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Biomedical subjects

G S Kumar

Publications and source records attributed to G S Kumar.

At least 19 recordsLinked to original sources
PubMed2004

Primary intraosseous odontogenic carcinoma with osteoid/dentinoid formation.

Primary intraosseous carcinoma/odontogenic carcinomas are extremely rare malignant odontogenic tumours that are thought to arise from residues of odontogenic epithelium. An unusual case of primary intraosseous carcinoma arising de novo in a previously unreported site of posterior maxilla is described. The tumour was characterized by sheets of pleomorphic round-to-ovoid cells and marked osteoid/dentinoid formation with foci of globular mineralization.

Adolescent↗
PubMed2003

Permanent maxillary central incisor impaction: report of two cases.

Impaction of the permanent maxillary central incisor is rare. Trauma to the primary maxillary anterior teeth is the most common cause. Two case reports of impacted permanent maxillary central incisors with a history of trauma to the primary maxillary anterior teeth are presented. In one case there was radiographic evidence of complete arrest of root formation, and in the other case the root was dilacerated from the cervical third and the enamel surface was rough.

Adult↗
PubMed2000

Synthesis and characterization of a peptide identified as a functional element in alphaA-crystallin.

Eye lens alpha-crystallin is a member of the small heat shock protein (sHSP) family and forms large multimeric structures. Earlier studies have shown that it can act like a molecular chaperone and form a stable complex with partially unfolded proteins. We have observed that prior binding of the hydrophobic protein melittin to alpha-crystallin diminishes its chaperone-like activity toward denaturing alcohol dehydrogenase, suggesting the presence of mutually exclusive sites for these proteins in alpha-crystallin. To investigate the mechanism of the interaction between alpha-crystallin and substrate proteins, we determined the melittin-binding sites in alpha-crystallin by cross-linking studies. Localization of melittin-binding sites in alpha-crystallin resulted in the identification of RTLGPFYPSR and FVIFLDVKHFSPEDLTVK of alphaA-crystallin and FSVNLDVK of alphaB-crystallin as the chaperone sites. Of these sites, FVIFLDVKHFSPEDLTVK and FSVNLDVK were identified earlier as 1,1'-bi(4-anilino) naphthalene-5,5'-disulfonic acid (bis-ANS)-binding hydrophobic sites. Here we also report the synthesis and characterization of the peptide, KFVIFLDVKHFSPEDLTVK, having the melittin as well as bis-ANS-binding sequence of alphaA-crystallin. We show that this peptide has characteristics similar to that of alphaA-crystallin by in vitro thermal aggregation assay, gel filtration study, CD spectroscopy, and bis-ANS interaction studies. The peptide sequence corresponds to the beta3 and beta4 region present in the alpha-crystallin domain of sHSP 16.5. We hypothesize that the alpha-crystallin domain in other sHSPs may have a similar function and would likely possess the anti-aggregation property even when separated from the native protein.

Alcohol Dehydrogenase↗
PubMed1999

Metabolic suppression of platelet-type 12-lipoxygenase in human uterine cervix with invasive carcinoma.

Several types of lipoxygenases with various functions occur in mammalian cells. Although the presence of 12-lipoxygenase activity has been reported in uterine tissues, neither its type nor its biological functions have yet been established. Moreover, the putative role of uterine 12-lipoxygenase in cervical cancer has not been addressed before. Homogenates of uterine tissues from women without cancer and from patients with invasive cervical carcinoma were incubated with (1-(14)C)-arachidonic acid under various conditions and the labelled reaction products were analyzed both by thin-layer chromatography and by high-pressure liquid chromatography. 12-Lipoxygenase protein was estimated by Western blot using anti-serum against recombinant human platelet-type 12-lipoxygenase. Highest concentrations and activities of 12-lipoxygenase were found in the exocervix. The formation of 12S-hydroxy-5Z,8Z,10E, 14Z-eicosatetraenoic acid (12-HETE) was stimulated by micromolar concentrations of 13S-hydroperoxy-9Z,11E-octadecadienoic acid, suggesting metabolic control of the 12-lipoxygenase activity via the hydroperoxide tone. Immunohistochemical investigation revealed that the enzyme is mainly located in the squamous epithelium, and is of platelet-type. Significantly lower values for the 12-HETE formation were found in samples from patients with invasive cervical carcinoma, whereas the amount of immunochemically detectable 12-lipoxygenase protein was unaltered. At the same time the expression levels of the bcl-2 gene were enhanced. Thus, it is concluded that during carcinogenesis the hydroperoxide-reducing capacity of the uterine cervix tissue is enhanced, possibly mediated by bcl-2 protein, and in turn metabolically suppresses the 12-lipoxygenase activity. Furthermore, the data suggest an anti-carcinogenic action of 12-lipoxygenase in human cervix, in contrast to its reported pro-carcinogenic action in breast cancer.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1999

9-Deoxopodophyllotoxin derivatives as anti-cancer agents.

Several 9-deoxo-9-substituted podophyllotoxin derivatives were synthesised starting from naturally occuring podophyllotoxin and their anti-cancer activity was evaluated against in vitro human cancer cell line assay. It was observed that these compounds do possess good anti-cancer activity particularly against ovarian, renal and lung cancer cell lines.

Antineoplastic Agents↗
PubMed1999

A RNA polymerase with transcriptional activity at 0 degrees C from the Antarctic bacterium Pseudomonas syringae.

A DNA-dependent RNA polymerase was purified from the Antarctic psychrotrophic bacterium Pseudomonas syringae. The RNA polymerase showed a typical eubacterial subunit composition with beta, beta', alpha2 and sigma subunits. The subunits cross-reacted with antibodies raised against holoenzyme and the individual subunits of the RNA polymerase of Escherichia coli. However, the enzyme was considered unique, since unlike the RNA polymerase of mesophilic E. coli it exhibited significant and consistent transcriptional activity (10-15%) even at 0 degrees C. But, similar to the enzyme from the mesophilic bacterium, the RNA polymerase from P. syringae exhibited optimum activity at 37 degrees C. The study also demonstrates that the RNA polymerase of P. syringae could preferentially transcribe the cold-inducible gene cspA of E. coli only at lower temperatures (0-22 degrees C). The polymerase was also observed to be relatively more rifampicin-resistant during transcription at lower temperature.

Adaptation, Biological↗
PubMed1999

Spectroscopic studies on the interaction of aristololactam-beta-D-glucoside with DNA and RNA double and triple helices: A comparative study.

The interaction of aristololactam-beta-D-glucoside (ADG), a DNA intercalating alkaloid, with the DNA triplexes, poly(dT). poly(dA)xpoly(dT) and poly(dC).poly(dG)xpoly(dC+), and the RNA triplex poly(rU).poly(rA)xpoly(rU) was investigated by circular dichroic, UV melting profile, spectrophotometric, and spectrofluorimetric techniques. Comparative interaction with the corresponding Watson-Crick duplexes has also been examined under identical experimental conditions. Triplex formation has been confirmed from biphasic thermal melting profiles and analysis of temperature-dependent circular dichroic measurements. The binding of ADG to triplexes and duplexes is characterized by the typical hypochromic and bathochromic effects in the absorption spectrum, quenching of steady-state fluorescence intensity, a decrease in fluorescence quantum yield, an increase or decrease of thermal melting temperatures, and perturbation in the circular dichroic spectrum. Scatchard analysis indicates that ADG binds both to the triplexes and the duplexes in a noncooperative manner. Binding parameters obtained from spectrophotometric measurements are best fit by the neighbor exclusion model. The binding affinity of ADG to the DNA triplexes is substantially stronger than to the RNA triplex. Thermal melting study further indicates that ADG stabilizes the Hoogsteen base-paired third strand of the DNA triplexes whereas it destabilizes the same strand of RNA triplex but stabilizes its Watson-Crick strands. Comparative data reveal that ADG exhibits a stronger binding to the triple helical structures than to the respective double helical structures.

Alkaloids↗
PubMed1999

Conversions of the left-handed form and the protonated form of DNA back to the bound right-handed form by sanguinarine and ethidium: a comparative study.

The interaction of sanguinarine and ethidium with right-handed (B-form), left-handed (Z-form) and left-handed protonated (designated as H(L)-form) structures of poly(dG-dC).poly(dG-dC) and poly(dG-me5dC).poly(dG-me5dC) was investigated by measuring the circular dichroism and UV absorption spectral analysis. Both sanguinarine and ethidium bind strongly to the B-form DNA and convert the Z-form and the H(L)-form back to the bound right-handed form. Circular dichroic data also show that the conformation at the binding site is right-handed, even though adjacent regions of the polymer have a left-handed conformation either in Z-form or in H(L)-form. Both the rate and extent of B-form to Z-form transition were decreased by sanguinarine and ethidium under ionic conditions that otherwise favour the left-handed conformation of the polynucleotides. The rate of decrease is faster in the case of ethidium as compared to that of sanguinarine. Scatchard analysis of the spectrophotometric data shows that sanguinarine binds strongly to both the polynucleotides in a non-cooperative manner under B-form conditions, in sharp contrast to the highly-cooperative binding under Z-form and H(L)-form conditions. Correlation of binding isotherms with circular dichroism data indicates that the cooperative binding of sanguinarine under the Z-form and the H(L)-form conditions is associated with a sequential conversion of the polymer from a left-handed to a bound right-handed conformation. Determination of bound alkaloid concentration by spectroscopic titration technique and the measurement of circular dichroic spectra have enabled us to calculate the number of base pairs of Z-form and H(L)-form that adopt a right-handed conformation for each bound alkaloid. Analysis reveals that 2-3 base pairs (bp) of Z-form of poly(dG-dC).poly(dG-dC) and poly(dG-me5dC).poly(dG-me5dC) switch to the right-handed form for each bound sanguinarine, while approximately same number of base pairs switch to the bound right-handed form in complexes with H(L)-form of these polynucleotides. Comparative binding analysis shows that ethidium also converts approximately 2 bp of Z-form or H(L)-form to bound right-handed form under same experimental conditions. Since sanguinarine binds preferentially to alternating GC sequences, which are capable of undergoing the B to Z or B to H(L) transition, these effects may be an important part in understanding its extensive biological activities.

Journal Article↗
PubMed1999

Transcriptional activity at supraoptimal temperature of growth in the antarctic psychrotrophic bacterium Pseudomonas syringae.

Transcriptional activity was monitored in cells of the Antarctic psychrotrophic bacterium Pseudomonas syringae (Lz4W), which does not grow above 30 degrees C. It was observed that the bacterium was capable of synthesising RNA at a temperature range of 0-37 degrees C, both in vitro and in vivo. The net incorporation of the radioactive precursor, [3H]uridine, into RNA was found to be affected at 37 degrees C. A pulse-chase experiment following a 32P labeling of RNA in vivo indicated that the ribosomal RNAs (rRNAs) degrade faster at and above 30 degrees C. It was also found that the increased ribonuclease (RNase) activity at high temperature might be responsible for this degradation. The attack on ribosomal RNAs by RNase took place after their assembly into ribosomal particles. It is suggested that the degradation of rRNAs at supraoptimal temperatures might be a detrimental factor for growth above 30 degrees C.

Animals↗
PubMed1999

Field's stain--a rapid staining method for Acanthamoeba spp.

Acanthamoeba sp. is a free-living amoeba known to cause chronic central nervous system infection or eye infection in humans. Many cases remain undetected for want of a good detection system. We report for the first time a rapid staining method to facilitate the identification of Acanthamoeba sp. using the modified Field's staining technique. A. castellanii, which was used in the present experiment, is maintained in our laboratory in mycological peptone medium (Gibco). The cultures were pooled together and smears were made on glass slides for staining purposes. Different types of stains such as Field's stain, modified Field's stain, Wright's stain, Giemsa stain, Ziehl-Neelsen stain, and trichrome stain were used to determine the best stain for the identification of this amoeba. The concentration of various stains and the duration of staining were varied to provide the best color and contrast for each stain. Acanthamoeba was also obtained from the brain of experimentally infected mice and was stained with various stains as mentioned above to determine the best stain for use in identifying the presence of this parasite in experimentally infected animals. The modified Field's stain gives a very good color contrast as compared with other stains. Furthermore, it takes only 20 s to be carried out using the least number of reagents, making it suitable for both laboratory and field use.

Acanthamoeba↗
PubMed1998

Identification of 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid binding sequences in alpha-crystallin.

The hydrophobic binding sites in alpha-crystallin were evaluated using fluorescent probes 1,1'-bi(4-anilino)naphthalenesulfonic acid (bis-ANS), 8-anilino-1-naphthalene sulfonate (ANS), and 1-azidonaphthalene 5-sulfonate (1,5-AZNS). The photolysis of bis-ANS-alpha-crystallin complex resulted in incorporation of the probe to both alphaA- and alphaB-subunits. Prior binding of denatured alcohol dehydrogenase to alpha-crystallin significantly decreased the photoincorporation of bis-ANS to alpha-crystallin. Localization of bis-ANS incorporated into alphaA-crystallin resulted in the identification of residues QSLFR and HFSPEDLTVK as the fluorophore binding regions. In alphaB-crystallin, sequences DRFSVNLNVK and VLGDVIEVHGK were found to be the bis-ANS binding regions. Of the bis-ANS binding sequences, HFSPEDLTVK of alphaA-crystallin and DRFSVNLNVK and VLGDVIEVHGK of alphaB-crystallin were earlier identified as part of the sequences involved in their interaction with target proteins during the molecular chaperone-like action. The hydrophobic probe, 1,5-AZNS, also interacted with both subunits of alpha-crystallin. Localization of 1,5-AZNS binding site in alphaB-crystallin lead to the identification of HFSPEEK sequence as the interacting site in this subunit of alpha-crystallin. Glycated alpha-crystallin displayed decreased ANS fluorescence and loss of chaperone-like function, suggesting the involvement of glycation site as well as ANS binding site in chaperone-like activity display.

Amino Acid Sequence↗
PubMed1998

Interaction of 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid with alpha-crystallin.

The hydrophobic sites in alpha-crystallin were evaluated using a fluorescent probe 1,1'-bi(4-anilino)naphthalenesulfonic acid (bis-ANS). Approximately one binding site/subunit of alpha-crystallin at 25 degrees C was estimated by equilibrium binding and Scatchard analysis (Kd = 1.1 microM). Based on fluorescence titration, the dissociation constant was 0.95 microM. The number of bis-ANS binding sites nearly doubled upon heat treatment of the protein at 60 degrees C. Likewise, the exposure of alpha-crystallin to 2-3 M urea resulted in increased binding of bis-ANS. Above 3 M urea there was a rapid loss in the fluorescence indicating the loss of interaction between bis-ANS and protein. The alpha-crystallin refolded from 6 M urea showed tryptophan fluorescence emission similar to the native alpha-crystallin. However, the refolded alpha-crystallin showed a 60% increase in bis-ANS binding, suggesting distinct changes on the protein surface resulting from exposure to urea similar to the changes occurring due to heat treatment. The fluorescence of tryptophan in native alpha-crystallin was quenched by the addition of bis-ANS. The quenching was inversely related to the amount of bis-ANS bound to alpha-crystallin. Additionally, the binding of bis-ANS reduced the chaperone-like activity of the protein. Photolysis of bis-ANS-alpha-crystallin complex resulted in incorporation of the probe to both A- and B-subunits, indicating that both subunits in native alpha-crystallin contribute to the surface hydrophobicity of the protein.

Anilino Naphthalenesulfonates↗
PubMed1998

Formation of a major DNA adduct of the mitomycin metabolite 2,7-diaminomitosene in EMT6 mouse mammary tumor cells treated with mitomycin C.

Treatment of EMT6 mouse mammary tumor cells with [3H]mitomycin C (MC) results in the formation of six major DNA adducts, as described earlier using an HPLC assay of 3H-labeled products of enzymatic hydrolysis of DNA isolated from MC-treated cells. Four of these adducts were identified as monofunctional and bifunctional guanine-N2 adducts in the minor groove of DNA. In order to establish relationships between individual types of MC-DNA adducts and biological responses it is necessary to identify all of the adducts formed in cells. To this end we have now identified a predominant, previously unknown adduct formed in MC-treated EMT6 cells as a derivative not of MC, but of 2,7-diaminomitosene (2,7-DAM), the major bioreductive metabolite of MC. Rigorous proof demonstrates that it is a DNA major groove, guanine-N7 adduct of 2,7-DAM, linked at C-10 to DNA. The adduct is relatively stable at ambient temperature, but is readily depurinated upon heating. Its isolation from MC-treated cells indicates that MC is reductively metabolized to 2,7-DAM, which then undergoes further reductive activation to alkylate DNA, along with the parent MC. Low MC:DNA ratios were identified as a critical factor promoting 2,7-DAM adduct formation in an in vitro model calf thymus DNA/ MC/reductase model system, as well as in MC-treated EMT6 cells. The 2,7-DAM-guanine-N7 DNA adduct appears to be relatively noncytotoxic, as indicated by the dramatically lower cytotoxicity of 2,7-DAM in comparison with MC in EMT6 cells. Like MC, 2,7-DAM exhibited slightly greater cytotoxicity to cells treated under hypoxic as compared to aerobic conditions. However, 2,7-DAM was markedly less cytotoxic than MC under both aerobic and hypoxic conditions. Thus, metabolic reduction of MC to 2,7-DAM represents a detoxification process. The differential effects of MC-DNA and 2,7-DAM-DNA adducts support the concept that specific structural features of the DNA damage may play a critical role in the cytotoxic response to a DNA-targeted chemotherapeutic agent.

Animals↗
PubMed1998

Interaction of the antitumour alkaloid coralyne with duplex deoxyribonucleic acid structures: spectroscopic and viscometric studies.

The interaction of coralyne, an antitumour alkaloid with natural and synthetic duplex DNAs was investigated under conditions where the drug existed fully as a true monomer for the first time using spectrophotometric, spectrofluorimetric, circular dichroic and viscometric techniques. The absorption spectrum of coralyne monomer showed hypochromic and bathochromic effects on binding to duplex DNAs. This effect was used to determine the binding parameters of coralyne. The binding constants for four natural DNAs and four synthetic polynucleotides obtained from spectrophotometric titration, according to an excluded site model, using McGhee-von Hippel analysis, were all in the range of (0.38-9.8) x 10(5) M-1, and showed a relatively high specificity for the GC rich ML DNA and the alternating GC polynucleotide. The binding of coralyne decreased with increasing ionic strength, indicating that the binding affinity has a strong electrostatic component. Coralyne stabilized all the DNAs against thermal strand separation. The intense steady state fluorescence of coralyne was effectively quenched on binding to DNAs and the quantitative data on the Stern-Volmer quenching constant obtained was sequence dependent, being maximum with the GC rich DNA and alternating GC polymer. Circular dichriosm studies further evidenced for a strong perturbation of the B-conformation of DNAs consequent to coralyne binding with the concomitant development of extrinsic circular dichroic bands for the bound drug molecules suggesting their strong intercalated geometry in duplex DNAs. Further tests of intercalation using viscosity measurements on linear and covalently closed plasmid DNA conclusively proved the strong intercalation of coralyne in duplex DNA. Binding of the closely related natural alkaloid, berberine under these conditions showed considerably lower affinity to duplex DNAs in all experiments. Taken together, these results suggest that coralyne binds strongly to duplex DNAs by a mechanism of intercalation with specificity towards alternating GC duplex structure.

Animals↗
PubMed1997

Production of 3R-hydroxy-polyenoic fatty acids by the yeast Dipodascopsis uninucleata.

Various fatty acids were fed to the yeast Dipodascopsis uninucleata UOFS Y 128, and the extracted samples were analyzed for the accumulation of 3-hydroxy metabolites with the help of electron impact gas chromatography-mass spectrometry. Fatty acids containing of 5Z,8Z-diene system (5Z,8Z,11Z-eicosatrienoic, 5Z,8Z,11Z,14Z-eicosatetraenoic, and 5Z,8Z,11Z,14Z,17Z-eicosapentaenoic acids) yielded the corresponding 3-hydroxy-all-Z-eicosapolyenoic acids. Moreover, linoleic acid (9Z,12Z-octadecadienoic acid) and 11Z,14Z,17Z-eicosatrienoic acid were converted to the 3-hydorxylated metabolites of shorter chain length, e,g., 3-hydroxy-5Z,8Z-tetradecadienoic acid and 3-hydroxy-5Z,8Z,11Z-tetradecatrienoic acid, respectively. In contrast, no accumulation of a 3-hydroxy metabolite was observed with oleic acid (9Z-octadecenoic acid), linolelaidic acid (9E,12E-octadecadienoic acid), gamma-linolenic acid (6Z,9Z,12Z-octadecatrienoic acid), and eicosanoic acid as substrate. These findings pinpoint that the 3-hydroxylation of a fatty acid in Dipodascopsis uninucleata requires a 5Z,8Z-diene system either directly or following initial incomplete beta-oxidation. Following analysis of the enantiomer composition, the arachidonic acid metabolite was identified as 3R-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid, which rules out a normal beta-oxidation as biosynthetic route to this new class of oxylipins.

Arachidonic Acids↗
PubMed1997

Single cell gel electrophoresis on peripheral blood leukocytes of patients with oral squamous cell carcinoma.

The single cell gel electrophoresis (SCGE) assay, also known as the comet assay, is a cytogenetic technique for measuring and analyzing DNA single stranded breaks (SSB) and/or alkali labile sites within individual cells. Peripheral blood leukocytes of 22 oral squamous cell carcinoma (OSCC) patients were subjected to SCGE and the DNA damage levels (SSB) were quantified with respect to clinical staging and histopathologic grading. Highly statistically significant differences in DNA damage levels were found between normal subjects and patients with OSCC of the same age group. DNA damage levels were altered in all clinical stages and histopathological grades of oral squamous cell carcinoma. The differences were generally significant between all the clinical stages of OSCC, while in histopathologic grading the results were significant only between grades I and III. The results support the concept of a systemic host response in malignancy.

Adult↗
PubMed1995

Free radical-dependent suppression of growth of mouse myeloma cells by alpha-linolenic and eicosapentaenoic acids in vitro.

Alpha-linolenic acid (ALA) and eicosapentaenoic acid (EPA) exhibited potent cytotoxic action on SP 2/0 mouse myeloma cells in vitro. Both SOD and vitamin E could inhibit the action of ALA and EPA indicating a role for reactive oxygen species and lipid peroxides. In addition, both ALA and EPA enhanced the formation of superoxide anion, hydrogen peroxide and lipid peroxides, and caused a reduction in the levels of antioxidant enzymes: SOD, catalase and glutathione peroxidase and induced significant damage to DNA in SP 2/0 cells. Thus, ALA and EPA inhibit antioxidant defenses of the cell and damage the DNA, which can ultimately lead to tumor cell lysis.

Animals↗