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Biomedical subjects

P Ghosh

Publications and source records attributed to P Ghosh.

At least 127 records · Page 7Linked to original sources
PubMed1998

Effect of chemical nutrients on aconitase activity during citric acid fermentation by a mutant strain of Aspergillus niger.

An ethyleneimine and UV irradiated mutant strain Aspergillus niger AB 1801 was explored for its aconitase activity, citric acid production and cell growth during citric acid fermentation. 10% sucrose and 0.25% urea served as the best carbon and nitrogen sources, respectively. Fe2+ stimulated aconitase activity and Co2+ (upto 5 micrograms/mL) and Ni2+ (10 micrograms/mL) strongly depressed enzyme activity. Boric acid (0.5 mg/L) had very good stimulatory response towards citric acid production. Aconitase activity has been found to vary inversely with the citric acid production upto a certain limit after which the activity was absent in the mycelial extract. Cell growth varied with the production parameter.

Aconitate Hydratase↗
PubMed1997

Interaction of NF-kappaB and NFAT with the interferon-gamma promoter.

Interferon-gamma (IFN-gamma) is a pleiotropic lymphokine whose production is restricted to activated T cells and NK cells. Along with other cytokines, IFN-gamma gene expression is inhibited by the immunosuppressant cyclosporin A. We have previously identified an intronic enhancer region (C3) of the IFN-gamma gene that binds the NF-kappaB protein c-Rel and that shows partial DNA sequence homology with the cyclosporin A-sensitive NFAT binding site and the 3'-half of the NF-kappaB consensus site. Sequence analysis of the IFN-gamma promoter revealed the presence of two additional C3-related elements (C3-1P and C3-3P). In addition, an NF-kappaB site (IFN-gamma kappaB) was identified within the promoter region. Based on this observation, we have analyzed the potential role of NF-kappaB and NFAT family members in regulating IFN-gamma transcription. Electrophoretic mobility shift assay analysis demonstrated that after T cell activation, the p50 and p65 NF-kappaB subunits bind specifically to the newly identified IFN-gamma kappaB and C3-related sites. In addition, we identified the NFAT proteins as a component of the inducible complexes that bind to the C3-3P site. Site-directed mutagenesis and transfection studies demonstrate that calcineurin-inducible transcriptional factors enhance the transcriptional activity of the IFN-gamma promoter through the cyclosporin-sensitive C3-3P site, whereas NF-kappaB proteins functionally interact with the C3-related sites. In addition, when located downstream to the beta-galactosidase gene driven by the IFN-gamma promoter, the intronic C3 site worked in concert with both the IFN-gamma kappaB and the C3-3P site to enhance gene transcription. These results demonstrate that the coordinate activities of NFAT and NF-kappaB proteins are involved in the molecular mechanisms controlling IFN-gamma gene transcription.

Base Sequence↗
PubMed1997

Purification and partial characterization of a cellular carotenoid-binding protein from ferret liver.

A cellular carotenoid-binding protein was purified to homogeneity from beta-carotene-fed ferret liver utilizing the following steps: ammonium sulfate precipitation, ion exchange, gel filtration, and affinity chromatography. The final purification was 607-fold. [14C]beta-Carotene co-purified with the binding protein throughout the purification procedures. SDS-PAGE of the purified protein showed a single band with an apparent molecular mass of 67 kDa. Scatchard analysis of the specific binding of the purified protein to beta-carotene showed two classes of binding sites, a high affinity site with an apparent Kd of 56 x 10(-9) M and a low affinity site with a Kd of 32 x 10(-6) M. The Bmax for beta-carotene binding to the high affinity site was 1 mol/mol, while that for the low affinity site was 145 mol/mol. The absorption spectrum of the complex showed a 32-nm bathochromic shift in lambdamax with minor peaks at 460 and 516 nm. Except for alpha-carotene and cryptoxanthin, none of the model carotenoids or retinol competed with beta-carotene binding to the protein. Thus, a specific carotenoid-binding protein of 67 kDa has been characterized in mammalian liver with a high degree of specificity for binding only carotenoids with at least one unsubstituted beta-ionone ring.

Animals↗
PubMed1997

Inhibitors of both nuclear factor-kappaB and activator protein-1 activation block the neoplastic transformation response.

Cross-coupling of active protein-1 (AP-1) and nuclear factor (NF)-kappaB has been reported. In the present study, we investigated the possibility that both of these two transcription factors might contribute to the process of tumor promoter-induced transformation. To establish a stable reporter cell system, two reporter genes were stably transfected into a JB6 mouse tumor promotion-sensitive (P+) cell line: a luciferase reporter controlled by a collagenase AP-1 sequence and a chloramphenicol acetyltransferase reporter controlled by an interleukin 6 NF-kappaB sequence. This double-reporter cell line maintained the phenotype of tumor promotion sensitivity and was able to report basal or induced AP-1 and NF-kappaB transactivation. The cytokine tumor promoter tumor necrosis factor (TNF)-alpha transactivated NF-kappaB and AP-1 for both DNA binding and transcriptional activity. Pyrrolidine dithiocarbamate, an antioxidant that acts as an NF-kappaB inhibitor, efficiently inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA) or TNF-alpha induced NF-kappaB as well as AP-1 transactivation and cell transformation, suggesting dependency of transformation on both transcription factors. The AP-1 transrepressing-retinoid SR11302 transrepressed AP-1 and cell transformation when these were TPA induced but not when TNF-alpha induced, indicating different signaling pathways for TNF-alpha and TPA. Supershift electrophoresis mobility shift assay revealed that Jun B and c-Jun were absent from the AP-1/DNA complex following TNF-alpha but present following TPA treatment. Together, these results suggest that both AP-1 and NF-kappaB activation may be required for transformation whether induced by TPA or by TNF, and the differential sensitivity of TPA and TNF-alpha-induced transformation to inhibition by a retinoid might be explained by differences in the composition of the DNA-bound AP-1 complexes.

Animals↗
PubMed1997

Sequential development of structural and functional alterations in T cells from tumor-bearing mice.

The TCR alpha beta or -gamma delta chains bind the peptide ligand, whereas the associated CD3 delta epsilon gamma and TCR zeta subunits couple the TCR to intracellular signal transduction components. Recently, several groups have described marked alterations in signal transduction elements in T cells from cancer patients or in mice bearing tumor for a few weeks (>26 days). The sequence in which these alterations develop is unknown. The aim of this study was to explore the kinetics of the development of alterations in signal transduction molecules (TCR zeta chain, NF kappaB family proteins, and tyrosine kinase p56(lck)) in mice bearing MC38 colon adenocarcinoma. The results demonstrate that alterations in NF kappaB family proteins, specifically the failure of p65 translocation to the nucleus, occur earlier and more frequently than the decrease in zeta-chain. These defects are paralleled by an impaired ability to produce Th1 cytokines (IL-2 and IFN-gamma). These initial changes are followed by the eventual loss of TCR zeta chain and p56(lck) and a marked decrease in cytotoxic function. An increased rate of lysosomal degradation is one of the mechanisms responsible for the loss of zeta-chain.

Adenocarcinoma↗
PubMed1997

Crystal structure of colicin Ia.

The ion-channel forming colicins A, B, E1, Ia, Ib and N all kill bacterial cells selectively by co-opting bacterial active-transport pathways and forming voltage-gated ion conducting channels across the plasma membrane of the target bacterium. The crystal structure of colicin Ia reveals a molecule 210 A long with three distinct functional domains arranged along a backbone of two extraordinarily long alpha-helices. A central domain at the bend of the hairpin-like structure mediates specific recognition and binding to an outer-membrane receptor. A second domain mediates translocation across the outer membrane via the TonB transport pathway; the TonB-box recognition element of colicin Ia is on one side of three 80 A-long helices arranged as a helical sheet. A third domain is made up of 10 alpha-helices which form a voltage-activated and voltage-gated ion conducting channel across the plasma membrane of the target cell. The two 160 A-long alpha-helices that link the receptor-binding domain to the other domains enable the colicin Ia molecule to span the periplasmic space and contact both the outer and plasma membranes simultaneously during function.

Biological Transport↗
PubMed1997

Identification of a Stat-6-responsive element in the promoter of the human interleukin-4 gene.

Interleukin (IL)-4 is an immunomodulatory cytokine produced by a number of cell types including T cells, basophils, and mast cells. This pleiotropic cytokine has a number of immunoregulatory functions; however, the molecular mechanisms controlling the transcription of this gene are not yet completely understood. Several studies have implicated a possible autoregulatory mechanism for its own expression. Here, we have identified a Stat-6-responsive element (Stat-6RE) in the promoter of the human IL-4 gene. Utilizing electrophoretic mobility shift analysis, we have demonstrated the presence of two specific IL-4-responsive DNA-protein complexes in nuclear extracts of both human Th1 and Th2 clones. Phytohemagglutinin-blasted peripheral blood T cells also generated an inducible complex in response to stimulation with IL-4 and the IL-4-like cytokine IL-13. Transient transfection of the murine pre-B cell line BA/F3 stably transfected with the full-length human IL-4 receptor alpha chain demonstrated the ability of multicopy Stat-6RE to initiate transcription from a heterologous promoter upon IL-4 or IL-13 stimulation. These results indicate a possible autocrine mechanism for the regulation of IL-4 gene transcription through the Stat-6RE as well as a possible mechanism for IL-13 regulation of the human IL-4 promoter.

Animals↗
PubMed1997

The serine proteinase inhibitory proteins of the chondrodystrophoid (beagle) and non-chondrodystrophoid (greyhound) canine intervertebral disc.

Trypsin inhibitory proteins of low buoyant density (p < or = 1.35 g/mL) fractions were prepared by CsCl density gradient ultracentrifugation of 4 M guanidinium hydrochloride extracts of lumbar beagle and greyhound annulus fibrosus and nucleus pulposus from animals aged 1 to 6 years. Affinity blotting with biotinylated trypsin was used to identify active trypsin inhibitory protein species; these species were also identified immunologically by Western blotting using antibodies against bovine pancreatic trypsin inhibitor (BPTI), and human inter-alpha-trypsin inhibitor (ITI). None of the trypsin inhibitory species evident in Western blots were reactive with anti-human alpha1-proteinase inhibitor (alpha-1-PI), alpha2-macroglobulin or secretory leucocyte proteinase inhibitor. The greyhound intervertebral disc samples generally had higher levels of active trypsin inhibitor species per unit weight of tissue extracted, and a more extensive range of inhibitor species. Inhibitor species of 30, 32, 34 kDa were identified in both beagle and greyhound intervertebral disc samples; these species were generally most prominent in the annulus fibrosus samples. In contrast, the nucleus pulposus samples contained relatively large trypsin inhibitor species; the anti-BPTI detected an inhibitor species of approximately 85-90 kDa; anti-ITI detected species of 120-250 kDa; biotinylated trypsin detected species of 60-110 kDa. A small molecular mass trypsin inhibitor species of 6 kDa, which was of similar mobility to BPTI, was also detected in annulus fibrosus samples; however, this species did not react with anti-BPTI.

Age of Onset↗
PubMed1997

Cholesterogenesis, lipogenesis, cholesterol degradation to bile acids, and histopathology of the liver in LA/N-cp obese rats.

Various lipid parameters were determined in lean control and LA/NIH-corpulent (LA/N-cp) rats, a normotensive strain showing metabolic characteristics associated with human Type IV hyperlipidemia. Hepatic and plasma total cholesterol, high density lipoproteins (HDL) cholesterol and triglycerides were significantly higher in the obese group than in the control group. Depending upon whether the data were expressed as per gram tissue or per organ, the rates of de novo fatty acid synthesis in the liver and adipose tissue were higher by 61% to 127% (P < .05) and 79% to 355% (P < .05), respectively, in the obese group compared with the lean control group. Similarly, hepatic rate of cholesterol synthesis was higher by 46% to 107% (P < .05) in the obese animals compared with the lean ones. In vivo hepatic rate of HDL2 cholesterol degradation to bile acids was lower in the obese group by 48% to 63% (P < .05). This was confirmed in the perfused liver in spite of the fact that cholesterol uptake from HDL2 was 3- to 4-fold higher in the obese group. These changes in lipid parameters of the obese animals were neither caused by hyperphagia because they were pair-fed with the control group nor caused by increased rate of food consumption because they were meal-fed. At the same time, all these lipid parameters were 17% to 20% higher in ad libitum-fed obese than in pair-fed obese group. Histopathological evaluation of the livers in the obese and control groups also showed prominent lipid droplets in the cytoplasm of the obese liver but not in the lean control liver. Thus, the possible causes of obesity in the LA/N-cp obese rats are higher synthetic rates of lipids coupled with lower rate of degradation of cholesterol to bile acids.

Animals↗
PubMed1997

Intercellular communication in rapidly proliferating and differentiated C6 glioma cells in culture.

Glial cells in the brain are known to provide structural and functional supports to neurons. To sustain such a supportive role, they have developed cell-to-cell communicating gap junctional channels. The authors studied the effect of dbcAMP on gap junctional channels mediated communication in C6 cells, a rat glioma cell line. Quantitative assessment of coupled cells under microscope after microinjection of a fluorescent dye was taken as a measure of junctional permeability. An enhanced coupling between cells was observed following dbcAMP treatment and this elevated coupling was found to be dependent on the duration of exposure of cells to dbcAMP. The studies have focused on a subtle shift in the spatial organization of the functional channels to the processes of dbcAMP induced differentiated cells from the cell cytoplasms and membranes of dbcAMP untreated cells. Immunofluorescence study with affinity purified antibody against gap junction further confirmed the spatial distribution of gap junctional protein(s) in the processes and also showed an increase in the density of the protein at the intercellular spaces in dbcAMP induced differentiated C6 glioma cells.

Animals↗
PubMed1997

Hyaluronan-phospholipid interactions.

Hyaluronan-phospholipid interactions have been studied in vitro by negative staining and rotary shadowing electron microscopy. Hyaluronan (HA) molecules of different molecular weights (around 170,000; 740,000, and 1.9 x 10(6) Da) were added to phospholipid suspensions (DPPC or egg lecithin) that were in the form of either unilamellar particles or multilamellar vesicles. Suspensions were then gently stirred and incubated at different temperatures from 24 hr up to 7 days. After 24 hr, at temperatures just above the melting point of the phospholipid used, both unilamellar particles and multilamellar vesicles were already shown to change their organization in the presence of HA, giving rise to the formation of (1) huge perforated membrane-like structures lying on the substrate; (2) 12-nm-thick "cylinders" (rollers) with a tendency to aggregate and to form sheets. These structures were seen only in the presence of high-molecular-weight HA, whereas low-molecular-weight HA (170 kDa) induced fragmentation of liposomes and formation of a few short rollers. These data show that phospholipids and HA interact and suggest they may also do so in vivo within the joint cavity, where both chemical species are present, giving rise to complexes which might exhibit peculiar lubricating and protective properties. It is also proposed that such interactions may not be as efficient in arthritic joints, where HA is degraded to low-molecular-weight fragments.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1997

Elevated synthesis of biglycan and decorin in an ovine annular lesion model of experimental disc degeneration.

The aim of this study was to extend our earlier observations on the changes that occur in the proteoglycans (PGs) of discs subjected to experimental injury to the annulus fibrosus (AF). We employed the alginate bead culture method to examine the metabolism of the dermatan sulphate (DS) containing PGs by cells derived from different regions of ovine discs that had been subjected to experimental annular injury. This was compared with the metabolism of the DS-PGs by cells isolated from equivalent regions of normal sham-operated discs. Six months after induction of the annular lesion, AF cells isolated from the lesion produced significantly higher levels of decorin and biglycan in alginate bead culture than did cells from equivalent zones of the controls. Decorin and biglycan were identified in culture media samples by immunoblotting, using specific antibodies (6-B-6, LF-96), and also by positive identification of their de-glycosylated core proteins. The core protein of the DS-PGs has been shown to inhibit type I/II collagen fibrillogenesis, to negatively regulate the action of transforming growth factor-beta (TGF-beta) and to diminish cellular proliferation in vitro; events which may be detrimental to tissue repair. The findings are therefore consistent with our previous observation the annular lesions in the avascular inner annulus have no capacity to heal.

Animals↗
PubMed1997

Use of an improved internal-standard method in the quantitative sterol analyses of phytoplankton and oysters.

Most work reporting the sterol composition of living organisms has not been done quantitatively, although good quantitative data are available for fatty acids and many other cellular components using an internal-standard method that compensates for errors during gas chromatographic analysis. In this paper, we report on the use of 7-stigmastenyl acetate as an internal standard for sterol analysis in two species of phytoplankton and oysters produced with two different diets. This internal-standard method provides an internal standard for this entire process of analysis, not just the gas chromatographic analysis. When analyzing 50-microgram samples of cholesterol acetate after hydrolysis and acetylation, about 30% of the sample was lost, resulting in a 30% error using the older external-standard method. Using the internal-standard method, the analysis error was less than 2%. Losses of sterol during analysis apparently are greater with plant and animal samples than with pure sterol standards. This internal-standard method was shown to be extremely useful, especially for samples with less than 500 micrograms of sterol. Finally, the standard error in sterol analysis is much lower when the internal-standard method is used, allowing statistical distinctions that are not possible otherwise. Use of 7-stigmastenyl acetate as an internal standard offers several advantages over the use of cholestane.

Animals↗
PubMed1997

Molecular regulation of cytokine gene expression: interferon-gamma as a model system.

The regulation of IFN-gamma transcription appears to be quite complex. In addition to the interaction of numerous regions of the genomic DNA with multiple DNA binding protein family members, DNA methylation may serve to act as an early determinant of the capacity of a cell to initiate transcription. Transcriptional activation occurs in response to both soluble extracellular signals and cell contact, and it appears quite likely that this activation may result from the interaction of different families of DNA binding proteins with different enhancer elements. Furthermore, because chronic IFN-gamma transcription and subsequent expression would likely be detrimental to the host (see 81), mechanisms have evolved to quench expression at both transcriptional and posttranscriptional levels. Given the complexity of cell-to cell interactions in the immune system, it is reasonable to expect that additional mechanisms regulating IFN-gamma transcription, involving previously identified or as yet unidentified DNA binding proteins, remain to be defined.

Animals↗
PubMed1997

The effect of strenuous versus moderate exercise on the metabolism of proteoglycans in articular cartilage from different weight-bearing regions of the equine third carpal bone.

Articular cartilage degeneration in the middle carpal joint is a common problem in racing horses. This study evaluated the effect of exercise on the in-vitro synthesis of the large aggregating proteoglycans (aggrecan) and two small proteoglycans, biglycan and decorin, in articular cartilage taken from three weight bearing regions of the third carpal bone of horses which were subjected to moderate or strenuous exercise. Twelve Standardbred horses free from clinical and radiographic disease of the middle carpal joint were subjected to an 8 week moderate exercise program. The horses were then randomly assigned to two groups: group A--continued moderate exercise and group B--strenuous exercise for 17 weeks. Horses were then rested for 16 weeks. Full-depth articular cartilage explants from the dorsal radial facet (DRF), dorsal intermediate facet (DIF) and palmar condyle (PC) of the third carpal bone were collected and cultured. Cartilage proteoglycan content and release into culture media were measured. Newly synthesized proteoglycans were labeled with 35SO4(2-) for 48 h and analyzed by size exclusion and hydrophobic chromatography, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and autoradiography. Histologic sections of adjacent osteochondral regions were evaluated for evidence of arthritic change. No histologic abnormalities or differences in proteoglycan content were detected in any of the articular cartilage regions examined. There was however, a significant reduction (P < 0.05) in aggrecan synthesis and a concomitant increase in decorin synthesis (P < 0.05) in articular cartilage from the DRF of group B animals. There was no change in biglycan synthesis, aggrecan hydrodynamic size or ability to aggregate in any articular cartilage region. This study has demonstrated that strenuous exercise in horses can lead to a disturbance in the biosynthesis of proteoglycans in articular cartilage regions subjected to high contact stresses (DRF). These metabolic abnormalities, which persisted for 16 weeks after cessation of exercise, could have deleterious effects on the biomechanical properties of the tissue. We suggest that the observed alteration in articular cartilage metabolism in CRF cartilage of strenuously exercised horses could represent a predisposing factor for cartilage degeneration and osteoarthritis at a later stage.

Aggrecans↗
PubMed1997

Variation in proteoglycan metabolism by articular chondrocytes in different joint regions is determined by post-natal mechanical loading.

In this study we investigated the hypothesis that cartilage from defined regions of ovine stifle joints, which were subjected to differing mechanical stresses, contained phenotypically distinct chondrocyte populations. Chondrocyte phenotypes were identified by the relative biosynthesis of the proteoglycans (PGs) aggrecan, biglycan and decorin. Articular cartilage (AC) from adult and neonatal ovine stifle joints were examined. Cells were cultured as both full-depth AC explants and in alginate beads after their isolation from the AC matrix. When chondrocytes from the various topographical regions of adult ovine knee joints were cultured as explants they demonstrated a consistent difference with regard to the metabolism of aggrecan and decorin. Significantly, this topographically-dependent phenotypic expression of PGs was preserved when the chondrocytes were cultured in alginate beads. In adult joints, chondrocytes from the central region of the tibial plateau not covered by the meniscus, which is subjected to high mechanical loads in-vivo, synthesized less aggrecan but more decorin than cells from regions covered by the meniscus. When chondrocytes from identical AC regions of neonatal ovine joints were cultured as explants, no topographical difference in aggrecan nor decorin metabolism could be detected. The results of this study, in association with the existing literature, lead us to propose that post-natal mechanical loading of AC could select for chondrocyte clones or induce a lasting modulation of chondrocyte phenotypic expression in different joint regions. Such cellular changes could result in the synthesis of PG populations that confer properties to AC most suited to resist the variable mechanical stresses in the different joint regions. This study serves to emphasize the importance of using cartilage from identical joint areas when examining PG metabolism by chondrocytes. Further investigation into the relationship between mechanical loading, regional chondrocyte phenotype selection and the response of these cells to anabolic and catabolic factors may provide important insights into the focal nature of AC degeneration in osteoarthritis.

Aggrecans↗
PubMed1997

Expression of NFAT-family proteins in normal human T cells.

NFAT proteins constitute a family of transcription factors involved in mediating signal transduction. Using a panel of specific antisera in immunoprecipitation assays, we found that NFATp (135 kDa) is constitutively expressed in normal human T cells, while synthesis of NFATc (predominant form of 86 kDa) is induced by ionomycin treatment. NFAT4/x was very weakly expressed in unstimulated cells, and its level did not increase upon treatment with activating agents. NFAT3 protein was not observed under any conditions. Higher-molecular-weight species of NFATc (of 110 and 140 kDa) were also detected. In addition, translation of NFATc mRNA apparently initiates at two different AUG codons, giving rise to proteins that differ in size by 36 amino acids. Additional size heterogeneity of both NFATc and NFATp results from phosphorylation. In contrast to ionomycin treatment, exposure of cells to phorbol myristate acetate (PMA) plus anti-CD28 did not induce NFATc, indicating that under these conditions, interleukin-2 synthesis by these cells is apparently independent of NFATc. In DNA binding assays, both PMA plus anti-CD28 and PMA plus ionomycin resulted in nuclear NFAT. Surprisingly, the PMA-ionomycin-induced synthesis of NFATc that was detected by immunoprecipitation was not mirrored in the DNA binding assays: nearly all of the activity was due to NFATp. This is the first study of expression of all family members at the protein level in normal human T cells.

CD28 Antigens↗