Biomedical subjects

G S Kumar

Publications and source records attributed to G S Kumar.

At least 37 records · Page 2Linked to original sources

PubMed1995

Binding of 2,7-diaminomitosene to DNA: model for the precovalent recognition of DNA by activated mitomycin C.

Mitomycin C (MC), mitomycin A, porfiromycin, BMY-25067, and BMY-25287, antitumor antibiotics collectively termed "mitosanes", were found to have no appreciable binding affinity to various natural and synthetic DNAs, as tested by UV spectrophotometry and equilibrium dialysis. Further tests of DNA binding applied to MC including thermal melting measurements, displacement of ethidium fluorescence, and unwinding of closed circular DNA were similarly negative. In contrast, 2,7-diaminomitosene (2,7-DAM), a major end product of the reductive activation of MC, binds to the same series of DNAs by all of these criteria. In the presence of DNA its UV absorbance at the 313 nm maximum decreased and underwent a slight red shift. This effect was used for determining DNA binding constants (Kb) by the spectrophotometric titration method. At pH 6.0 the Kbs of three natural DNAs with varying GC content, as well as poly(dA-dT).poly(dA-dT), and poly(dG-dC).poly(dG-dC), were all in the range of (1.2-5.3) x 10(4) (M nucleotide)-1, with no apparent specificity of binding. Poly(dG-m5dC).poly(dG-m5dC) displayed a slightly higher Kb ((7.5-8.4) x 10(4)). Binding of other, closely related mitosenes was tested to calf thymus DNA by equilibrium dialysis. Neither the presence of a 1-OH substituent, removal of the 10-carbamoyl group, nor methylation of the 2-amino group modifies the binding affinity of the mitosenes significantly. The 1-phosphate substituent abolishes binding. The binding of 2,7-DAM to DNA increased with decreasing pH and decreasing ionic strength. It was determined that 2,7-DAM is protonated at the 2-amino group with a pKa = 7.55, and this correlated well with the observed pH dependence of the binding, indicating that the binding affinity has a strong electrostatic component. This was confirmed by the finding that the extrapolated Kb to 1 M Na+ concentration diminishes to only 10% of the value of Kb at 0.01 M Na+ concentration. Viscosity tests showed conclusively that 2,7-DAM intercalates in DNA, in a nonspecific manner. DNA binding by 2,7-DAM is shown to be a close model of the binding of the reduced activated form of MC, previously characterized indirectly [Teng, S. P., Woodson, S. A., and Crothers, D. M. (1989) Biochemistry 28, 3901-3907]. The nonspecific precovalent binding of the active form may serve in the cell to concentrate the drug at its critical target, DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Structural and function modification of DNA by mitomycin C. Mechanism of the DNA sequence specificity of mitomycins.

Mitomycin C (MC) is a clinically used antitumor agent, which upon reductive activation activates and cross-links DNA. The covalent products of alkylation and cross-linking by the unnatural synthetic enantiomer of MC (ent-MC) were isolated as drug-deoxyguanosine monoadducts and drug-deoxyguanosine bisadducts and were fully characterized structurally. Specificity of alkylation and cross-linking of guanines in the CpG.CpG sequence was observed by ent-MC, similarly to that observed previously by MC. These findings define the mechanism of recognition of the CpG.CpG sequence of DNA by the mitomycins in the minor groove. In contrast, the natural MC metabolite, 2,7-diaminomitosene (2,7-DAM) which lacks the aziridine alkylator function is shown to recognize and alkylate guanines only the GpG.CpC sequence in the major groove, by a different mechanism. Thoughts on the molecular evolution of the basic mitomycin structure as a very efficient lethal DNA cross-linker are discussed.

Antibiotics, Antineoplastic↗

PubMed1994

Suppression of human T-cell growth in vitro by cis-unsaturated fatty acids: relationship to free radicals and lipid peroxidation.

Cis-unsaturated fatty acids such as dihomogamma-linolenic acid (DGLA), arachidonic acid (AA) and eicosapentaenoic acid (EPA), which form precursors to 1, 2 and 3 series prostaglandins (PGs), have been shown to suppress human T-cell growth in vitro by a prostaglandin E (PGE)-independent mechanism. In an earlier study, we showed that these fatty acids can induce free radical generation in human neutrophils and tumor cells. Here we show that cis-unsaturated fatty acids augment free radical generation and lipid peroxidation in human T-cells. The growth suppressive action of cis-unsaturated fatty acids on human T-cells could be blocked by anti-oxidant, vitamin E and the superoxide anion quencher superoxide dismutase. These results suggest that c-UFAs-induced cell growth suppression is a free radical dependent process.

8,11,14-Eicosatrienoic Acid↗

PubMed1994

Effect of prostaglandins and their precursors on the proliferation of human lymphocytes and their secretion of tumor necrosis factor and various interleukins.

Cytokines, released by T cells, participate in inflammation and produce tissue injury. Excess production of cytokines such as interleukins (ILs) and tumor necrosis factor (TNF) is believed to be involved in the pathobiology of conditions such as septicemia and septic shock, collagen vascular diseases, glomerulonephritis etc. On the other hand, prostaglandins (PGs) are known to modulate inflammation, immune response, and T-cell response to antigens. But relatively little information is available on the effects of PGs and PG precursors on the release of cytokines. Here the authors present data which suggests that PGs including thromboxane B2 (TXB2) and their precursors such as dihomo-gamma linolenic acid (DGLA), arachidonic acid (AA) and eicosapentaenoic acid (EPA) can inhibit T-cell proliferation and influence their ability to secrete IL-2, IL-4, IL-6 and TNF in vitro. These results may have relevance to the use of PG-precursors in various inflammatory conditions including collagen vascular diseases.

8,11,14-Eicosatrienoic Acid↗

PubMed1994

DNA polymorphism under the influence of low pH and low temperature.

The polymorphic behaviour on the conformation of a alternating GC polymer and its methylated analogue has been studied under the influence of low pH, low temperature and low ionic strength from the measurements of UV-absorption and circular dichroic spectroscopy. Studies indicate that both the polymers isomerize to a stable left handed type conformations. The duplex nature of these conformations were inferred from thermal denaturation curves and the temperature dependence of the CD spectra. In natural DNA, the influence of low pH and low temperature also shows a defined conformational change, characterized by two positive CD bands. This conformational status is achieved in all DNAs irrespective of base composition or sequence of base pairs. Further evidence to this altered polymorphic state of natural DNAs is inferred from ethidium binding study.

Animals↗

PubMed1994

Phosphorylation of membrane proteins in response to temperature in an Antarctic Pseudomonas syringae.

Temperature-dependent phosphorylation and dephosphorylation of membrane proteins was studied in vitro in a number of psychrotrophic Antarctic bacteria which grow between 0 and 30 degrees C. One of them, a Pseudomonas syringae isolate, was studied in detail and was found to have three membrane proteins of molecular mass 30, 65 and 85 kDa which were phosphorylated differently in response to low and high temperatures. The 65 kDa protein was phosphorylated only at lower temperatures (between 0 and 15 degrees C). The 30 kDa protein was phosphorylated more at higher temperatures and was possibly a histidine kinase. This protein was present in all the psychrotrophic Pseudomonas species studied and in Sphingobacterium antarcticus. A possible role for these proteins in sensing environmental temperature is proposed.

Adenosine Triphosphate↗

PubMed1994

Phosphorylation of lipopolysaccharides in the Antarctic psychrotroph Pseudomonas syringae: a possible role in temperature adaptation.

Phosphorylation of lipopolysaccharide (LPS) from a psychrotrophic bacterium, Pseudomonas syringae, from Antarctica was studied by using sucrose gradient-separated membrane fractions. The bacterium was found to possess an LPS kinase which could phosphorylate more LPS postsynthetically at higher temperatures. The phosphorylation was low at a lower temperature and was also found to occur in vivo. After phosphorylation of LPS in vitro, it was found that the major part of the radioactivity (> 85%) was associated with the core oligosaccharide region of the LPS. The phosphate groups of this region are probably involved in the binding of metal ions, which could be removed by EDTA. The cells grown at the lower temperature probably contained fewer divalent cations because of the smaller amount of phosphate and thereby were more sensitive to EDTA. The cells were also more sensitive to cationic antibiotics at the lower temperature. A possible role of this differential phosphorylation of LPS in modulating the function of the outer membrane as a permeability barrier in the psychrotroph is discussed.

Adaptation, Physiological↗

PubMed1994

Synthesis and secretion of lipoproteins by primary cultures of rat hepatocytes.

Synthesis and secretion of VLDL and LDL by primary cultures of rat hepatocytes maintained in serum free medium have been studied. A time-dependent increase found in the [3H]leucine labelled lipoproteins which floated at a density of 1.006 g/ml indicate the secretion of VLDL into the medium. That the hepatocytes also secrete. LDL is shown by floatation of [3H]leucine labelled lipoproteins by sequential centrifugation at a density range of 1.006-1.06 g/ml. Electrophoretic and immunoprecipitation analysis show that about 60% and 65% respectively of 3H-radioactivity is associated with apoB in the two fraction of lipoproteins. At about 12hr 70-75% lipoproteins in the culture medium is in the VLDL density range and 25-30% is in the LDL density range. Conversion of secreted VLDL to LDL has also been shown by incubating hepatocytes with pre-labelled lipoproteins when there is a decrease in the fraction of VLDL range with a corresponding increase in the fraction of the LDL density range. Addition of glycosaminoglycans such as hyaluronic acid, chondroitin sulphate, and heparin into the medium cause significant increase in the synthesis and secretion of [3H]apoB into the medium indicating a possible secretory control of apoB by local reuptake.

Animals↗

PubMed1993

Thermodynamics of the interaction of berberine with DNA.

The interaction of berberine [7,8,13,13a-tetrahydro-9-10-dimethoxy-2,3-(methylene dioxy)-berberinium] with calf thymus DNA has been studied by spectrophotometry in buffers of various salt concentrations and temperatures. Binding parameters obtained are best fit by the neighbour exclusion model. The salt and temperature dependence of the binding constants are used to estimate thermodynamic parameters involved in the complex formation of berberine with DNA. The binding process is exothermic over the entire range and the values of enthalpy and entropy change are strongly dependent on the salt concentration. The negative enthalpy and positive entropy changes compensate one another to produce a relatively small Gibbs' free energy change. Possible molecular contribution to the enthalpy and entropy changes is discussed.

Animals↗

PubMed1993

Synthesis and secretion of apo B containing lipoproteins by primary cultures of hepatocytes isolated from rats fed atherogenic diet.

The effect of experimentally induced atherosclerosis on the synthesis and secretion of lipoproteins in the density range of very low density lipoproteins (VLDL) and low density lipoproteins (LDL) have been studied using primary cultures of rat hepatocytes. Rats fed atherogenic diet showed higher levels of lipids associated with serum VLDL and LDL fraction, aorta and liver when compared with animals fed normal diet. Incorporation of [3H]leucine into apo B associated with the cell layer and secreted by hepatocytes from rats fed atherogenic diet was significantly more when compared with normal hepatocytes. [14C]Acetate incorporation studies showed that the synthesis of cholesterol was lower in hepatocytes from atherogenic diet fed rats, but more of the newly synthesised cholesterol was found in the secreted VLDL; secretion of lipids, particularly triglycerides, unesterified cholesterol and cholesterol in the lipoproteins in the density range of VLDL and LDL was significantly more in these hepatocytes. The relative distribution of [3H]-radioactivity in the LDL density range was 57% in hepatocytes from atherogenic diet fed animals as compared with 28% in controls, suggesting a relatively higher production of lipoproteins in the LDL density range than VLDL by these cells. These results indicate that the hypercholesterolemia in atherogenic diet fed animals may among other factors be caused by increased synthesis of apo B by liver cells and resultant increase in the secretion of apo B containing lipoproteins.

Acetates↗

PubMed1993

Prostacyclin is a potent anti-mutagen.

Prostacyclin (PGI2) prevented genetic damage to the bone marrow cells of mice induced by gamma-radiation, benzo(a)pyrene(BP) and cis-platinum(cis-DDP). Carba-PGI2, an analogue of PGI2, was also effective against cis-DDP-induced mutagenicity. In a time-course study it was observed that the geno-protective action of PGI2, can last as long as 24 hr. 6-keto-PGF1 alpha, a major metabolite of PGI2 and c-AMP, a second messenger, were ineffective in bringing about this beneficial action. PGI2 did not influence free radical generation induced by phorbol myristate acetate in human peripheral leukocytes. This suggests that the genoprotective action of PGI2 is not mediated by its metabolite 6-keto-PGF1 alpha and the second messenger cyclic-AMP and is not due to any action on free radical generation. This geno-protective action of PGI2 would be futile if it interfered with the tumoricidal action of cis-DDP. It was observed that the cytotoxic action of cis-DDP against Meth-A tumor cells was not interfered with by PGI2 and carba-PGI2 both in vitro and in vivo. This description of the geno-protective action of PGI2 is important in the development of new strategies in cancer chemotherapy since, it is likely that anticancer drugs, at least cis-DDP can be given along with PGI2 to prevent genetic damage to normal cells without interfering with their tumoricidal action.

6-Ketoprostaglandin F1 alpha↗

PubMed1993

Sensitivity of non-cooperative binding parameters of ligand-DNA complex by computer analysis.

A simple user-friendly computer programme has been developed to operate on an IBM-PC compatible machine to aid in the process of curve fitting using the McGhee and von Hippel equation [J. Mol Biol, 86 (1974), 469-489] for the analysis of ligand-DNA interactions and the experimental data on berberine-calf thymus DNA and berberine-poly(dI-dC). poly(dI-dC) for non-cooperative binding. A sensitivity analysis on binding constant (K0) and the number of binding sites (N0) show that a small variation in the latter remarkably alters the fitting curve. At a level of 1% change in N0 from the best fit value, the standard deviation grows by almost 4%, while, on the other hand, a 1% change in K0 affects the same value only by less than 0.4%.

Berberine↗

PubMed1992

Cytotoxic action of cis-unsaturated fatty acids on human cervical carcinoma (HeLa) cells: relationship to free radicals and lipid peroxidation and its modulation by calmodulin antagonists.

Specific fatty acids such as linoleic acid (LA), gamma-linolenic acid (GLA), dihomo gamma linolenic acid (DGLA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) showed cytotoxicity towards human cervical (HeLa) cells in vitro. Cyclo-oxygenase inhibitor, indomethacin; lipoxygenase inhibitor, nordihydroguiaretic acid (NDGA); anti-oxidant, vitamin E; and calmodulin antagonists, trifluoperazine (TFP) and chlorpromazine (CPZ) blocked the cytotoxic action of these fatty acids. GLA-induced free radical generation and lipid peroxidation were also inhibited by indomethacin, NDGA, vitamin E, TFP and CPZ. Both indomethacin and NDGA also showed significant anti-oxidant property. These results suggest that fatty acid-induced cytotoxic action against HeLa cells is a free radical dependent process and that it can be modulated by calmodulin antagonists. These results are in contrast to those observed by us earlier with human breast cancer cells where in it was found that the tumoricidal action of fatty acids can be blocked by anti-oxidants but not by cyclo-oxygenase (CO) and lipoxygenase (LO) inhibitors. From these results it can be suggested that though free radicals are the mediators of the tumoricidal action of fatty acids, the mechanism of their production may be different in different types of tumor cells.

Calmodulin↗

PubMed1992

Extracellular protease from the antarctic yeast Candida humicola.

The psychrotrophic, dimorphic yeast Candida humicola, isolated from Antarctic soil, secretes an acidic protease into the medium. The secretion of this protease by C. humicola was found to be dependent on the composition of the medium. In YPD or yeast nitrogen base medium containing either amino acids or ammonium sulfate as the nitrogen source, the activity of the protease in the medium was low (basal level). However, when yeast nitrogen base medium was depleted of amino acids or ammonium sulfate and supplemented with proteins, the activity of the enzyme increased. The secretion of the enzyme was greater during exponential growth at low temperatures than during growth at higher temperatures. The purified protease had a molecular mass of 36,000 Da and was inhibited by pepstatin, iodoacetamide, and sodium dodecyl sulfate. Despite the prevalent cold temperatures in Antarctica, this extracellular protease of the psychrotrophic yeast C. humicola was active at temperatures ranging from 0 to 45 degrees C, with an optimum activity at 37 degrees C.

Adaptation, Physiological↗

PubMed1992

Synthesis and secretion of VLDL by rat hepatocytes--modulation by cholesterol and phospholipids.

The synthesis and secretion of apoB, the major protein component of very low density lipoprotein (VLDL) and low density lipoprotein (LDL), were studied using rat hepatocytes maintained in primary culture. Supplementation of hepatocytes with rat serum VLDL and LDL increased the production of apoB while delipidated lipoproteins had no significant effect, suggesting a role for lipids in the production of apoB. Addition of cholesterol to the culture medium also increased the production of apoB in a concentration-dependent manner. Pulse labelling followed by chase in presence of cholesterol indicated enhancement in apoB secretion. Mevinolin which inhibits cholesterol synthesis significantly reduced the secretion of apoB. The presence of phosphatidylcholine and phosphatidylethanolamine in the culture medium also increased the secretion of apoB into the medium. These data suggest that availability of lipids, particularly cholesterol, is an important determinant of apoB synthesis and secretion as VLDL.

Animals↗

PubMed1992

Metabolism of very low density lipoproteins--effect of sardine oil.

The effect of feeding fish oil on the metabolism of lipoproteins was studied in rats. Rats were fed diet containing 10% sardine or groundnut oil for 6 weeks. There was a significant decrease in the total cholesterol, phospholipids and triglycerides as well as the amount of the lipids associated with VLDL and LDL in serum in fish oil-fed rats. The synthesis and secretion of lipoproteins particularly apoB containing lipoproteins by primary cultures of hepatocytes from these rats were studied by 14(C)-acetate or 3(H)-leucine labelling. Primary cultures of hepatocytes derived from sardine oil-fed rats showed reduced incorporation of 3(H)-leucine into apoB containing lipoproteins secreted into the medium when compared to those fed groundnut oil, indicating a decreased synthesis and secretion of apoB. This was further confirmed by significantly lower incorporation of 14(C)-radioactivity into total and individual lipids of VLDL secreted into the medium, as well as that associated with different lipids in cell layer. The activity of lipoprotein lipase in adipose tissue and aorta was significantly higher in rats fed sardine oil which may cause an increased clearance of triglyceride-rich lipoproteins from circulation. These results indicate that the fish oil exerts hypolipidemic effect particularly by decreasing the synthesis and secretion of VLDL by liver and possibly by an increased clearance of triglyceride-rich lipoproteins from circulation.

Administration, Oral↗

PubMed1992

Conformational aspects of poly(dI-dC).poly(dI-dC) and poly(dG-dC).poly(dG-dC) on binding of the alkaloid, berberine chloride.

Interaction of berberine chloride with poly(dI-dC).poly(dI-dC) and poly(dG-dC).poly(dG-dC) has been studied by various spectroscopic methods. Comparative data on binding parameters, fluorescence enhancement and Tm measurements indicate a stronger binding of berberine to the IC polymer than to the GC one. Conformational changes as monitored by circular dichroic spectra indicate change of the unusual circular dichroism of poly(IC) to a conformation similar to A-conformation, while in poly(GC) only a B-conformational change is observed. Extrinsic CD bands developed in the visible absorption region of berberine on interaction with poly(IC) are of much higher intensity compared to poly(GC). It is concluded that berberine induces a conformational change similar to the A-conformation on binding to poly(IC).

Berberine↗

PubMed1991

Reactive oxygen species, lipid peroxides and essential fatty acids in patients with rheumatoid arthritis and systemic lupus erythematosus.

We studied free radical generation, lipid peroxidation and the levels of essential fatty acids and of their metabolites in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Both superoxide and hydrogen peroxide generation by peripheral leukocytes but not malondialdehyde levels, as measured by thiobarbituric acid assay, were found to be significantly enhanced both in RA and SLE. Fatty acid analysis of the plasma PL fraction revealed that both LA and ALA metabolites are significantly decreased in RA and SLE compared to controls. These results suggest that essential fatty acid metabolism is altered in RA and SLE.

Adult↗