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Biomedical subjects

R Ghosh

Publications and source records attributed to R Ghosh.

At least 55 records · Page 3Linked to original sources
PubMed1997

Two-dimensional crystallization of the light-harvesting I-reaction centre photounit from Rhodospirillum rubrum.

A stoichiometric unit of the light-harvesting complex I and the reaction centre (LHI-RC complex) has been isolated from a carotenoid-less mutant of the purple non-sulphur bacterium Rhodospirillum rubrum by mild solubilization of photosynthetic membranes with the phospholipid detergent diheptylphosphatidylcholine. Dialysis of the isolated LHI-RC complexes in the presence of added dioleoyl-sn-phosphatidylcholine produced ordered two-dimensional crystals. Digital image processing revealed that the LHI-RC are packed together in a square lattice (a = b = 16.3 nm). The dimensions of the LHI ring are essentially identical with those determined from two-dimensional (2D) crystals of purified carotenoid-containing light-harvesting I complexes after analysis by cryo-electron microscopic techniques or from negatively stained 2D crystals of purified LHI complexes from a carotenoid-less mutant. Each LHI ring of the LHI-RC complex contains a central diffuse stain-excluding region, which is assigned to the reaction centre. The analysis of the LHI-RC 2D crystals strongly suggests that the geometry and subunit stoichiometry of the LHI ring is unaffected by the presence of a reaction centre that can probably assume various orientations within the ring.

Bacterial Proteins↗
PubMed1997

Impact of carbofuran in the oocyte maturation of catfish, Heteropenustes fossilis (Bloch).

Investigations were undertaken to observe the influence of technical grade carbofuran (CF) on the egg maturational processes of catfish, Heteropneustes fossilis (Bloch). Fish were exposed to sublethal doses (0.5, 1, and 2 mg/L) of CF for 30 days at 25 +/- 1 degrees C. Control fish were maintained in water. The gonado-somatic index (G.S. I. = ovarian weight x 100/body weight) of the fish, treated with 1 or 2 mg/L CF, was decreased significantly compared to that of the controls. The inhibition was almost at the same level in these doses. CF at the dose of 0.5 mg/L was found to be ineffective. From the histomorphological observations of the ovary, it was observed that CF altered both the area and the percentage occurrence of the various types of primary oocytes in the ovary compared to that of the control fish. The stage I primary oocytes were predominantly higher in CF-treated fish than stage II and stage III which was reversed in control animals. The degeneration of follicular walls, connective tissues and vacuolization in the ooplasm of the stage II and III oocytes were observed in CF-treated fish (0.5-2 mg/L). It appears that CF at sublethal concentrations inhibits oocyte maturational processes in catfish.

Animals↗
PubMed1996

Evidence for a novel DNA damage binding protein in human cells.

We describe a novel DNA damage binding activity in nuclear extracts from a normal human fibroblast cell strain. This protein was identified using electrophoretic mobility shift assays of immunopurified UV-irradiated oligonucleotide substrates containing a single, site-specific cyclobutane pyrimidine dimer or a pyrimidine (6-4) pyrimidinone photoproduct. Compared with the (6-4) photoproduct, which displayed similar levels of binding in double and single-stranded substrates, the protein showed somewhat lower affinity for the cyclobutane dimer in a single-stranded oligonucleotide and negligible binding in double-stranded DNA. The specificity and magnitude of binding was similar in cells with normal excision repair (GM637) and repair-deficient cells from xeroderma pigmentosum groups A (XP12RO) and E (XP2RO). An apparent molecular mass of 66 kDa consisting of two subunits of approximately 22 and approximately 44 kDa was determined by Southwestern analysis. Cell cycle studies using centrifugal cell elutriation indicated that the binding activity was significantly greater in G1 phase compared with S phase in a human lymphoblast cell line. Gel supershift analysis using an anti-replication protein A antibody showed that the binding protein was not antigenically related to the human single-stranded binding protein. Taken together, these data suggest that this activity represents a novel DNA damage binding protein that, in addition to a putative role in excision repair, may also function in cell cycle or gene regulation.

Base Sequence↗
PubMed1996

Nutrition and urinary calcium stone formation in northwestern India: a case control study.

The nutrient intake of 69 stone formers (SFs) from three subsets of the local population (urban 22, rural tribal 22 and rural nontribal 25) and 69 age, sex, weight and socioeconomically matched control subjects (NSs) (urban 20, rural tribal 22 and rural nontribal 27) was studied. Simultaneously their times 24-h urine samples collected over a similar period were analyzed. In general caloric and protein intake was low in all the groups but was strikingly low in the rural subjects. Intake of all nutrients was lowest in the tribal group. Although no difference was observed in diet between NSs and SFs in the same population subjects. SFs had higher urinary excretion of oxalic acid and calcium and lower excretion of citric acid and excreted more saturated urine. Notably magnesium intake was normal in both NSs and SFs, but mean excretion of magnesium was lower than normal in all the groups, suggesting its defective absorption. The influence of dietary intake of protein, carbohydrate, fat, fiber, calcium and oxalic acid on urinary excretion of calcium, oxalic acid, uric acid, inorganic phosphorus, magnesium and citric acid was examined using the chi-square test. No association was observed, thus suggesting that this low nutrient intake did not influence the lithogenic process. Thus, the overall observations suggest: (a) poor nutrition, (b) no effect of diet on urinary stone disease, (c) no difference in the nutrient intake between NSs and SFs and (d) a higher excretion of promoters and a lower excretion of inhibitors in SFs than in NSs.

Adult↗
PubMed1996

Optical fiber-based in situ spectroscopy of pigmented single colonies.

We have adapted a commercially available fiber-optic spectroradiometer with diode array detection to record reflection and absorption spectra from single, 1-mm-diameter bacterial colonies. A careful assessment of the performance of the spectroradiometer for this application is reported. In a model study employing colonies from various phototrophic bacteria, we show that the reflectance spectra are reliable within the range of 450 to 820 nm, whereas the transmission spectra yield accurate peak intensities and absorption maxima from 400 to 900 nm. For screening of populations of about 10(sup4) colonies, fiber-optic transmission spectroscopy provides an attractive and inexpensive alternative to present techniques based on charge-coupled device imaging technology.

Journal Article↗
PubMed1996

Induction of apoptosis by ionizing radiation in Chinese hamster V79 cells and a radioresistant cell strain derived from V79.

DNA fragmentation into nucleosome ladder, a hall mark of apoptosis, could be obtained by as low as 0.58 Gy of gamma irradiation within 6 hr of irradiation which increased appreciably after 48 hr in V79 cells. In the same condition condensation of the nucleus and marginalization of the cytoplasm the characteristic morphology of apoptotic death were observed. Unirradiated controls had approximately 2% apoptotic cells. When cells were irradiated with 0.58 Gy, approximately 10% of the cells had the apoptotic morphology. This number increased to approximately 29% at 3.5 Gy dose. At a higher dose, apoptotic and necrotic cells were visualized. In radio resistant cells higher doses were required to induce morphological changes. The results indicated that gamma irradiation can induce apoptosis in Chinese hamster V79, fibroblast cell line and the radioresistant cell strain derived from V79 cells is also resistant to induction of apoptosis.

Animals↗
PubMed1995

Crystallographic structure of a PLP-dependent ornithine decarboxylase from Lactobacillus 30a to 3.0 A resolution.

Ornithine decarboxylase from Lactobacillus 30a (L30a OrnDC) is representative of the large, pyridoxal-5'-phosphate-dependent decarboxylases that act on lysine, arginine or ornithine. The crystal structure of the L30a OrnDC has been solved to 3.0 A resolution using MIR phases in combination with density modification (space group P6; a = 195.6 A, c = 97.6 A; dimer of 1460 amino acid residues/asymmetric unit; VM = 3.26 A3/Da). The refined crystallographic R-value was 0.219 (Rfree = 0.268) using 2-fold restraints with a 4 sigma cutoff and 8.0 to 3.0 A resolution data. Six dimers related by C6 symmetry compose the enzymatically active dodecamer (approximately 10(6) Da). Each monomer of L30a OrnDC can be described in terms of five sequential folding domains. The amino-terminal domain, residues 1 to 107, consists of a five-stranded beta-sheet termed the "wing" domain. Two wing domains of each dimer project inward towards the center of the dodecamer and contribute to dodecamer stabilization. The "linker" domain, residues 108 to 160, consists of short alpha-helices separated by a loop that fills in the PLP pocket. The third domain, residues 161 to 413, is an alpha/beta domain containing a seven stranded beta-sheet that resembles the PLP-binding domain of the aspartate aminotransferases. The fourth domain, residues 414 to 569, resembles the "small" domain of the aspartate aminotransferases, but is significantly larger due to insertions. The remaining carboxy-terminal domain, residues 570 to 730, is organized into multiple antiparallel loops and seven alpha-helices that help form a deep channel leading to the PLP-binding site.

Amino Acid Sequence↗
PubMed1995

Molecular determinants of plasma cholesteryl ester transfer protein binding to high density lipoproteins.

The plasma cholesteryl ester transfer protein (CETP) mediates the transfer of neutral lipids between lipoproteins and is associated with high density lipoproteins (HDL). To understand the mechanism of interaction of CETP with HDL, we studied the binding of pure recombinant CETP to 1-palmitoyl-2-oleoylphosphatidylcholine (POPC)/apoA-I discoidal particles. Separating bound from free CETP using native gradient gel electrophoresis, complexes of CETP with 10-nm hydrodynamic diameter discoidal particles migrated with a diameter of 12-16 nm, compared with approximately 7.5 nm for CETP. At lower ratios of CETP to discs, CETP bound to discs without displacement of apoA-I. CETP alone was unable to generate discoidal complexes. Cross-linking and fluorescence resonance energy transfer experiments indicated that CETP bound to discs as monomers. Cross-linking of CETP to apoA-I in discs suggested proximity of apoA-I and CETP. By negative-stain electron microscopy, discoidal complexes containing CETP and CETP monoclonal antibody showed localization of antibody molecules to the disc edge, suggesting that CETP was bound to the disc edge. The binding of CETP to discs of different composition or size was studied. Discs (10-nm Stokes diameter) prepared with either apoA-I or apoA-II had a similar Kd (120 nM). Inclusion of 1 mol % cholesteryl oleate, 5 mol % cholesterol, or 6 mol % phosphatidylinositol increased the binding affinity of CETP 3-10 times (20-30 nM). In comparison, plasma HDL3 had a Kd of approximately 450 nM. For POPC/apoA-I discs, 10-nm discs bound CETP with much higher affinity than smaller 7.8-nm discs (Kd = 1-2 microM). 7.7-nm hydrodynamic diameter POPC/apoA-I spherical particles containing either triolein or cholesteryl oleate in their core bound CETP with higher affinity (Kd = 50-100 nM) than 7.8-nm POPC/apoA-I discs. Thus, CETP appears to bind to the perimeter of discoidal particles, possibly in a process in which flexible segments in apoA-I or apoA-II accommodate CETP at the disc edge. The binding of CETP to HDL is markedly influenced by overall particle size and shape and by lipid composition, and the increased binding affinity for cholesterol- and cholesteryl ester-containing discs suggests a higher affinity of CETP for nascent than mature HDL.

Animals↗
PubMed1995

Functional reconstitution of the human placental transferrin receptor into phospholipid bilayers leads to long tubular structures proceeding from the vesicle surface.

We have reconstituted the human placental transferrin receptor (hTfR) into phospholipid vesicles using either dialysis, dilution, or gel filtration. Several different detergents and phospholipids and a variety of lipid-to-protein weight ratios were tested. Preformed vesicles as well as detergent-solubilized phospholipids were used. Reconstituted mixtures were fractionated by sucrose density gradient centrifugation and screened for ferritransferrin binding activity, and peak fractions were analyzed by electron microscopy. The efficiency of reconstitution was strongly influenced by the choice of the phospholipids used and the reconstitution method. Best results were obtained when hTfR was dissolved with octylpolyoxyethylene, mixed with detergent-solubilized soy bean lecithin, and reconstituted by slow dialysis. The data show that hTfR capable of binding ferritransferrin was reconstituted into vesicles with an irregular surface and many protrusions. In addition the reconstitution of hTfR resulted in the formation of tubular structures proceeding from the vesicle surface, which may serve as an in vitro model for future studies on the relevance of self-assembly processes for cellular endocytosis.

Centrifugation, Density Gradient↗
PubMed1995

Prolactin modulates oxytocin mRNA during lactation by its action on the hypothalamo-neurohypophyseal axis.

Since prolactin (PRL) can increase electrically stimulated oxytocin (OT) release by a direct action on the neurohypophysis, experiments were done to test the effect of PRL on OT mRNA content of explants of the hypothalamo-neurohypophyseal system (HNS) obtained from Day 10 lactating Sprague-Dawley rats, to determine if PRL could alter OT mRNA by a direct effect on the HNS. The effect of PRL was evaluated alone and in conjunction with bicuculline, an antagonist at gamma amino butyric acid receptors, in order to provide a concomitant stimulus for OT release. Neither PRL nor bicuculline alone altered OT or VP release. However, the simultaneous administration of bicuculline and PRL caused a statistically significant increase in the release of OT and in OT mRNA content of the explants (P < 0.05). On the other hand, PRL did not cause vasopressin (VP) release nor an increase in VP mRNA in these explants even in the presence of bicuculline, thus ruling out a direct effect of PRL on the HNS VP neurons. In conclusion, during lactation, PRL, in combination with other stimuli can increase OT mRNA content in the hypothalamus by a direct action on the hypothalamo-neurohypophyseal system. This effect may be secondary to the release of OT. This effect is specific to OT, because PRL did not alter either the release of VP or VP mRNA content.

Animals↗
PubMed1995

The 8.5 A projection map of the light-harvesting complex I from Rhodospirillum rubrum reveals a ring composed of 16 subunits.

Two-dimensional crystals from light-harvesting complex I (LHC I) of the purple non-sulfur bacterium Rhodospirillum rubrum have been reconstituted from detergent-solubilized protein complexes. Frozen-hydrated samples have been analysed by electron microscopy. The crystals diffract beyond 8 A and a projection map was calculated to 8.5 A. The projection map shows 16 subunits in a 116 A diameter ring with a 68 A hole in the centre. These dimensions are sufficient to incorporate a reaction centre in vivo. Within each subunit, density for the alpha- and the beta-polypeptide chains is clearly resolved, and the density for the bacteriochlorophylls can be assigned. The experimentally determined structure contradicts models of the LHC I presented so far.

Crystallography↗
PubMed1995

Structural motifs for pyridoxal-5'-phosphate binding in decarboxylases: an analysis based on the crystal structure of the Lactobacillus 30a ornithine decarboxylase.

Two of the five domains in the structure of the ornithine decarboxylase (OrnDC) from Lactobacillus 30a share similar structural folds around the pyridoxal-5'-phosphate (PLP)-binding pocket with the aspartate aminotransferases (AspATs). Sequence comparisons focusing on conserved residues of the aligned structures reveal that this structural motif is also present in a number of other PLP-dependent enzymes including the histidine, dopa, tryptophan, glutamate, and glycine decarboxylases as well as tryptophanase and serine-hydroxymethyl transferase. However, this motif is not present in eukaryotic OrnDCs, the diaminopimelate decarboxylases, nor the Escherichia coli or oat arginine decarboxylases. The identification and comparison of residues involved in defining the different classes are discussed.

Amino Acid Sequence↗
PubMed1995

Supernatant medium from UV-irradiated cells influences the cytotoxicity and mutagenicity of V79 cells.

It is known that UV light induces the secretion of some proteins into the extracellular medium. We have carried out experiments to study how the supernatant medium from UV-irradiated cells affects the cytotoxicity and mutagenicity of V79 cells exposed to different damaging agents. So we exposed exponentially growing cells to 20 J/m2 of UV light and then harvested the supernatant medium after 22 h. This supernatant medium was then used to treat a fresh batch of cells for 2 h. After the treatment with this supernatant medium the cells were subsequently exposed to UV light, gamma-rays, hydrogen peroxide or MNNG. We found that exposure to this medium had a protective effect on the survival levels for UV light, gamma-rays and hydrogen peroxide while MNNG-induced killing remained unaffected. With UV light and gamma-rays we found that mutation induction at all doses was increased. Cycloheximide could inhibit this protection and the increase in mutation frequencies was also suppressed. The results indicated a protective role for the UV-induced factor(s). They were probably involved directly or they triggered repair process(es) that were related to oxidative stress.

Animals↗
PubMed1995

The vaccinia virus D5 protein, which is required for DNA replication, is a nucleic acid-independent nucleoside triphosphatase.

The vaccinia virus D5 gene encodes a 90-kDa protein that is transiently expressed at early times after infection. Temperature-sensitive mutants with lesions in the D5 gene exhibit a fast-stop DNA- phenotype and are also impaired in homologous recombination. Here we report the overexpression of the D5 protein within the context of a vaccinia virus infection and its purification to apparent homogeneity. The purified protein has an intrinsic nucleoside triphosphatase activity which is independent of, and not stimulated by, any common nucleic acid cofactors. All eight common ribo- and deoxyribonucleoside triphosphates are hydrolyzed to the diphosphate form in the presence of a divalent cation. Implications for the role of D5 in viral DNA replication are addressed.

Acid Anhydride Hydrolases↗
PubMed1995

Role of prolactin and gonadal steroids in regulation of oxytocin mRNA during lactation.

The role of suckling, prolactin, and gonadal steroids in regulating hypothalamic oxytocin (OT) mRNA content during the 1st and 2nd wk of lactation was evaluated. On day 4 of lactation, OT mRNA content decreased in rats removed from their litter for 24 h compared with suckled controls. Either suckling (in the absence of prolactin release) or prolactin (in the absence of suckling) maintained OT mRNA content at this stage of lactation. In contrast, at day 11 of lactation, OT mRNA content remained unchanged in rats deprived of pups for 24 h. Ovariectomy did not compromise the ability of day 11 nonsuckled rats to maintain OT mRNA content; however, the increased water intake, plasma prolactin, plasma vasopressin (VP), plasma renin concentration, and hypothalamic VP mRNA content at day 11 compared with day 4 suggest a role for fluid balance in determining OT mRNA content during the 2nd wk of lactation. Thus, at day 4, suckling is a major determinant of OT mRNA content as a result of both direct activation of neuronal afferents to the OT neurons and stimulation of prolactin release. This is in contrast to day 11, when fluid balance may predominate in regulation of OT mRNA.

Animals↗