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Biomedical subjects

R Ghosh

Publications and source records attributed to R Ghosh.

At least 73 records · Page 4Linked to original sources
PubMed1994

An improved procedure and new vectors for transposon Tn5 mutagenesis of the phototrophic bacterium Rhodospirillum rubrum.

A detailed examination of vectors and procedures used for Tn5 mutagenesis of the phototrophic purple non-sulfur bacterium Rhodospirillum rubrum has been performed. The mobilizable Tn5 suicide vectors currently available show a frequency of Tn5 mutagenesis for R. rubrum of approx. 10(-7)-10(-8), approx. 100-1000-fold lower than observed for the related bacteria Rhodobacter capsulatus and Rhodobacter sphaeroides. Using the blue-to-red reversion of a blue-green mutant, R. rubrum ST6, containing a single Tn5 lesion in one of the early genes for carotenoid biosynthesis, we have shown that the frequency of precise excision of a chromosomally inserted Tn5 element, to restore the wild-type phenotype in the absence of selection, is 10(-6). We have constructed three new suicide vectors for Tn5 mutagenesis, where the transposase encoded by the IS50R element was placed in the same (pSUPEG11, pSUPEG21) or in the opposing (pSUPEG22) orientation from the weak promoter of the RK2-derived tetR gene. With the vector pSUPEG11, the frequency of Tn5 mutagenesis was increased to 10(-5), approx. 100-fold higher than observed previously.

Conjugation, Genetic↗
PubMed1994

Optimization of the Sistrom Culture Medium for Large-Scale Batch Cultivation of Rhodospirillum rubrum under Semiaerobic Conditions with Maximal Yield of Photosynthetic Membranes.

The defined medium A of W. R. Sistrom (W. R. Sistrom, J. Gen. Microbiol. 22:77-85, 1960) has been modified to allow the growth of Rhodospirillum rubrum in large-scale batch cultures under dark, semiaerobic conditions. The simultaneous use of two substrates, NH(4)-succinate (46 mM) and fructose (0.3%), which are utilized in aerobic and fermentative metabolism, respectively, leads to very high cell densities with a maximal yield of photosynthetic membranes.

Journal Article↗
PubMed1994

The role of the cellular antioxidant defense in oxidant carcinogenesis.

Oxidant carcinogens interact with multiple cellular targets including membranes, proteins, and nucleic acids. They cause structural damage to DNA and have the potential to mutate cancer-related genes. At the same time, oxidants activate signal transduction pathways and alter the expression of growth- and differentiation-related genes. Indeed, the carcinogenic action of oxidants results from the superposition of these genetic and epigenetic effects. All cells possess elaborate antioxidant defense systems that consist of interacting low and high molecular weight components. Among them, superoxide dismutases (SOD), glutathione peroxidases (GPx), and catalase (CAT) play a central role. Our studies with mouse epidermal cells demonstrate that the balance between several antioxidant enzymes rather than the activity of a single component determines the degree of protection. Unexpectedly, increased levels of Cu,Zn-SOD alone in stable transfectants resulted in sensitization to oxidative chromosomal aberrations and DNA strand breaks. However, a concomitant increase in CAT or GPx in double transfectants corrected or overcorrected the hypersensitivity of the SOD clones depending on the ratios of activities CAT/SOD or GPx/SOD. The cellular antioxidant capacity also affected oxidant induction of the growth-related immediate early protooncogene c-fos. Increases in CAT or SOD reduced the accumulation of c-fos message, albeit for different reasons. The cellular antioxidant defense also affects the action of UVB light (290-320 nm) that represents the most potent carcinogenic wavelength range of the solar spectrum. UVB light is known to exert its action in part through oxidative mechanisms. Increases in CAT and GPx protected mouse epidermal cells from UVB-induced DNA breakage.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

A novel effect of selenium on streptozotocin-induced diabetic mice.

The effect of oral administration of sodium selenite on glucose homoeostasis was studied in male Swiss albino mice 6 weeks after they were made diabetic with streptozotocin. Diabetes caused hyperglycaemia (2.5-fold), a marked decrease (4.5-fold) in liver glycogen, a 4-fold increase in the glucose-6-phosphatase activity and significant decrease in plasma insulin levels and protein kinase activity. Although selenium administration in control animals showed no significant effect on various parameters measured, selenite treatment of diabetic mice restored these parameters to near control values. Thus the results show insulin-like in vivo action of selenium in diabetic mice.

Animals↗
PubMed1993

Two-dimensional crystallization of the light-harvesting complex from Rhodospirillum rubrum.

Homogeneous detergent-solubilized B873 light-harvesting complexes from a carotenoid-less mutant of the purple non-sulfur bacterium, Rhodospirillum rubrum G9, were reassembled spontaneously into two-dimensional (2D) hexagonal arrays during extensive and controlled dialysis. As the complexes contain only 1 to 2 mol phospholipid per mol alpha beta dimer, the arrays formed by a self assembly process are primary due to protein-protein interactions. The hexagonal lattices were analyzed by negative stain electron microscopy and digital image processing. They exhibited a unit cell size of 12.3 nm, in close agreement with the particle diameter of the active photo-unit in native chromatophore membranes. The unit cell contains a central 5 nm stain-filled depression, embraced by a ring with an outer diameter of 10 nm.

Crystallization↗
PubMed1993

Mechanism of induction of c-fos by ultraviolet B (290-320 nm) in mouse JB6 epidermal cells.

The UVB (290-320 nm) portion of the solar spectrum possesses the highest activity for the induction of skin cancer and has the capacity to stimulate epidermal proliferation. We report that UVB is a transcriptional inducer of the c-fos protooncogene in mouse JB6 epidermal cells. Induction is biphasic with an immediate early peak at 30-60 min and a second broader peak 8 h following irradiation. The immediate early phase is suppressed by inhibitors of nuclear adenosine diphosphoribose transferase. For UVB induction, the formation of full-length messages is less efficient than of early, short messages, while both types of messages are produced at similar rates following serum stimulation. Experiments with stable transfectants with reporter constructs linked to 5'-upstream sequences of c-fos indicate that UVB and serum stimulation both require the sequences from -345 to -285 which contain the joint DSE-AP-1 enhancer motifs for efficient induction. Mobility shift data reveal that the complement of c-Fos and c-Jun proteins which bind to the fos-AP-1 octanucleotide decrease immediately following irradiation. Increased binding of Fos and Jun is observed 8-24 h later. UVB did not cause an observable change in the nuclear proteins which bind to the dyad symmetry element oligonucleotide in vitro. Fos protein was detected among the binding proteins. We propose that the two phases of UVB-induced c-fos expression occur by quite different mechanisms. The immediate early phase is inhibited by adenosine diphosphoribose transferase inhibitors because poly-ADP ribosylation of chromosomal proteins is required for the resealing of UVB-induced DNA strand breaks which otherwise retard message elongation. The production of an autocrine factor may be responsible for the late phase of c-fos induction.

Animals↗
PubMed1993

Characterisation of methotrexate-resistant clones.

Three methotrexate (MTX)-resistant clones M1, M2 and M3 have been isolated from Chinese hamster V79 cells and characterised for aneuploidy, chromosomal aberrations, sister-chromatid exchange (SCE) mutation and transfection. Amplification of the dihydrofolate reductase (DHFR) gene in these clones has been established from (a) direct measurement of DHFR activity, (b) existence of double minute chromosomes and (c) homogeneously staining region (HSR) in chromosome number 2 by G-banding technique. Clone M1 on further exposure to gradually increasing concentrations of MTX gave rise to two more clones M4 and M5, resistant to 1200 nM and 2400 nM MTX, respectively. The levels of folate reductase activity in clones M4 and M5 were 21.90 units per 10(7) cells and 33.30 units per 10(7) cells, whereas the value was 13.90 units per 10(7) cells in clone M1 and 1.1 units per 10(7) cells in normal V79 cells. Increased chromosomal aberrations were observed in each of these clones in comparison to those in normal V79 cells. There was a correlation between the increase in DHFR activity and the increase in the level of background SCE as well as the increase in aneuploidy. However, background mutation frequency at the HGPRT locus remained unaffected though transfection frequency decreased.

Animals↗
PubMed1993

Gene expression of the B875 light-harvesting prepolypeptides from Rhodospirillum rubrum in Escherichia coli.

The gene coding for the prepolypeptides of alpha and beta, obtained as a 429 bp fragment from chromosomal DNA of Rhodospirillum rubrum S1 by polymerase chain reaction amplification, were cloned in tandem into the high-level expression vector pOTSNco 12 for expression in Escherichia coli. The vector pOTSNco12 is a derivative of the pAS vector system, which contains the strong lambda PL promotor and is under tight control by the cI857 repressor encoded by the expression strain AR58. Induction of transcription from the lambda PL promotor is achieved by shifting the growth temperature from 32 to 42 degrees C. Expression of the gene products was monitored by sodium dodecylsulfate polyacrylamide gel electrophoresis and western blotting. The expressed B875 light-harvesting prepolypeptides were located in the E. coli inner membrane and could not be removed by washing with high salt. The amount of expressed B875 light-harvesting prepolypeptides was estimated to be about 0.1% of the total soluble protein.

Amino Acid Sequence↗
PubMed1993

UVB-induced DNA breaks interfere with transcriptional induction of c-fos.

Oxidative stress may play an important role in the carcinogenic action of UVB light (290 to 320 nm). UVB light induces the growth-related immediate-early gene c-fos in JB6 mouse epidermal cells, but at the same time it causes structural damage to DNA, in particular DNA strand breakage. We have studied the effect of the modulation of the frequencies of DNA breaks on the transcriptional induction of c-fos by changing the cellular antioxidant defense or by inhibiting break repair. Reduction of UVB-induced DNA breakage in a stable transfectant with an increased complement of glutathione peroxidase enhanced the induction of c-fos. In contrast, c-fos induction was diminished in stable transfectants with Cu,Zn-superoxide dismutase. Increasing the stationary concentration of UVB-induced DNA breaks by inhibition of repair in the presence of the adenosine diphosphoribose (ADPR)-transferase inhibitor 3-amino-benzamide suppressed the induction of c-fos. We conclude that DNA breaks which are induced by UVB via oxidative processes interfere with the transcriptional induction of c-fos. DNA breaks appear to exert a long-range effect on chromatin conformation which is incompatible with efficient transcription. This notion is supported by the observation that inhibition of break rejoining by 3-amino-benzamide suppressed the UVB induction of the endogenous c-fos gene and of a stably integrated construct containing the c-fos regulatory sequences linked to a reporter gene. In contrast, the induction of the same construct was not inhibited when it remained extrachromosomal in transient transfection experiments.

Animals↗
PubMed1993

Protective role of vanadium in the survival of hosts during the growth of a transplantable murine lymphoma and its profound effects on the rates and patterns of biotransformation.

Vanadium, as ammonium mononium monovanadate, has been found to have anticarcinogenic effect in host mice bearing a transplantable ascitic lymphoma. The potentiating effect of vanadium was supported by biochemical analysis of autopsies relating to markers like microsomal cytochrome P-450, UDP glucuronyl transferase (UDPGT) and cytosolic glutathione-S-transferase (GSHT) in the hepatic tissue of the hosts, which showed substantial alterations in the extent of tumor regression as compared to their lymphoma and normal counterparts, prolonging the survival of the hosts.

Animals↗
PubMed1993

Effect of dietary methionine and inorganic sulfate with and without calcium supplementation, on urinary calcium excretion of guinea pigs (Cavia porcellus).

Supplementation of sodium sulfate and DL-methionine along with the standard diet to guinea pigs nearly doubled the urinary calcium in 6 weeks. This was probably due to decreased tubular reabsorption of calcium which was complexed with sulfate in the tubular lumen. A mild calcium load didn't further enhance calcium excretion in sodium sulfate supplemented group, but did so in methionine supplemented group. It may be due to methionine which might have increased the intestinal absorption of calcium. Both of these compounds increased citric acid excretion and decreased magnesium excretion.

Animals↗
PubMed1992

Crystal structures explain functional properties of two E. coli porins.

Porins form aqueous channels that aid the diffusion of small hydrophilic molecules across the outer membrane of Gram-negative bacteria. The crystal structures of matrix porin and phosphoporin both reveal trimers of identical subunits, each subunit consisting of a 16-stranded anti-parallel beta-barrel containing a pore. A long loop inside the barrel contributes to a constriction of the channel where the charge distribution affects ion selectivity. The structures explain at the molecular level functional characteristics and their alterations by known mutations.

Amino Acid Sequence↗
PubMed1991

Response of V79 cells to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment: inhibition of poly(ADP-ribose) and topoisomerase activity.

MNNG-induced killing of V79 cells has been found to be enhanced on inhibition of topoisomerase II activity by nalidixic acid and poly(ADP-ribose) polymerase synthesis by benzamide. Using these 2 inhibitors in conjunction after MNNG treatment, some overlap in the functions of these 2 enzymes was observed. Nalidixic acid and benzamide were found to suppress the yields of mutations and SCEs induced by MNNG. Benzamide was more effective in suppressing the mutation yield whereas nalidixic acid was more effective in suppressing SCEs. A model based on the relative requirement of topoisomerase and poly(ADP-ribose) for the repair of different types of damage has been proposed to explain the results.

Adenosine Diphosphate Ribose↗