Biomedical subjects
Publications and source records attributed to R Bhat.
Gonadotropins independent precocious puberty (GIPP) in male is characterized by early appearance of sexual hairs and phallic growth but without testicular enlargement. We report a case of GIPP with testicular enlargement who was diagnosed to have testotoxicosis and successfully managed with spironolactone.
BACKGROUND: Diminished lung function appears to be a risk factor for respiratory syncytial virus (RSV) infection/bronchiolitis in term born infants. AIMS: To determine if diminished lung function prior to neonatal unit discharge was associated with subsequent symptomatic RSV lower respiratory tract infection (LRTI) and respiratory morbidity in prematurely born infants. METHODS: Of 39 infants in a tertiary neonatal intensive care unit (median gestational age 28 weeks, range 23-31), 20 had bronchopulmonary dysplasia. Lung function (compliance and resistance of the respiratory system (C(rs) and R(rs)) and functional residual capacity (FRC)) was measured on the neonatal unit at 36 weeks postmenstrual age (PMA). Following neonatal unit discharge, nasopharyngeal aspirates were obtained on every occasion, at home or in hospital, an infant had an LRTI. RSV was identified by immunofluorescence and/or culture. RESULTS: The 15 infants who suffered a symptomatic RSV LRTI had a higher mean R(rs) and suffered more wheeze at follow up than the rest of the cohort. Regression analysis showed that a high R(rs) was significantly associated with a symptomatic RSV LRTI; significant factors for cough were a high R(rs) and a symptomatic RSV LRTI, and for wheeze were a high R(rs). CONCLUSION: Prematurely born infants, who had a symptomatic RSV LRTI and/or respiratory morbidity at follow up, had worse lung function prior to neonatal unit discharge.
We have recently purified from meconium-instilled rabbit lungs a novel serine proteinase inhibitor, with an apparent molecular mass of 50 kDa, which we assign to be alpha1-antitripsin. We hypothesize that serpin may attenuate pulmonary inflammation and improve surfactant function after meconium aspiration. Alpha1-antitripsin is a member of the proteinase inhibitor (serpin) superfamily and inhibitor of neutrophil elastase, and it can be identified as a member of the family by its amino acid sequence due to the high degree of conserved residues. Alpha1-antitripsin is synthesized by epithelial cells, macrophages, monocytes, and neutrophils. Deficiency in alpha1-antitripsin leads to exposure of lungs to uncontrolled proteolytic attack from neutrophil elastase or other damaging factors culminating in lung destruction and cell apoptosis. We hypothesize that accumulation of alpha1-antitripsin in the lungs serves as a predisposed protection against meconium-induced lung injury. In this paper, we show how this knowledge can lead to the development of novel therapeutic approaches for treatment of MAS.
We describe a 3-year-old male patient with the ichthyosis follicularis, alopecia and photophobia (IFAP) syndrome, who developed cutaneous and ocular involvement in infancy. In addition, he had growth retardation and borderline intelligence; no other systemic involvement was found on detailed investigation. A moderate response to acitretin therapy (1 mg/kg) administered for 6 months was observed, with improvement in cutaneous features and corneal erosions and no change in alopecia or photophobia.
The tauopathies are a group of disorders characterised by aggregation of the microtubule-associated protein tau and include Alzheimer's disease (AD) and the fronto-temporal dementias (FTD). We have used Drosophila to analyse how tau abnormalities cause neurodegeneration. By selectively co-expressing wild-type human tau (0N3R isoform) and a GFP vesicle marker in motorneurons, we examined the consequences of tau overexpression on axonal transport in vivo. The results show that overexpression of tau disrupts axonal transport causing vesicle aggregation and this is associated with loss of locomotor function. All these effects occur without neuron death. Co-expression of constitutively active glycogen-synthase kinase-3beta (GSK-3beta) enhances and two GSK-3beta inhibitors, lithium and AR-A014418, reverse both the axon transport and locomotor phenotypes, suggesting that the pathological effects of tau are phosphorylation dependent. These data show that tau abnormalities significantly disrupt neuronal function, in a phosphorylation-dependent manner, before the classical pathological hallmarks are evident and also suggest that the inhibition of GSK-3beta might have potential therapeutic benefits in tauopathies.
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We studied the uptake, transport and metabolism of cocaine in human intestine using the colonic T-84 monolayers as a model. T-84 cells were grown in DMEM/Ham's F-12 medium containing 6% newborn calf serum at 37 degrees C on 1.0 microm collagen inserts. The cells develop into a polarized monolayer with the apical surface facing the upper chamber and the basolateral surface facing the lower. The monolayers develop a transepithelial resistance of > or = 600 ohms cm2 in 7 days. Varying concentrations of cocaine HCI was added in a serum free medium to the luminal side only, and after 30 min and 60 min samples from the luminal and serosal aspect were removed for analysis. Cocaine and its metabolites were measured by GC/MS. Cocaine transport across T-84 monolayers increased linearly with increasing concentration of cocaine, with no significant difference between 30 min and 60 min of exposure. Transepithelial resistance did not change even at 800 ng of cocaine suggesting no effect on monolayer viability. The metabolites, benzoylecgonine (BE) and ecgonine methyl ester (EME) but not norcocaine were detected in both luminal and serosal side. The concentrations of BE and EME in the luminal side were significantly higher than in the serosal. Combined recovery of cocaine, BE and EME from luminal and serosal sides were 52 - 80% of total added cocaine. While fresh medium did not metabolize cocaine, media previously exposed to the monolayer (cell-free medium) caused a significant breakdown into BE and EME, suggesting that esterases may be released into the medium. These results indicate transfer of cocaine across this model of intestinal epithelial cell line is by simple diffusion and is concentration dependent. These studies imply that cocaine in swallowed amniotic fluid can be absorbed by the fetal gastrointestinal tract.
Anthrax is caused by Gram positive bacterium Bacillus anthracis. Pathogenesis is result of production of three protein components, protective antigen (PA), lethal factor (LF), and edema factor (EF). PA in combination with LF (lethal toxin) is lethal to animals, while PA in combination with EF (edema toxin), causes edema. PA, LF, and EF are very thermolabile. Differential scanning calorimetry (DSC) was used to unravel the energetics of LF denaturation as a function of pH ranging from 7.8 to 5.5. Transition temperature (T(m)) of LF was found to be approximately equal to 42 degrees C and onset of denaturation occurs at approximately equal to 30 degrees C. The ratio of calorimetric to van't Hoff's enthalpy was nearly equal to unity at pH 7.0, indicative of presence of single structural domain in LF at pH 7.0, unlike PA which has been structurally observed to consist of 4 domains. It was found by cytotoxicity studies using J774A.1 macrophage like cells that LF was most stable at pH approximately 6.5. This paper reports for the first time the denaturation of LF at different pH values at 37 degrees C and tries to establish a correlation between denaturation and loss of LF activity at different pH values.
Estrogen receptor-beta (ERbeta) is a recently discovered member of the steroid hormone superfamily. Because its distribution is distinct from that of the classical estrogen receptor, and it is expressed in several nonclassical estrogen target tissues (e.g. prostate and bladder), its role in mediating the action of estrogen is unclear. One approach to elucidating the function of this receptor is to identify genes that it regulates. Using differential display, we profiled the messenger RNAs regulated by 17beta-estradiol in SAOS-2 and LNCaPLN3 cells overexpressing ERbeta. Follow-up studies used cells expressing either ERalpha or ERbeta. One gene, metallothionein II, was regulated by both receptor subtypes in LNCaPLN3 cells, but only by ERbeta in SAOS-2 cells. Because cycloheximide blocks this response, induction is probably mediated through regulation of at least one other protein. Identification of endogenous genes that are regulated differentially by ERalpha and ERbeta will be valuable tools in elucidating the function of ERbeta and the mechanisms by which these two receptors regulate transcription.
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UNLABELLED: Meconium aspiration syndrome (MAS) is a major cause of newborn mortality and morbidity. In this study we investigated the inflammatory responses and morphological changes in the newborn lung to debris-free meconium instillation. We developed a model for studies of MAS using 2-week-old rabbit pups. Cell death was assessed by DNA staining and detection of DNA fragmentation by in situ end labeling. Cell death was seen in association with an increase of inflammatory cytokines levels, studied by ELISA. Necrotic cells were detected by staining of lavage cells with ethidium bromide and 4',6'-diamino-2'-phenylidon. Meconium instillation resulted selectively in loss of airway and alveolar epithelial cells followed by cell death, which increased with time. Necrotic cells looked smaller and damaged with maximal counts at 24 h after instillation. CONCLUSION: Meconium instillation into lungs caused massive cell death, possibly by apoptosis, and necrosis that may have been activated by the inflammatory cytokine production.
DNA-based immunizations have been used to elicit cellular immunity to hepatitis C virus (HCV) proteins in mice. Mice were immunized by intramuscular or intradermal injections of plasmid DNA derived from a near-full-length HCV genotype 1b genomic clone (pRC/B2) or individual genomic clones. These immunizations induced cytotoxic T lymphocytes (CTLs), as revealed in standard chromium-release assays that used syngeneic peptide-pulsed or transfected target cells. These assays identified four CTL epitopes within the capsid, E1, and E2 regions of the polyprotein sequence of HCV genotype 1a that were cross-reactive with HCV genotype 1b. Additionally, CTLs derived from mice immunized with either NS3 or NS5 specifically lysed target cells sensitized to either the genotype 1a or 1b gene products. Nucleic acid immunizations also generated humoral immunity to HCV proteins, as detected by anti-HCV reactivity to NS3 and capsid in ELISAs and immunoblot assays.
Supply factors, depicted by input market conditions and government regulations, and demand factors, depicted by financing mechanisms and utilization patterns, are likely to determine the shape and character of private medical practice. The interaction of this complex set of factors will have considerable implications for the cost access and quality of services offered by this sector. Understanding these characteristics from a provider perspective is imperative to influence the behaviour of providers in this sector. This paper describes some of the important characteristics of private medical practice using a case study of an urban district in India, Ahmedabad, and analyzes their implications. Using survey data of 130 private doctors in the allopathic system, the paper describes broad characteristics of private medical practice using parameters such as growth of private practice, patient load and referrals within the sector, payment methods and determinants, patient concerns, and risks associated with private practice. The paper presents views on the prevalence of various undesirable practices in the private medical sector. It also discusses the awareness of providers about selected important regulations. The findings suggest that growing capital intensity due to cost of location, medical equipment and technology, and financial sources of capital investments are some unfavourable environmental factors experienced by private providers. The findings also indicate a high prevalence of various undesirable practices and low awareness of the objectives of important legislation among practicing doctors. Lack of awareness of important and relevant legislation raises serious questions about the implementation of these laws. The paper identifies the strong need for instituting and implementing an effective continuing medical education programme for practicing doctors, and linking it with their registration and continuation of their license to practice. The paper also suggests that cost of health care, access and quality problems will worsen with the growth of the private sector. The public policy response to check some of the undesirable consequences of this growth is critical and should focus on strengthening the existing institutional mechanisms to protect patients, developing and implementing an appropriate regulatory framework and strengthening the public health care delivery system. The study also discusses various other policy implications arising.
The stability of proteins is known to be affected significantly in the presence of high concentration of salts and is highly pH dependent. Extensive studies have been carried out on the stability of proteins in the presence of simple electrolytes and evaluated in terms of preferential interactions and increase in the surface tension of the medium. We have carried out an in-depth study of the effects of a series of carboxylic acid salts: ethylene diamine tetra acetate, butane tetra carboxylate, propane tricarballylate, citrate, succinate, tartarate, malonate, and gluconate on the thermal stability of five different proteins that vary in their physico-chemical properties: RNase A, cytochrome c, trypsin inhibitor, myoglobin, and lysozyme. Surface tension measurements of aqueous solutions of the salts indicate an increase in the surface tension of the medium that is very strongly correlated with the increase in the thermal stability of proteins. There is also a linear correlation of the increase in thermal stability with the number of carboxylic groups in the salt. Thermal stability has been found to increase by as much as 22 C at 1 M concentration of salt. Such a high thermal stability at identical concentrations has not been reported before. The differences in the heat capacities of denaturation, deltaCp for RNase A, deduced from the transition curves obtained in the presence of varying concentrations of GdmCl and that of carboxylic acid salts as a function of pH, indicate that the nature of the solvent medium and its interactions with the two end states of the protein control the thermodynamics of protein denaturation. Among the physico-chemical properties of proteins, there seems to be an interplay of the hydrophobic and electrostatic interactions that lead to an overall stabilizing effect. Increase in surface free energy of the solvent medium upon addition of the carboxylic acid salts appears to be the dominant factor in governing the thermal stability of proteins.
The cytochrome P-4501A1 (CYP1A1) gene is regulated by several trans-acting factors including the 4 S polycyclic aromatic hydrocarbon (PAH)-binding protein, which has recently been identified as glycine N-methyltransferase (GNMT) (Raha, A., Wagner, C., Macdonald, R. G., and Bresnick, E. (1994) J. Biol. Chem. 269, 5750-5756). The role of GNMT as a 4 S PAH-binding protein in mediating the induction of cytochrome P-4501A1 has been investigated further. GNMT cDNA, which was cloned into a pMAMneo vector containing the Rous sarcoma virus promoter and the neomycin resistance gene, was stably transfected into D422 Chinese hamster ovary (CHO) cells. Several positive clones were selected by reverse transcription-polymerase chain reaction and assayed for the expression of recombinant protein. Western blot analysis indicated the expression of significant levels of the 4 S protein in the stably transfected CHO cells (CHO-GNMT). Cytosolic preparations from the CHO-GNMT showed high benzo[a]pyrene (B[a]P) binding but no 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) binding activity when compared with clones transfected with the pMAMneo vector alone (CHO-neo) or the parental CHO cells. Challanging the CHO-GNMT cells with 4 microM B[a]P resulted in elevated levels of CYP1A1 mRNA. Equally effective in inducing CYP1A1 mRNA were benzo[e]pyrene and 3-methylcholanthrene. On the other hand, TCDD did not induce CYP1A1 gene expression in these cells. B[a]P-treated CHO-GNMT, expressing the 4 S protein, also showed CYP1A1 protein by Western blotting and exhibited ethoxyresorufin-O-deethylase activity; neither the CHO-neo or parental CHO cells were positive for any of these measures. No Ah receptor message or protein was detectable in the parental CHO, CHO-neo, or CHO-GNMT cells. Furthermore, no XRE binding activity was observed in TCDD-treated cytosolic preparations or nuclear extracts from CHO-GNMT cells that were treated with TCDD. These studies unequivocally establish that GNMT is a PAH-binding protein that can mediate the induction of CYP1A1 by PAHs such as B[a]P through an Ah receptor-independent pathway.
In the rat, cytochrome P4501A1 gene expression is thought to be regulated by several trans-acting factors including the 4S polycyclic aromatic hydrocarbon (PAH)-binding protein, which was recently identified as glycine N-methyltransferase [Raha, A., Wagner, C., MacDonald, R. G., and Bresnick, E. (1994) J. Biol. Chem. 268, 5750-5756]. Glycine N-methyltransferase (GNMT) is one of those unique proteins which exhibit diversity in function. Different subunit configurations are involved in its enzymatic role as a methyltransferase and as PAH-binding receptor. Here we report a systematic study of the oligomeric state of GNMT in the presence of benzo[a]pyrene (B[a]P) in vivo and in vitro. We have used chemical cross-linking and denaturing polyacrylamide gel electrophoresis to show that the B[a]P-binding unit of GNMT is a homodimer. We recently reported that phosphorylation is involved in the interaction of B[a]P with the 4S PAH-binding protein [Bhat, R., Weaver, J. A., Wagner, C., Bodwell, J. E., and Bresnick, E. (1996) J. Biol. Chem. 271, 32551-32556]. In the present study, this observation has been amplified by using bacterially expressed GNMT, which was post-translationally modified by a reticulocyte lysate and ATP-generating system. This modification was also accompanied by the formation of homodimers in the presence of B[a]P. These results indicate that post-translational modification is involved in determining the final configuration, i.e., dimeric form, of GNMT which then acts as a PAH-binding receptor.