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Biomedical subjects

S Bhattacharya

Publications and source records attributed to S Bhattacharya.

At least 181 records · Page 10Linked to original sources
PubMed1999

Functional compensation by particular nucleotide substitutions of a critical G*U wobble base-pair during aminoacylation of transfer RNA.

Expression of the genetic code depends on precise tRNA aminoacylation by cognate aminoacyl-tRNA synthetase enzymes. The G.U wobble base-pair in the acceptor helix of Escherichia coli alanine tRNA is the primary aminoacylation determinant of this molecule. Previous work on the process of synthetase recognition of the G.U pair showed that replacing G.U by a G.C Watson-Crick base-pair inactivates alanine acceptance by the tRNA, but that C.A and G.A wobble pair replacements preserve acceptance. Work by another group reported that the effects of a G.C replacement were reversed by a distal wobble base-pair in the anticodon helix. This result is potentially interesting because it suggests that distant regions in alanine tRNA are functionally coupled during synthetase recognition and more generally because recognition determinants of many other tRNAs lie in both the acceptor helix and anticodon helix region. Here, we have conducted an extensive in vivo analysis of the distal wobble pair in alanine tRNA and report that it does not behave like a compensating mutation. Restoration of alanine acceptance was not detected even when the synthetase enzyme was overproduced. We discuss the previous experimental evidence and suggest how the distal wobble pair was incorrectly analyzed. The available data indicate that all principal recognition determinants of alanine tRNA lie in the molecule's acceptor helix.

Acylation↗
PubMed1999

Backbone dynamics of apocytochrome b5 in its native, partially folded state.

The backbone dynamics in the native state of apocytochrome b5 were studied using 15N nuclear magnetic spin relaxation measurements. The field (11.7 and 14.1 T) and temperature (10-25 degrees C) dependence of the relaxation parameters (R1, R2, and R1rho) and the 1H-15N NOE established that the protein undergoes multiple time scale internal motions related to the secondary structure. The relaxation data were analyzed with the reduced spectral density mapping approach and within the extended model-free framework. The apoprotein was confirmed to contain a disordered heme-binding loop of approximately 30 residues with dynamics on the sub-nanosecond time scale (0.6 < S2 < 0.7, 100 ps < taue < 500 ps). This loop is attached to a structured hydrophobic core, rigid on the picosecond time scale (S2 > 0.75, taue < 50 ps). The inability to fit the data for several residues with the model-free protocol revealed the presence of correlated motion. An exchange contribution was detected in the transverse relaxation rate (R2) of all residues. The differential temperature response of R2 along the backbone supported slower exchange rates for residues in the loop (tauex > 300 micros) than for the folded polypeptide chain (tauex < 150 micros). The distribution of the reduced spectral densities at the 1H and 15N frequencies followed the dynamic trend and predicted the slowing of the internal motions at 10 degrees C. Comparison of the dynamics with those of the holoprotein [Dangi, B., Sarma, S., Yan, C., Banville, D. L., and Guiles, R. D. (1998) Biochemistry 37, 8289-8302] demonstrated that binding of the heme alters the time scale of motions both in the heme-binding loop and in the structured hydrophobic core.

Animals↗
PubMed1999

Functional role of p35srj, a novel p300/CBP binding protein, during transactivation by HIF-1.

Recruitment of p300/CBP by the hypoxia-inducible factor, HIF-1, is essential for the transcriptional response to hypoxia and requires an interaction between the p300/CBP CH1 region and HIF-1alpha. A new p300-CH1 interacting protein, p35srj, has been identified and cloned. p35srj is an alternatively spliced isoform of MRG1, a human protein of unknown function. Virtually all endogenous p35srj is bound to p300/CBP in vivo, and it inhibits HIF-1 transactivation by blocking the HIF-1alpha/p300 CH1 interaction. p35srj did not affect transactivation by transcription factors that bind p300/CBP outside the CH1 region. Endogenous p35srj is up-regulated markedly by the HIF-1 activators hypoxia or deferoxamine, suggesting that it could operate in a negative-feedback loop. In keeping with this notion, a p300 CH1 mutant domain, defective in HIF-1 but not p35srj binding, enhanced endogenous HIF-1 function. In hypoxic cells, p35srj may regulate HIF-1 transactivation by controlling access of HIF-1alpha to p300/CBP, and may keep a significant portion of p300/CBP available for interaction with other transcription factors by partially sequestering and functionally compartmentalizing cellular p300/CBP.

Alternative Splicing↗
PubMed1999

Evolution of genetic abnormalities in hepatocellular carcinomas demonstrated by DNA fingerprinting.

Hepatocellular carcinomas (HCC) often contain subpopulations of cells showing heterogeneous differentiation within each tumour. The majority of HCCs first appear as well-differentiated lesions and proliferate with gradual dedifferentiation. The present study was designed to investigate the clonal diversity which is seen with progression in neoplasms. The degree of genomic heterogeneity of HCC nodules was assessed using the arbitrarily primed-polymerase chain reaction technique. Two or more sectors of 31 HCC nodules were needle-microdissected and amplified with two different arbitrary primers in appropriate conditions. In every HCC less than 6 mm in diameter (n=18, range 3-6 mm, mean diameter 4.7 mm), all sectors of each of these lesions had the same DNA fingerprint. All HCC nodules greater than 6 mm diameter (n=13, range 7-30 mm, mean diameter 15.4 mm) showed distinct DNA fingerprints in each sector sampled (p< 0. 05, compared with size less than 6 mm in diameter). When synchronous HCCs were present, no two tumour nodules had the same DNA fingerprint. These results suggest that a process of clonal evolution occurs in expanding HCC, with neoplasms more than 6 mm in diameter developing as multiple clones. The advent of laser capture microdissection technology makes such analysis much more rapid and easily applied. Studies of clonality in HCCs, including borderline cases, are made possible by the combination of these novel techniques.

Carcinoma, Hepatocellular↗
PubMed1999

Thyroid hormone regulation of perch ovarian 3beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase activity: involvement of a 52-kDa protein.

Ovarian follicles were collected from perch belonging to the prespawning (vitellogenic) stage and incubated in vitro for 5 h in the absence (control) and presence of 3, 5, 3'-triiodothyronine (T3). Addition of increasing concentrations of T3 from 12.5 to 100 ng/ml caused a linear increase of 3beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase (3 beta-HSD) activity to 50 ng and then it leveled off indicating a saturation of enzyme activity with 50 ng T3. T3 stimulation of 3beta-HSD activity could be blocked by cycloheximide indicating the involvement of T3-induced protein (TIP) isolated and purified earlier from this laboratory. Addition of fish TIP purified from perch ovarian follicle (fTIP) or rat granulosa cell TIP to ovarian follicular incubation at a dose of 5 microg/ml significantly increased (P < 0.01) 3beta-HSD activity. To observe whether TIP acts directly on the enzyme or not, 3beta-HSD from perch ovarian follicle was purified to homogeneity by the following steps: (i) Sephadex G 75 gel filtration, (ii) DEAE-Sephacel chromatography, and (iii) NAD-affinity column chromatography. Purified 3beta-HSD gave a clear single band on an SDS gel and its molecular weight is 45 kDa. Addition of fTIP to an assay mixture containing purified 3beta-HSD resulted in a fourfold increase of the enzyme activity. fTIP alone did not show enzyme activity when incubated with the radiolabeled substrate. Addition of T3 (50 ng) to the 3beta-HSD assay mixture had no effect on the enzyme activity. Determination of Vmax and Km of the purified enzyme in the absence (control) and presence of fTIP demonstrated a considerable increase of 3beta-HSD affinity and rate of enzyme reaction.

Animals↗
PubMed1999

Lack of a chromosomal copy of the circular rDNA plasmid of Entamoeba histolytica.

A number of small circular DNAs constitute a part of the genome of Entamoeba histolytica. Among them, the 24.5 kb circular DNA encoding rRNA (EhR1) is the most abundant. Pulsed field gel electrophoresis was used to determine if a chromosomal copy of EhR1 exists and what fraction of the total genome is circular. The results show that the chromosomes of E. histolytica are linear, and that no copy of EhR1 could be detected in any of the linear chromosomes.

Animals↗
PubMed1999

Stimulation of nitric oxide synthesis and protective role of insulin in acute thrombosis in vivo.

Administration of physiologic amounts of insulin in mice (200 microunits/g body weight) resulted in 9 fold increase of basal nitric oxide level from 0.51+/-0.1224 nmol/ml (mean+/-SD, n=12) to 4.45+/-0.645 nmol/ml after 30min of the injection of the hormone. Since NO is a potent inhibitor of platelet aggregation both in vitro and in vivo, we tested the possibility whether the administration of the hormone would result in the in vivo inhibition of thrombosis through the increase of NO level in the circulation. It was found that administration of insulin (200 microunits/g body weight) in mice protected >90%(p<0.00001, n=500) of these animals from death due to thrombosis in the coronary arteries induced by ADP injection in the heart. This effect of insulin in vivo was found to be directly related to the hormone induced increase of NO level in the system. The thromboprotective effect of insulin could not be achieved by using either prostacyclin, a well known antithrombotic agent or its stable probe prostaglandin E1 instead of insulin. The efficacy of insulin was neither related to the blood glucose level nor was the consequence of the hypoglycemic effect of the hormone. In contrast, inhibition of insulin induced increase of NO level resulted in the complete loss of the thromboprotective effect of the hormone. These results suggest that insulin besides being a hypoglycemic hormone could also be a potent antithrombotic humoral factor.

Adenosine Diphosphate↗
PubMed1999

Lens biology: development and human cataractogenesis.

Cataract, or opacification of the lens of the eye, is the commonest cause of visual impairment world-wide. It is only treatable at present by surgical removal. Recent advances in our understanding of the genetics of human cataract, in particular the inherited congenital form, together with the development of an array of animal models have provided valuable new insights into normal vertebrate lens biology and the mechanisms that underlie cataract formation. In this article, we review the current state of research in these areas and discuss thinking regarding the relationship between the phenotypes observed and the underlying genotype in inherited cataract.

Animals↗
PubMed1999

Differential regulation of Leydig cell 3beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase activity by gonadotropin and thyroid hormone in a freshwater perch, Anabas testudineus (Bloch).

Leydig cells were isolated from the perch testes belonging to the pre-spawning stage by collagenase treatment and mechanical separation followed by percoll gradient. They were incubated in vitro either for 5 h or at different times in the absence (control) or presence of piscine gonadotropin (GTH, 2 microg (1 x 10(6) cells)(-1)) or 3,5,3'-triiodothyronine (T3, 50 ng (1 x 10(6) cells(-1)) or T3-induced protein (TIP, 2 microg (1 x 10(6) cells)(-1)). 3Beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase (3beta-HSD) activity was determined by the conversion of [3H]delta5-dehydroepiandrosterone (DHEA) to [3H]delta4-androstenedione or [3H]delta5-pregnenolone to [3H]delta4-progesterone (P4) or by spectrophotometric estimation of NADH formation from NAD. T3 significantly increased (P < 0.01) both delta5-DHEA to delta4-androstenedione and delta5-pregnenolone to delta4-P4 conversion in Leydig cells indicating stimulation of 3beta-HSD activity. T3 stimulation of 3beta-HSD activity could be inhibited by cycloheximide (50 microg ml(-1)) suggesting the involvement of T3-induced protein (TIP) which was isolated and purified earlier in this laboratory from goat Leydig cells [15]. Addition of TIP or GTH significantly stimulated Leydig cell 3beta-HSD activity (P < 0.01). However, there was a difference between TIP and GTH stimulation in time kinetic study where TIP enhanced 3beta-HSD activity at 1 h (P < 0.05), reached its peak at 3 h (P < 0.01) and then plateaued till 8 h. GTH, on the other hand, did not show any stimulation of 3beta-HSD activity for 2 h, stimulation was marked only at 3 h (P < 0.05), reached a peak at 6 h (P < 0.01) and then leveled off. Determination of Km and Vmax of the enzyme showed an increase in the velocity of reaction by GTH with unaltered Km. TIP increased both velocity and affinity of the enzyme. GTH significantly increased the synthesis of 3beta-HSD protein at 3 h (P < 0.01) reaching maximal stimulation at 6 h which clearly coincided with the enzyme activity. In contrast, TIP had no effect on 3beta-HSD protein synthesis, but its direct addition to 3beta-HSD enzyme preparation in vitro caused significant augmentation of the enzyme activity (P < 0.01) suggesting thereby its modulatory effect on the enzyme. Results, therefore, show that although both T3 and GTH stimulated perch testicular Leydig cell 3beta-HSD activity, T3 effect was not direct but mediated via TIP and there is a clear distinction between GTH and TIP stimulation. GTH increased the enzyme activity by stimulating 3beta-HSD protein synthesis while TIP acts directly on the enzyme modulating it from less active to more active state.

Animals↗
PubMed1999

A proton magnetic resonance spectroscopy study of the striatum and cerebral cortex in Parkinson's disease.

Animal studies have suggested an increased striatal glutamate activity in Parkinson's disease models, although this has not been substantiated in magnetic resonance spectroscopy studies in patients. Our initial aim was to assess glutamate and glutamine levels in the striatum of patients with idiopathic Parkinson's disease, using multivoxel proton magnetic resonance spectroscopy techniques. Since data were collected from other areas of the brain without a priori selection, information on the cortex was also obtained. Twelve healthy volunteers, seven dyskinetic and five non-dyskinetic patients were studied. Peak area ratios of choline-containing compounds (Cho), glutamine and glutamate (Glx) and N-acetyl moieties including N-acetylaspartate (NAx), relative to creatine (Cr) were calculated. Spectra were analysed from the corpus striatum, the occipital cortex and the temporo-parietal cortex. The median Glx/Cr ratio was unaltered in the striatal spectra of Parkinson's disease patients compared to healthy controls. However, the more severely affected patients had significantly reduced NAx/Cr ratios in spectra localised to the temporo-parietal cortex, compared to healthy controls. Furthermore, the entire patient population had significantly reduced Cho/Cr ratios in spectra from the temporo-parietal cortex, compared to the reference population. We found no evidence of increased striatal glutamate in either dyskinetic or non-dyskinetic Parkinson's disease. However, the low NAx/Cr and Cho/Cr ratios in the temporo-parietal cortex may indicate the presence of subclinical cortical dysfunction.

Aged↗
PubMed1999

Connexin46 mutations in autosomal dominant congenital cataract.

Loci for autosomal dominant "zonular pulverulent" cataract have been mapped to chromosomes 1q (CZP1) and 13q (CZP3). Here we report genetic refinement of the CZP3 locus and identify underlying mutations in the gene for gap-junction protein alpha-3 (GJA3), or connexin46 (Cx46). Linkage analysis gave a significantly positive two-point LOD score (Z) at marker D13S175 (maximum Z [Zmax]=>7.0; maximum recombination frequency [thetamax] =0). Haplotyping indicated that CZP3 probably lies in the genetic interval D13S1236-D13S175-D13S1316-cen-13pter, close to GJA3. Sequencing of a genomic clone isolated from the CZP3 candidate region identified an open reading frame coding for a protein of 435 amino acids (47,435 D) that shared approximately 88% homology with rat Cx46. Mutation analysis of GJA3 in two families with CZP3 detected distinct sequence changes that were not present in a panel of 105 normal, unrelated individuals. In family B, an A-->G transition resulted in an asparagine-to-serine substitution at codon 63 (N63S) and introduced a novel MwoI restriction site. In family E, insertion of a C at nucleotide 1137 (1137insC) introduced a novel BstXI site, causing a frameshift at codon 380. Restriction analysis confirmed that the novel MwoI and BstXI sites cosegregated with the disease in families B and E, respectively. This study identifies GJA3 as the sixth member of the connexin gene family to be implicated in human disease, and it highlights the physiological importance of gap-junction communication in the development of a transparent eye lens.

Amino Acid Sequence↗
PubMed1999

Detection of prostatic specific membrane antigen messenger RNA using immunobead reverse transcriptase polymerase chain reaction.

The present study was performed to detect circulating prostatic carcinoma (PC) cells using a novel three-step immunobead reverse transcriptase (RT) polymerase chain reaction (PCR) assay for prostatic specific membrane antigen (PSMA) messenger RNA (mRNA). The sensitivity and specificity of this technique was assessed and the incidence of immunobead RT-PCR positivity correlated with progressive metastatic disease and serum prostatic specific antigen (PSA) levels. Fifty peripheral blood (PB) samples from 46 patients with PC were incubated with magnetic beads coated with Ber-EP4 antibody directed against the human epithelial antigen a membrane antigen widely expressed by epithelial cells. The epithelial cell-enriched magnetic fraction was then subjected to mRNA isolation using oligo-deoxythymidine (dT) magnetic beads. Nested RT-PCR for PSMA was performed on the mRNA oligo-dT complex and the identity of the RT-PCR products was confirmed by Southern blotting. Twenty-one PB samples from 8 control subjects without PC were also evaluated. Three-step immunobead PSMA RT-PCR was able to detect one PC cell per 1 mL of PB. The positivity rate of the RT-PCR assay was significantly higher (11 of 25; 44%) in patients with metastatic tumor than in patients with non-metastatic disease (1 of 21; 5%) (P = 0.003). In patients with metastatic PC, RT-PCR positivity was much higher in patients with progressive disease (10 of 13; 77%) than in patients with responding or stable disease (1 of 12; 8%) (P = 0.001). There was a statistically significant correlation between immunobead PSMA PCR positivity and high levels of serum PSA (P = 0.005). All control subjects without PC tested negative for PSMA PCR. The three-step immunobead RT-PCR for PSMA can detect circulating PC cells with high specificity and sensitivity. Preliminary data show a strong correlation between immunobead PCR positivity, the presence of progressive metastatic disease, and high levels of serum PSA.

Antigens, Surface↗
PubMed1999

Review: polymerase chain reaction detection of micrometastases and circulating tumor cells: application to melanoma, prostate, and thyroid carcinomas.

The main strategy used for the detection of circulating tumor cells (CTC) and micrometastases in solid tumors is the polymerase chain reaction (PCR) amplification of tissue specific messenger RNA present in the tumor cells. PCR was more sensitive than conventional techniques, allowing the identification of one tumor cell diluted into 1 mL of blood. PCR was shown to be specific in most studies related to the detection of CTC and marrow micrometastases in melanoma and prostate carcinoma (PC). PCR positivity for thyroid markers was reported in the blood of control subjects. Large variations in the PCR positivity rates and the prognostic value of these assays have been encountered in PC and melanoma. There was a correlation between PCR and stage in some but not all the studies. Despite these discrepancies, many investigators have shown PCR to be predictive of outcome in PC and especially in melanoma. PCR in blood and bone marrow was an independent predictor of overall and disease-free survival in melanoma patients rendered surgically free of disease. These tests may help better stratify patients for radical surgeries and adjuvant therapy. Large prospective and interlaboratory studies are needed to confirm the accuracy and prognostic value of these assays.

Humans↗
PubMed1999

Conformational properties of native sperm whale apomyoglobin in solution.

Apomyoglobin from sperm whale is often used for studies of ligand binding, protein folding, and protein stability. In an effort to describe its conformational properties in solution, homonuclear and heteronuclear (13C and 15N) NMR methods were applied to the protein in its native state. Assignments were confirmed for nuclear Overhauser effects (NOEs) involving side chain and backbone protons in the folded regions of the structure. These NOEs were used to derive distance restraints. The shifts induced by the hydrophobic dye 8-anilino-1-naphthalenesulfonic acid (ANS) were inspected in the regions remote from its binding site and served as an indicator of conformational flexibility. 3JalphaH-NH values were obtained to assess dihedral angle averaging and to provide additional restraints. A family of structures was calculated with X-PLOR and an ab initio simulated annealing protocol using holomyoglobin as a template. Where the structure appeared well defined by chemical shift, line width, ANS perturbation, and density of NOEs, the low resolution model of apomyoglobin provides a valid approximation for the structure. The new model offers an improved representation of the folded regions of the protein, which encompass the A, B, E, helices as well as parts of the G and H helices. Regions that are less well defined at this stage of calculations include the CD corner and the end of the H-helix. The EF-F-FG segment remains uncharacterized.

Animals↗
PubMed1999

Clinical and genetic heterogeneity in autosomal dominant cataract.

AIMS: To determine the different morphologies of autosomal dominant cataract (ADC), assess the intra- and interfamilial variation in cataract morphology, and undertake a genetic linkage study to identify loci for genes causing ADC and detect the underlying mutation. METHODS: Patients were recruited from the ocular genetic database at Moorfields Eye Hospital. All individuals underwent an eye examination with particular attention to the lens including anterior segment photography where possible. Blood samples were taken for DNA extraction and genetic linkage analysis was carried out using polymorphic microsatellite markers. RESULTS: 292 individuals from 16 large pedigrees with ADC were examined, of whom 161 were found to be affected. The cataract phenotypes could all be described as one of the eight following morphologies-anterior polar, posterior polar, nuclear, lamellar, coralliform, blue dot (cerulean), cortical, and pulverulent. The phenotypes varied in severity but the morphology was consistent within each pedigree, except in those affected by the pulverulent cataract, which showed considerable intrafamilial variation. Positive linkage was obtained in five families; in two families linkage was demonstrated to new loci and in three families linkage was demonstrated to previously described loci. In one of the families the underlying mutation was isolated. Exclusion data were obtained on five families. CONCLUSIONS: Although there is considerable clinical heterogeneity in ADC, the phenotype is usually consistent within families. There is extensive genetic heterogeneity and specific cataract phenotypes appear to be associated with mutations at more than one chromosome locus. In cases where the genetic mutation has been identified the molecular biology and clinical phenotype are closely associated.

Adolescent↗
PubMed1999

Genomic heterogeneity in synchronous hepatocellular carcinomas.

BACKGROUND: Hepatocellular carcinoma (HCC) arising in cirrhosis is frequently multifocal. Whether HCC develops monoclonally or multiclonally is an unresolved question. Of the multiple tumour nodules present in many patients, it has not been established whether the smaller lesions represent intrahepatic metastases or de novo cancers. AIMS: To assess the degree of genomic heterogeneity in synchronous HCCs in cirrhosis. METHODS: The arbitrarily primed polymerase chain reaction technique was utilised to compare the DNA fingerprint of HCCs and regenerative nodules (RNs) removed from cirrhotic explant livers. RESULTS: Polymorphic genomic heterogeneity was noted in 54 HCCs and 31 RNs microdissected. Even satellite nodules in close proximity within the same segment of the liver were found to have distinct genomic patterns. CONCLUSION: Such genomic heterogeneity in synchronous HCCs may explain poor patient survival after surgical resection. If the smaller tumours are de novo lesions rather than metastases (as these data suggest), then current concepts regarding liver resection as a curative treatment modality for HCC may require reassessment.

Autoradiography↗