on the lack of standards for private practice, the uncertainty and risks of medicines, the effectiveness of the judiciary system, and the responsibility of proving negligence. How relevant are these concerns? Is the enactment of COPRA really appropriate to the medical sector? The paper argues that while this development is a welcome step, we need to comprehensively look into the various quality concerns. The effective implementation of COPRA presumes certain conditions, the most important being the availability of standards. Besides this, greater involvement of professional organizations is needed to ensure appropriate quality in private practice, since health and medical cases are very different from other goods and services. The paper discusses the results of a mailed survey and interview responses of 130 providers from the city of Ahmedabad, India. The questionnaire study was designed to assess the opinion of providers on various implications of the COPRA. We also analyze the data on cases filed with the Consumer Disputes and Redressal Commission in Gujarat since 1991. Four selected cases filed with the National Commission on Consumers Redressal are discussed in detail to illustrate various issues affecting the implementation of this Act.

Consumer Advocacy↗
PubMed1995

Changes in the concentration of endothelin-1 during development of hypertensive rats.

The concentration of endothelin-1 (ET-1) in the brain regions, heart, and throacic aorta of 1-, 4-, 6- and 8-week-old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined using radioimmunoassay. ET-1-like immunoreactivity in the brain regions of 1-week-old WKY and SHR rats was lower compared to older (6 and 8 weeks) rats. ET-1 levels in the central nervous system gradually increased with age in both SHR and WKY rats. However, the concentration of ET-1 in 8-week-old rats was lower in the brain regions of SHR compared to WKY rats. The concentration of ET-1 in the thoracic aorta of SHR (224 +/- 43 pg/g tissue) rats was lower than that of WKY (452 +/- 11 pg/g tissue) rats at 1 week of age. However, ET-1 levels gradually increased with age in SHR rats. By 8 weeks of age, levels of ET-1 in SHR (623 +/- 33 pg/g tissue) rats were higher compared to WKY (439 +/- 62 pg/g tissue) rats. In the heart, ET-1 levels were similar in WKY and SHR rats at 4 weeks of age, but at 8 weeks of age ET-1 levels were higher in SHR rats (364 +/- 33 pg/g tissue) compared to WKY rats (260 +/- 31 pg/g tissue). It appears that at 8 weeks of age when hypertension is fully expressed in rats, ET-1 levels are lower in the central nervous system and are higher in the thoracic aorta and heart of SHR compared to WKY rats.

Animals↗
PubMed1995

Renal actions of endothelin-1 in newborn piglets: dose-effect relation and the effects of receptor antagonist (BQ-123) and cyclooxygenase inhibitor (indomethacin).

Although the effects of endothelin-1 (ET-1) on intact or perfused adult kidney are well understood, its effects in the fetus and the newborn have not been well studied. We examined the effects of infusions of 25, 50, and 100 ng/kg of ET-1 per minute on mean blood pressure (MBP), cardiac index (CI), renal blood flow (RBF), glomerular filtration rate (GFR), and urine volume (UV) in 7- to 10-day-old piglets (n = 24). In addition, the effects of pretreatment with a receptor antagonist (BQ-123) and with a cyclooxygenase inhibitor (indomethacin) were studied in 12 separate piglets. ET-1 produced a dose- and level-dependent decrease in CI (60%), RBF (50% to 75%), GFR (66% to 80%) and MBP 15% to 17%. These changes returned to 75% to 80% of baseline 60 minutes after discontinuation of ET-1. Low-dose infusion (25 ng/kg) did not result in any changes in systemic or renal hemodynamics. Plasma half-life of ET-1 in piglets was 2.1 +/- 0.4 minutes. Pretreatment with the specific ETA receptor antagonist BQ-123 completely blocked the ET-1-induced systemic and renal hemodynamic changes. Indomethacin blocked the ET-1-induced rise in MBP but failed to block any renal changes. In fact, indomethacin accentuated the changes induced by ET-1, especially the changes in RBF, RVR, and GFR. Studies of receptor binding in the renal cortex and medulla showed that, in the cortex, the Ki value for ET-1 was 6.32 +/- 1.57, and for ET-3 it was 20.05 +/- 4.38 (p < 0.05); in the medulla, the Ki values were similar for both ET-1 and ET-3. These results indicate that in piglets the renal vascular bed is highly sensitive to ET-1, and its effects are predominantly mediated through ETA receptors.

Animals↗
PubMed1994

DNA breakage by tannic acid and Cu(II): sequence specificity of the reaction and involvement of active oxygen species.

Tannic acid has numerous chemical, food and pharmacological applications. In the presence of Cu(II) and molecular oxygen it was found to cause breakage of calf thymus DNA and supercoiled plasmid DNA. Treatment of lambda phage DNA with tannic acid protected cleavage with restriction endonucleases DraI and EcoRI* but not with SmaI and HaeIII. The results indicate that under the conditions used tannic acid preferably binds to AT base pairs. Restriction analysis of open circular form II plasmid DNA generated by tannic acid-Cu(II) treatment further showed that the strand breakage is caused at specific sites or sequences. In this reaction Cu(I) was shown to be an essential intermediate by using the Cu(I) sequestering reagents neocuproine and bathocuproine. By using job plots, we established that in the absence of DNA, six Cu(II) ions can be reduced by one tannic acid molecule. The involvement of active oxygen species in the reaction was established by the inhibition of DNA breakage by superoxide dismutase, thiourea, mannitol, formate and catalase.

Animals↗
PubMed1994

DNA breakage by tannic acid and Cu(II): generation of active oxygen species and biological activity of the reaction.

Tannic acid was shown to reduce oxygen to superoxide anion. In the presence of Cu(II), the hydroxyl radical and hydrogen peroxide were formed. Strand scission reaction was shown to account for the biological activity of tannic acid as assayed by bacteriophage inactivation. The inactivating activity occurs through the Fenton pathway for free radical production and subsequent DNA cleavage by these radicals.

Bacteriophage lambda↗
PubMed1994

Immunogenicity of high expression adenovirus-hepatitis B virus recombinant vaccines in dogs.

High yielding adenovirus (Ad)-hepatitis B recombinant (Ad-Hep B) viruses were prepared by insertion of the hepatitis B surface antigen (HBsAg) gene into the early region 3 (E3 region) of Ad4 or Ad7 vectors containing intact or largely deleted E3 regions. Both E3-deleted and non-deleted recombinants produced about six- to eight-fold higher amounts of HBsAg than previously reported recombinants. These recombinant viruses were evaluated for immunogenicity in dogs which sustain abortive lung infections by Ad4 and Ad7. Recombinants containing E3 deletions elicited 10- to 12-fold stronger anti-HBs primary responses than previously evaluated low yield recombinants. Further immunizations with heterotypic Ad-Hep B recombinants induced substantial anti-HBs booster responses as well as anti-'a' epitope responses. In contrast, recombinant viruses containing intact E3 regions induced only weak or negligible anti-HBs responses, although such viruses induced strong antibody responses to the Ad vectors. The immunogenicity of high-yielding Ad recombinants correlated with temporal expression of HBsAg and thus the dog represents a valuable model for evaluation of immune responses to heterologous proteins that are expressed early and that do not require efficient DNA replication. Recombinants expressing HBsAg late in the infectious cycle require further testing in the fully permissive chimpanzee model. This study establishes that the E3-deleted high yield Ad4 and Ad7 recombinants represent promising live oral hepatitis B vaccine candidates.

Adenovirus E3 Proteins↗
PubMed1994

Photoinactivation of bacteriophage lambda by kojic acid and Fe(III): role of oxygen radical intermediates in the reaction.

Kojic acid, a bacterial metabolite, intensively used in food and pharmaceutical industry has been reported to induce strand breaks in double stranded DNA. This genotoxic action of kojic acid was tested directly by using bacteriophage inactivation. The inactivating activity is mediated through DNA strand scissions by oxygen free radical intermediates. This was tested by quenching of the inactivation reaction in presence of radical scavengers. These results indicate the usefulness of a simple system to test various risk factors in dietary components.

Azides↗
PubMed1993

High-performance liquid chromatographic determination of morphine and its metabolites in plasma using diode-array detection.

An isocratic high-performance liquid chromatographic method has been developed for the determination of morphine, codeine, normorphine, morphine 3-glucuronide and morphine 6-glucuronide in plasma using a diol column and diode-array detection. Samples were extracted using solid-phase extraction with recoveries in excess of 90%. The limit of determination was 1 ng/ml for morphine, codeine and morphine 3-glucuronide, and 10 ng/ml for normorphine and morphine 6-glucuronide. Inter- and intra-day precision were better than 10%.

Chromatography, High Pressure Liquid↗