Biomedical subjects

S Ghosh

Publications and source records attributed to S Ghosh.

At least 343 records · Page 19Linked to original sources

PubMed1999

Rational design and synthesis of a novel anti-leukemic agent targeting Bruton's tyrosine kinase (BTK), LFM-A13 [alpha-cyano-beta-hydroxy-beta-methyl-N-(2, 5-dibromophenyl)propenamide].

In a systematic effort to design potent inhibitors of the anti-apoptotic tyrosine kinase BTK (Bruton's tyrosine kinase) as anti-leukemic agents with apoptosis-promoting and chemosensitizing properties, we have constructed a three-dimensional homology model of the BTK kinase domain. Our modeling studies revealed a distinct rectangular binding pocket near the hinge region of the BTK kinase domain with Leu460, Tyr476, Arg525, and Asp539 residues occupying the corners of the rectangle. The dimensions of this rectangle are approximately 18 x 8 x 9 x 17 A, and the thickness of the pocket is approximately 7 A. Advanced docking procedures were employed for the rational design of leflunomide metabolite (LFM) analogs with a high likelihood to bind favorably to the catalytic site within the kinase domain of BTK. The lead compound LFM-A13, for which we calculated a Ki value of 1.4 microM, inhibited human BTK in vitro with an IC50 value of 17.2 +/- 0.8 microM. Similarly, LFM-A13 inhibited recombinant BTK expressed in a baculovirus expression vector system with an IC50 value of 2.5 microM. The energetically favorable position of LFM-A13 in the binding pocket is such that its aromatic ring is close to Tyr476, and its substituent group is sandwiched between residues Arg525 and Asp539. In addition, LFM-A13 is capable of favorable hydrogen bonding interactions with BTK via Asp539 and Arg525 residues. Besides its remarkable potency in BTK kinase assays, LFM-A13 was also discovered to be a highly specific inhibitor of BTK. Even at concentrations as high as 100 micrograms/ml (approximately 278 microM), this novel inhibitor did not affect the enzymatic activity of other protein tyrosine kinases, including JAK1, JAK3, HCK, epidermal growth factor receptor kinase, and insulin receptor kinase. In accordance with the anti-apoptotic function of BTK, treatment of BTK+ B-lineage leukemic cells with LFM-A13 enhanced their sensitivity to ceramide- or vincristine-induced apoptosis. To our knowledge, LFM-A13 is the first BTK-specific tyrosine kinase inhibitor and the first anti-leukemic agent targeting BTK.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed1999

Type 2 diabetes: evidence for linkage on chromosome 20 in 716 Finnish affected sib pairs.

We are conducting a genome scan at an average resolution of 10 centimorgans (cM) for type 2 diabetes susceptibility genes in 716 affected sib pairs from 477 Finnish families. To date, our best evidence for linkage is on chromosome 20 with potentially separable peaks located on both the long and short arms. The unweighted multipoint maximum logarithm of odds score (MLS) was 3.08 on 20p (location, chi = 19.5 cM) under an additive model, whereas the weighted MLS was 2.06 on 20q (chi = 57 cM, recurrence risk,lambda(s) = 1. 25, P = 0.009). Weighted logarithm of odds scores of 2.00 (chi = 69.5 cM, P = 0.010) and 1.92 (chi = 18.5 cM, P = 0.013) were also observed. Ordered subset analyses based on sibships with extreme mean values of diabetes-related quantitative traits yielded sets of families who contributed disproportionately to the peaks. Two-hour glucose levels in offspring of diabetic individuals gave a MLS of 2. 12 (P = 0.0018) at 9.5 cM. Evidence from this and other studies suggests at least two diabetes-susceptibility genes on chromosome 20. We have also screened the gene for maturity-onset diabetes of the young 1, hepatic nuclear factor 4-a (HNF-4alpha) in 64 affected sibships with evidence for high chromosomal sharing at its location on chromosome 20q. We found no evidence that sequence changes in this gene accounted for the linkage results we observed.

Adult↗

PubMed1999

Feasibility studies of large scale production of human anti-tetanus toxoid monoclonal antibodies.

The feasibility of large scale production of human anti-tetanus toxoid monoclonal antibody for therapeutic use was evaluated using a human heterohybridoma. The effects of duration of subculture, transition from static to agitated culture conditions and the level of serum concentration were studied. The level of antibody secreted by the clone decreased with increasing length of subculture and decreasing serum concentration. The clone exhibited heterogeneity in expression of surface IgG after 2 or 7 weeks of subculture in static culture conditions irrespective of the serum concentration. However, a prolonged duration of subculture (9 weeks) in 3% serum medium had an effect on the expression of surface IgG both in static and agitated culture conditions. With respect to total (surface and intracellular) IgG, two distinct cell populations were observed. On long term subculture (9 weeks) in low serum medium (3% FCS), there was a decrease in the population which was the high synthesizer. In addition, when these cells were cultivated in agitated spinner flasks, a defect in secretion of antibodies was observed. Thus a general fall in the amount of antibody in the supernatant of agitated cultures was due to decrease in antibody synthesis as well as the defect in secretion of antibodies.

Antibodies, Monoclonal↗

PubMed1999

On the application of phase relationships to complex structures. XXXVI: some experiments with a small protein without heavy atoms.

The direct-methods program MULTAN88 has been applied to a known protein, ribonuclease (RNAP1), containing 808 non-H atoms, including five S atoms, plus 83 ordered solvent water molecules. Phase sets with mean phase errors between 69 and 75 degrees were selected by modified figures of merit for trials with the full data at 1.17 A resolution and also with restricted data at 1.25 and 1.5 A resolution. These figures of merit had previously only been applied to protein structures containing heavy atoms, and this is the first demonstration of their usefulness with no heavy atom present. An initial set of 1091 phases from a 1.17 A trial was developed by an objective procedure to give the full structure with a residual of 0. 21, which agrees well with the published structure.

Crystallography, X-Ray↗

PubMed1999

The effect of phenotype variation on detection of linkage in the COGA data.

Error in phenotypic measurement can significantly compromise ability to detect linkage. We assessed the impact of introducing phenotypic measurement error on our ability to detect a quantitative trait locus in the Collaborative Study on the Genetics of Alcoholism (COGA) data. The impact of introducing three different types of errors was evaluated: 1) errors generated by sampling from a normal distribution; 2) errors generated by permuting phenotype values between subjects; and 3) errors generated by sampling from a uniform error distribution.

Alcoholism↗

PubMed1999

Identifying influential individuals in linkage analysis: application to a quantitative trait locus detected in the COGA data.

Once linkage is detected to a quantitative trait locus (QTL), the next step towards localizing the gene involved may be to identify those families, or individuals, in whom the putative mutations are segregating. In this paper, we describe a jackknife procedure for identifying individuals (and families) who contribute disproportionately to the linkage. Following initial detection of linkage to a QTL, the strategy involves sequentially removing each individual (or each family) from the analysis and recomputing the lod score associated with the linked region using data from all remaining subjects (or families). This procedure can be used to determine if particular observations have substantial impact on evidence for linkage. Identification of such observations may provide insights for further efforts to localize the QTL.

Alcoholism↗

PubMed1999

Application of an ordered subset analysis approach to the genetics of alcoholism.

For complex diseases, underlying etiologic heterogeneity may reduce power to detect linkage. Thus, methods to identify more homogeneous subgroups within a given sample in a linkage study may improve detection of putative susceptibility loci. In this study we describe an ordered subsetting approach that utilizes disease-related quantitative trait data to complement traditional linkage analysis. This approach uses family-based lod scores derived from the initial genome screen and a family-based descriptor of the trait of interest. The goal of the approach is to identify more homogeneous subgroups of the data by ranking families based on their quantitative trait data. Permutation testing is used to assess statistical significance. This approach can be adapted to a variety of linkage methods and may provide a means to dissect some of the underlying heterogeneity in complex disease genetics.

Alcoholism↗

PubMed1999

Methionine synthase: high-resolution mapping of the human gene and evaluation as a candidate locus for neural tube defects.

Periconceptual folate supplementation has been found to prevent the occurrence of many neural tube defects (NTDs). Consequently, genetic variation in folate metabolism genes is expected to contribute to the risk for neural tube defects. Methionine synthase catalyzes the vitamin B(12)-dependent conversion of homocysteine and 5-methyltetrahydrofolate to methionine and tetrahydrofolate. The observation that homocysteine and vitamin B(12) levels are independent predictors of NTD risk suggested that methionine synthase could be a candidate gene for NTDs. To assess the role of the MS gene in NTDs, we performed high-resolution physical mapping of the MS locus, isolated highly polymorphic markers linked to the MS gene, and tested for an association between specific MS alleles and NTDs. We mapped the MS gene to a position between 909 and 913 cR(10000) on chromosome 1 by radiation hybrid mapping. Polymorphic markers D1S1567 and D1S1568 map to locations no more than 900 and 194 kb from the MS gene, respectively. The segregation of these polymorphic markers was measured in 85 Irish NTD families. No allele of either marker showed a significant association with NTDs using the transmission disequilibrium test. A lack of association was also observed for the D1919G missense mutation within the gene. Our results suggest that inherited variation in the MS gene does not contribute to NTD risk in this population.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1999

Interstitial brachytherapy in carcinoma of the penis.

AIM: Keeping in line with the increasing emphasis on organ preservation, we at the Tata Memorial Hospital have evaluated the role of Ir-192 interstitial implant as regards local control, functional and cosmetic outcome in early as well as locally recurrent carcinoma of the distal penis. PATIENTS AND METHODS: From October 1988 to December 1996, 23 patients with histopathologically proven cancer of the penis were treated with radical radiation therapy using Ir-192 temporary interstitial implant. Our patients were in the age group of 20 to 60 years. The primary lesions were T1 in 7, T2 in 7 and recurrent in 9 patients. Only 7 patients had palpable groin nodes at presentation, all of which were pathologically negative. The median dose of implant was 50 Gy (range 40 to 60 Gy), using the LDR afterloading system and the Paris system of implant rules for dosimetry. Follow-up ranged from 4 to 117 months (median 24 months). RESULTS: At last follow-up 18 of the 23 patients remained locally controlled with implant alone. Three patients failed only locally, 2 locoregionally and 1 only at the groin. Of the 5 patients who failed locally, 4 were successfully salvaged with partial penectomy and remained controlled when last seen. Local control with implant alone at 8 years was 70% by life table analysis. The patients had excellent functional and cosmetic outcome. We did not record any case of skin or soft-tissue necrosis. Only 2 patients developed meatal stenosis, both of which were treated endoscopically. CONCLUSION: Our results lead us to interpret that interstitial brachytherapy with Ir-192 offers excellent local control rates with preservation of organ and function. Penectomy can be reserved as a means for effective salvage.

Adult↗

PubMed1999

Regulation of inducible gene expression by the transcription factor NF-kappaB.

The transcription factor NF-kappaB plays a critical role in regulating inducible gene expression in immune responses. The activation of NF-kappaB is regulated at multiple levels, probably reflecting a need to maintain a tight control of its activity. We have recently discovered that direct phosphorylation of NF-kappaB itself is essential for its transcriptional activity and that phosphorylation acts as a switch to determine association of nuclear NF-kappaB with histone acetylases vs deacetylases.

Animals↗

PubMed1999

The W64R variant of the beta3-adrenergic receptor is not associated with type II diabetes or obesity in a large Finnish sample.

Recent studies have suggested an association between Type II (non-insulin-dependent) diabetes mellitus-related phenotypes and a cytosine-to-thymidine substitution that results in the replacement of tryptophan by arginine at codon 64 (Trp64Arg or W64R) of the beta3-adrenergic receptor gene. Here, we present the results of possibly the largest association study to date on the variant in a sample of 526 families with a total of 1725 subjects, 1053 of whom had Type II diabetes. Preliminary calculations suggested that we had excellent power to detect the moderate associations which were reported in previous studies. No associations were found between the W64R variant and the following phenotypes in our sample: Type II diabetes, age at diagnosis for Type II diabetes, measures of obesity, fasting glucose, fasting insulin, minimal model variables, and systolic and diastolic blood pressures. In the analysis of plasma lipids, we detected an association between the variant and HDL ratios (HDL cholesterol/total cholesterol) (p = 0.013), which remained significant even after adjusting for sex, affection status and age. Since W64R homozygotes (n = 11) had the highest HDL ratios, however, heterozygotes had the lowest and the wild-type subjects had intermediate values, we conclude that the W64R variant is unlikely to reduce HDL ratios in a dose-dependent, pathogenic manner.

Adult↗

PubMed1999

Facilitation of neuronal activity in somatosensory and posterior parietal cortex during prehension.

In order to study prehension in a reproducible manner, we trained monkeys to perform a task in which rectangular, spherical, and cylindrical objects were grasped, lifted, held, and lowered in response to visual cues. The animal's hand movements were monitored using digital video, together with simultaneously recorded spike trains of neurons in primary somatosensory cortex (S-I) and posterior parietal cortex (PPC). Statistically significant task-related modulation of activity occurred in 78% of neurons tested in the hand area; twice as many cells were facilitated during object acquisition as were depressed. Cortical neurons receiving inputs from tactile receptors in glabrous skin of the fingers and palm, hairy skin of the hand dorsum, or deep receptors in muscles and joints of the hand modulated their firing rates during prehension in consistent and reproducible patterns. Spike trains of individual neurons differed in duration and amplitude of firing, the particular hand behavior(s) monitored, and their sensitivity to the shape of the grasped object. Neurons were classified by statistical analysis into groups whose spike trains were tuned to single task stages, spanned two successive stages, or were multiaction. The classes were not uniformly distributed in specific cytoarchitectonic fields, nor among particular somatosensory modalities. Sequential deformation of parts of the hand as the task progressed was reflected in successive responses of different members of this population. The earliest activity occurred in PPC, where 28% of neurons increased firing prior to hand contact with objects; such neurons may participate in anticipatory motor control programs. Activity shifted rostrally to S-I as the hand contacted the object and manipulated it. The shape of the grasped object had the strongest influence on PPC cells. The results suggest that parietal neurons monitor hand actions during prehension, as well as the physical properties of the grasped object, by shifting activity between populations responsive to hand shaping, grasping, and manipulatory behaviors.

Animals↗

PubMed1999

Identification of a novel inflammation-protective locus in the Fischer rat.

Inbred LEW/N rats are relatively susceptible, while histocompatible inbred F344/N rats are relatively resistant to development of a wide variety of inflammatory diseases in response to a range of pro-inflammatory stimuli. In a LEW/N vs. F344/N F2 intercross, we identified a quantitative trait locus (QTL) on Chr 10 that protects in a dominant fashion against the exudate volume component of innate inflammation in the F344/N rat, as well as a suggestive QTL on Chr 2 near the Fibrinogen cluster region. The exudate volume linkage region on Chr 10 may be similar to one of the multiple regions found to link to inflammatory arthritis phenotypes in other crosses. The suggestive linkage on Chr 2 has not been previously reported and does not seem to contribute to this phenotype in the same manner as the QTL on Chr 10. These findings are consistent with the hypothesis that the innate exudate volume trait is a sub-phenotype of more complex inflammatory phenotypes, such as arthritis, and genes within the Chr 10 linkage region could account for differences in this non-specific acute phase component of the inflammatory response. Since the rat Chr 10 exudate volume linkage region we have identified is syntenic with a region of human Chr 17 that has been shown to link to a variety of autoimmune/inflammatory diseases, including insulin-dependent diabetes mellitus, multiple sclerosis, and psoriasis, identification of genes within this linkage region will shed light on genes relevant to the earliest inflammatory component and to susceptibility and resistance to such human autoimmune/inflammatory diseases.

Animals↗

PubMed1999

Dietary effects of conjugated octadecatrienoic fatty acid (9 cis, 11 trans, 13 trans) levels on blood lipids and nonenzymatic in vitro lipid peroxidation in rats.

The present study examined the antioxidant activity of conjugated octadecatrienoic fatty acid (9 cis,11 trans,13 trans-18:3), alpha-eleostearic acid, of karela seed (Momordica charantia), fed to rats for 4 wk. The growth pattern of rats and the effect on plasma cholesterol and high density lipoprotein (HDL) cholesterol and peroxidation of plasma lipid, lipoprotein, eryhrocyte membrane, and liver lipid were measured. Rats were raised on diets containing sunflower oil mixed with three different levels of conjugated trienoic fatty acid (9c,11t,3t-18:3) 0.5, 2, and 10% by weight; the control group was raised with sunflower oil as dietary oil as the source of linoleic acid (9c,12c-18:2). The growth pattern of the three experimental groups of rats showed no significant difference compared to the control group of rats, but the group with 10% 9c,11t,13t-18:3 had slightly higher body weight than the control group of rats. Concentrations of total cholesterol, HDL-cholesterol, and non-HDL-cholesterol in plasma were similar in all four groups. Plasma lipid peroxidation was significantly lower in the case of 0.5% 9c,11t,13t-18:3 group than the control group and the 2 and 10% 9c,11t,13t-18:3 dietary groups as well. Lipoprotein oxidation susceptibility test with 0.5, 2, and 10% 9c,11t,13t-18:3 dietary groups was significantly less susceptible to lipoprotein peroxidation when compared with sunflower oil dietary group, and the dietary group with 0.5% 9c,11t,13t-18:3 showed least susceptibility. There was significant lowering in erythrocyte ghost membrane lipid peroxidation in the 0.5, 2, and 10% 9c,11t,13t-18:3 dietary groups compared to the sunflower oil groups. Nonenzymatic liver tissue lipid peroxidation was significantly lower in the group of rats raised on 0.5% 9c,11t,13t-18:3, but the groups on 2 and 10% 9c,11t,13t-18:3 acid did not show any significant difference compared with the control group of rats.

Animals↗

PubMed1999

A combination of ciprofloxacin and Rowachol does not prevent biliary stent occlusion.

BACKGROUND: Endoscopic insertion of biliary prostheses is now an established palliative treatment for frail and elderly patients with distal extrahepatic malignant biliary strictures who are unable to tolerate major surgery. The major limitation to long-term biliary stenting is late stent occlusion caused by bacterial adherence followed by encrustation with amorphous bacterial products. We studied the effect on the duration of stent patency of combination therapy with ciprofloxacin and Rowachol, a choleretic agent, in a group of patients who underwent stent insertion for extrahepatic malignant biliary strictures. METHODS: Patients with this diagnosis were randomized to either active treatment with ciprofloxacin (500 mg twice daily) and Rowachol (2 tablets 3 times daily) or no drug treatment (control group). They were followed up prospectively and the end points of the study were stent occlusion and patient survival. RESULTS: Forty-eight patients who had successful stent placement were recruited; 8 patients were excluded from the final analysis because of death in less than 1 month (5 patients) or stent replacement within a month because of persistent jaundice (3 patients). Twenty patients were randomized to the active and control groups. One patient in the active group withdrew because of nausea. The 2 groups were comparable with regard to age, gender, causes of extrahepatic malignant biliary strictures, and levels of serum alkaline phosphatase and bilirubin. There were significant reductions in mean serum bilirubin and alkaline phosphatase concentrations 1 month after stent placement. At the time of analysis, 12 patients were still alive (7 patients from the control group). Median survival was 23 weeks in both the active treatment group and the control group. There were 9 episodes (45%) of stent occlusions in the active treatment group and 10 episodes (50%) in the control group. The median time to stent occlusion was 23 (range 7 to 68) weeks for the active treatment group and 21 (range 6 to 56) weeks for the control group, p = 0.23. There was no significant difference in the duration of patient survival and of stent patency between the 2 groups. The lack of difference between treated and untreated patients in survival duration and stent patency was also shown if the results were analyzed on an intent-to-treat basis and the 8 patients who were excluded in the final analysis were also analyzed. CONCLUSIONS: Prophylactic therapy with ciprofloxacin and Rowachol does not prolong stent patency or patient survival.

Aged↗

PubMed1999

Cross-bred cattle protected against Hyalomma anatolicum anatolicum by larval antigens purified by immunoaffinity chromatography.

Antigens from larvae of Hyalomma anatolicum anatolicum were extracted and purified by immunoaffinity chromatography using immunoglobulin ligands from cross-bred animals immunized with soluble larval antigen. Affinity-purified antigen (Aff-TLE) and a total larval extract (TLE) were used to immunize cross-bred (Bos indicus x Bos taurus) cattle. The group immunized with Aff-TLE rejected 71.6% of larvae and 77.3% of nymphs. However, the rejection percentages were lower in the TLE-immunized group. No significant changes in the feeding period, moulting percentages or moulting period of engorged larvae and nymphs were recorded. There was, however, a significant decrease in the number of resultant nymphs p < 0.01) and adults (p < 0.01) in the ticks fed on the Aff-TLE-immunized group. The Aff-TLE antigen was 93.3% purified. SDS-PAGE analysis identified a 39 kDa protein, reported for the first time, as the antigen responsible for the induction of resistance in the host.

Animals↗

PubMed1999

TNF-alpha induced altered signaling mechanism in human neutrophil.

In the present study we investigated the TNF-alpha induced signal transduction mechanism in human neutrophil. Exogenously added TNF-alpha affects both PKC activity and its translocation from cytosol to the membrane. Endogenous protein phosphorylation pattern is inhibited in TNF-alpha induced neutrophil in Ca-dependent and Ca-independent manner, including a major 47 and 66 kDa cytosolic proteins, which may be implicated in superoxide anion generation. However TNF-alpha dose dependently enhances the expression of zeta-PKC isotype but not the beta-PKC. Morphology and cell cytotoxicity are studied in TNF-alpha treated neutrophil to understand the TNF-alpha induced cell death or apoptosis and these experiment is further confirmed by DNA fragmentation analysis. These results clearly demonstrate that TNF-alpha induces cellular death of human neutrophil at least in part by enhanced expression of Ca-independent zeta-PKC. These observations provide an insight towards understanding the function of zeta-PKC in apoptotic pathway.

Apoptosis↗

PubMed1999

Mutational analysis of Raf-1 cysteine rich domain: requirement for a cluster of basic aminoacids for interaction with phosphatidylserine.

Activation of Raf-1 kinase is preceded by a translocation of Raf-1 to the plasma membrane in response to external stimuli. The membrane localization of Raf-1 is facilitated through its interaction with activated Ras and with membrane phospholipids. Previous evidence suggests that the interaction of Raf-1 with Ras is mediated by two distinct domains within the N-terminal region of Raf-1 comprising amino acid residues 51-131 and residues 139-184, the latter of which codes for a zinc containing cysteine-rich domain. The cysteine-rich domain of Raf-1 is also reported to associate with other proteins, such as 14-3-3, and for selectively binding acidic phospholipids, particularly phosphatidylserine (PS). In the present study, we have investigated the consequences of progressive deletions and point mutations within the cysteine-rich domain of Raf-1 on its ability to bind PS. A reduced interaction with PS was observed in vitro for all deletion mutants of Raf-1 expressed either as full-length proteins or as fragments containing the isolated cysteine-rich domain. In particular, the cluster of basic amino acids R143, K144, and K148 appeared to be critical for interaction with PS, since substitution of all three residues to alanine resulted in a protein that failed to interact with liposomes enriched for PS. Expression of Raf-1 in vivo, containing point mutations in the cysteine-rich domain resulted in a truncated polypeptide that lacked both the Ras and PS binding sites and could no longer translocate to the plasma membrane upon serum stimulation. These results indicate that the basic residues 143, 144 and 148 in the anterior half of Raf-1 cysteine-rich domain play a role in the association with the lipid bilayer and possibly in protein stability, therefore they might contribute to Raf-1 localization and subsequent activation.

Amino Acids↗