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Biomedical subjects

S Mitra

Publications and source records attributed to S Mitra.

At least 91 records · Page 5Linked to original sources
PubMed2000

Characterization of a novel 8-oxoguanine-DNA glycosylase activity in Escherichia coli and identification of the enzyme as endonuclease VIII.

8-Oxoguanine (G*), induced by reactive oxygen species, is mutagenic because it mispairs with A. The major G*-DNA glycosylase (OGG), namely, OGG1 in eukaryotes, or MutM in Escherichia coli, excises G* when paired in DNA with C, G, and T, but not A, presumably because removal of G* from a G*.A pair would be mutagenic. However, repair of G* will prevent mutation when it is incorporated in the nascent strand opposite A. This could be carried out by a second OGG, OGG2, identified in yeast and human cells. We have characterized a new OGG activity in E. coli and then identified it to be endonuclease VIII (Nei), discovered as a damaged pyrimidine-specific DNA glycosylase. Nei shares sequence homology and reaction mechanism with MutM and is similar to human OGG2 in being able to excise G* when paired with A (or G). Kinetic analysis of wild type Nei showed that it has significant activity for excising G* relative to dihydrouracil. The presence of OGG2 type enzyme in both E. coli and eukaryotes, which is at least as efficient in excising G* from a G*.A (or G) pair as from a G*.C pair, supports the possibility of G* repair in the nascent DNA strand.

DNA Damage↗
PubMed2000

Deficiency of N-methylpurine-DNA-glycosylase expression in nonparenchymal cells, the target cell for vinyl chloride and vinyl fluoride.

The ability to repair promutagenic damage resulting from exposure to carcinogens is a critical factor in determining quantitative relationships in carcinogenesis, including the target cell for neoplasia. One major pathway for the repair of alkylating agent-induced DNA damage involves removal of alkylated bases by N-methylpurine-DNA-glycosylase (MPG), the first enzyme in base excision repair. We have measured the expression level of MPG mRNA in liver, lung, and kidney of Sprague-Dawley rats as a function of age. A quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) method was used to measure cellular MPG mRNA. MPG mRNA was readily detectable in each tissue analyzed and the age-dependent and tissue specific expressions were not statistically different. The lowest amount of mRNA was measured in preweanling liver and the highest amounts were found in preweanling lung and kidney. Since MPG is reported to be responsible for excision of 1,N(6)-ethenoadenine and N(2),3-ethenoguanine, two promutagenic DNA adducts of vinyl chloride (VC) and vinyl fluoride (VF), we examined the regulation of this enzyme after carcinogen exposure. Expression of MPG was induced in rat liver by these carcinogens. In order to determine the repair capacity in different cell populations of liver, we measured MPG gene expression in isolated hepatocytes and nonparenchymal cells (NPC). The amount of MPG mRNA was 4.5-5 times higher in hepatocytes than in NPC of control rats. Induction of MPG expression was observed in hepatocytes of VF exposed-rats but not in NPC. The expression of MPG in NPC was only 15% of that of the hepatocytes from exposed rats. Western blots of MPG protein confirmed the cell type differences, but did not show increased protein in exposed vs. control liver and hepatocytes. Since metabolism of VC and VF requires CYP2E1, an enzyme exhibiting much greater activity in hepatocytes, formation of etheno adducts preferentially occurs in hepatocytes. These data suggest that cellular differences in the repair of N-alkylpurines may be a critical mechanism in the development of cell specificity in VC carcinogenesis.

Aging↗
PubMed2000

Binding of camphor to Pseudomonas putida cytochrome p450(cam): steady-state and picosecond time-resolved fluorescence studies.

The binding of camphor to cytochrome P450(cam) has been investigated by steady-state and time-resolved tryptophan fluorescence spectroscopy to obtain information on the substrate access channel. The fluorescence quenching experiments show that some of the tryptophan residues undergo changes in their local environment on camphor binding. The time-resolved fluorescence decay profile gives four lifetime components in the range from 99 ps to 4.5 ns. The shortest lifetime component assigned to W42 lies close to the proposed camphor access channel. The results show that the fluorescence of W42 is greatly affected on binding of camphor, and supports dynamic fluctuations involved in the passage of camphor through the access channel as proposed earlier on the basis of crystallographic, molecular dynamics simulation and site-directed mutagenesis studies.

Camphor↗
PubMed2000

Mechanism of the inhibition of milk xanthine oxidase activity by metal ions: a transient kinetic study.

The nature and mechanism of the inhibition of the oxidoreductase activity of milk xanthine oxidase (XO) by Cu(2+), Hg(2+) and Ag(+) ions has been studied by steady state and stopped flow transient kinetic measurements. The results show that the nature of the inhibition is noncompetitive. The inhibition constants for Cu(2+) and Hg(2+) are in the micromolar and that for Ag(+) is in the nanomolar range. This suggests that the metal ions have strong affinity towards XO. pH dependence studies of the inhibition indicate that at least two ionisable groups of XO are involved in the binding of these metal ions. The effect of the interaction of the metal ions on the reductive and oxidative half reactions of XO has been investigated, and it is observed that the kinetic parameters of the reductive half reaction are not affected by these metal ions. However, the interaction of these metal ions with XO significantly affects the kinetic parameters of the oxidative half reaction. It is suggested that this may be the main cause for the inhibition of XO activity by the metal ions.

Animals↗
PubMed2000

Mitochondrial DNA damage and a hypoxic response are induced by CoCl(2) in rat neuronal PC12 cells.

Generation of reactive oxygen species (ROS) and activation of a transcriptional program that mimics the hypoxic response have been documented in cultured cells in the presence of cobalt chloride. We found that in the presence of hypoxia-mimicking concentrations of CoCl(2), mitochondrial but not nuclear DNA damage is induced in rat neuronal, PC12 cells. To our knowledge, this is the first documentation of induction of mitochondrial DNA (mtDNA) damage under these conditions. Likewise, we provide the first evidence for elevation of MYH, the mammalian homolog of the Escherichia coli MutY DNA glycosylase, in mammalian cells. Recently, the human MYH was implicated in repair of oxidative DNA damage and shown to carry a mitochondrial localization sequence. Here, an induction of mtDNA damage and a time-dependent increase in the MYH level were detected with exposure of cells to 100 microM CoCl(2). In addition, the levels of proteins involved in cellular responses to hypoxia, ROS and nuclear DNA damage; hypoxia-inducible factor 1alpha(HIF-1alpha), p53, p21 and PCNA were also modulated temporally. Earlier studies suggested that the mtDNA is a primary target for oxidative damage. Our findings extend these observations and suggest that activation of DNA repair processes is associated with the presence of mtDNA damage.

Animals↗
PubMed2000

Mutation of a unique aspartate residue abolishes the catalytic activity but not substrate binding of the mouse N-methylpurine-DNA glycosylase (MPG).

N-Methylpurine-DNA glycosylase (MPG) initiates base excision repair in DNA by removing a variety of alkylated purine adducts. Although Asp was identified as the active site residue in various DNA glycosylases based on the crystal structure, Glu-125 in human MPG (Glu-145 in mouse MPG) was recently proposed to be the catalytic residue. Mutational analysis for all Asp residues in a truncated, fully active MPG protein showed that only Asp-152 (Asp-132 in the human protein), which is located near the active site, is essential for catalytic activity. However, the substrate binding was not affected in the inactive Glu-152, Asn-152, and Ala-152 mutants. Furthermore, mutation of Asp-152 did not significantly affect the intrinsic tryptophan fluorescence of the enzyme and the far UV CD spectra, although a small change in the near UV CD spectra of the mutants suggests localized conformational change in the aromatic residues. We propose that in addition to Glu-145 in mouse MPG, which functions as the activator of a water molecule for nucleophilic attack, Asp-152 plays an essential role either by donating a proton to the substrate base and, thus, facilitating its release or by stabilizing the steric configuration of the active site pocket.

Amino Acid Sequence↗
PubMed2000

DNA-bound structures and mutants reveal abasic DNA binding by APE1 and DNA repair coordination [corrected].

Non-coding apurinic/apyrimidinic (AP) sites in DNA are continually created in cells both spontaneously and by damage-specific DNA glycosylases. The biologically critical human base excision repair enzyme APE1 cleaves the DNA sugar-phosphate backbone at a position 5' of AP sites to prime DNA repair synthesis. Here we report three co-crystal structures of human APE1 bound to abasic DNA which show that APE1 uses a rigid, pre-formed, positively charged surface to kink the DNA helix and engulf the AP-DNA strand. APE1 inserts loops into both the DNA major and minor grooves and binds a flipped-out AP site in a pocket that excludes DNA bases and racemized beta-anomer AP sites. Both the APE1 active-site geometry and a complex with cleaved AP-DNA and Mn2+ support a testable structure-based catalytic mechanism. Alanine substitutions of the residues that penetrate the DNA helix unexpectedly show that human APE1 is structurally optimized to retain the cleaved DNA product. These structural and mutational results show how APE1 probably displaces bound glycosylases and retains the nicked DNA product, suggesting that APE1 acts in vivo to coordinate the orderly transfer of unstable DNA damage intermediates between the excision and synthesis steps of DNA repair.

Aminopeptidases↗
PubMed2000

Acquired alkylating drug resistance of a human ovarian carcinoma cell line is unaffected by altered levels of pro- and anti-apoptotic proteins.

In a systematic study to elucidate the involvement of pro- and anti-apoptotic proteins in alkylating drug resistance of tumor cells, we utilized the A2780(100) line, that was selected by repeated exposure of A2780 cell line (human ovarian carcinoma line) to chlorambucil (CBL). A2780(100) was 5 - 10-fold more resistant to nitrogen mustards (IC50 of 50 - 60 microM) and other DNA crosslinking agents, e.g., cisplatin, and also to DNA topoisomerase inhibitor etoposide (ETO) than A2780. CBL (125 microM) induced extensive apoptosis in A2780 associated with mitochondrial damage but not in A2780(100). No significant differences were observed between A2780 and A2780(100) cells in the basal levels, or the enhanced levels in some cases after CBL treatment, of DNA repair proteins involved in repair of alkyl base adducts or in repair of DNA crosslinks or double strand break repair. However, the basal levels of anti-apoptotic proteins Bcl-xL and Mcl-1 were 4 - 8-fold higher in A2780(100) than in A2780 neither of which expressed Bcl-2. In contrast, the levels of pro-apoptotic Bax and Bak were 3 - 5-fold higher in the CBL-treated A2780 but not in A2780(100). ETO (5 microM) induced apoptosis in A2780 without altering the levels of Bax and Bak in these cells. At the same time, neither overexpression of Bcl-xL in A2780, nor its antisense expression in A2780(100), and nor overexpression of Bax in A2780(100), significantly affected drug sensitivity of either line. Our results suggest that a change in an early step in DNA damage processing which affects intracellular signaling, such as enhanced DNA double-strand break repair, could be the primary cause for development of resistance in A2780(100) cells to drugs which induce DNA crosslinks or double strand-breaks.

Antineoplastic Agents, Alkylating↗
PubMed2000

Choroidal infiltrates simulating fundal changes of acute leukemia during hematological recovery following high-dose chemotherapy in acute myelomonocytic leukemia in remission.

A young female patient of 18 who was diagnosed to have acute myelomonocytic leukemia (AML M4) developed choroidal infiltrate and fundal changes suggestive of acute leukemia deposits while she was in remission and was recovering from chemotherapy induced myelosuppression. The choroidal infiltrates were associated with peripheral blood and CSF monocytosis. The choroidal lesion resolved on its own in 2 week's time, when the peripheral and CSF monocytosis subsided. Interestingly this patient had pseudo-Chediak Higashi inclusions in leukemic blasts with normal karyotype.

Adolescent↗
PubMed2000

Leprosy and the anesthesiologist.

PURPOSE: To discuss particular aspects of leprosy (complications treatment, special population) that have implications for anesthetic management in leprous patients scheduled for surgery. SOURCE: MEDLINE and manual searches of relevant literature. Multiple MEDLINE searches (from 1966 onwards) were done, using LEPROSY as a common Medical Subject Heading (MeSH). Other headings used were: anesthesia, surgery, cardiovascular system, respiratory system, eye, skin, nervous system, genitalia, pathology, psychology and pregnancy. A large number of references were retrieved, but only 18 of them were relevant to our topic. Others were obtained by manual search and cross referencing. PRINCIPAL FINDINGS: Leprosy, especially lepromatous leprosy, is a systemic disease, affecting many organs and systems of the body, e.g., cardiovascular (cardiac dysautonomia), respiratory (impaired cough response, nasal obstruction), hepatobiliary (hepatitis), renal (nephritis), ocular (anesthesia), hematological (reduced red, white and platelet count) and osseous systems (bone resorption). CONCLUSION: Investigation of the systems likely to be affected by leprosy (e.g., complete hemogram, liver, lung and kidney function tests, Valsalva response, assessment of ocular anesthesia) should form part of a preanesthetic check up in patients with leprosy.

Anesthesia↗
PubMed2000

Photochemical oxygen consumption sensitized by a porphyrin phosphorescent probe in two model systems.

Phosphorescence quenching of certain metalloporphyrins is used to measure tissue and microvascular pO(2). Oxygen quenching of metalloporphyrin triplet states creates singlet oxygen, which is highly reactive in biological systems, and these oxygen-consuming reactions are capable of perturbing tissue oxygenation. Kinetics of photochemical oxygen consumption were measured for a Pd-porphyrin in two model systems in vitro over a range of irradiances (1.34-134 mW cm(-2)). For a given irradiance, and, after correction for differing porphyrin concentrations, rates of oxygen consumption were similar when the Pd-porphyrin was bound to bovine serum albumin and when it was taken up by tumor cells in spheroids. At irradiances comparable to those used in imaging superficial anatomy, rates of oxygen consumption were sufficiently low (2.5 microM s(-1)) that tissue oxygenation would be reduced by a maximum of 6%. An irradiance of 20 mW cm(-2), however, initiated a rate of oxygen consumption capable of reducing tissue pO(2) by at least 20-40%. These measured rates of consumption impose limitations on the use of phosphorescence quenching in thick tissues. The irreversible photobleaching of the Pd-porphyrin was also measured indirectly. The bleaching branching ratio, 23 M(-1), is significantly lower than that of porphyrin photodynamic agents.

Animals↗
PubMed2000

Sorption and desorption of the diketonitrile metabolite of isoxaflutole in soils.

Isoxaflutole is a new pre-emergence corn herbicide that undergoes rapid conversion to a diketonitrile derivative (DKN) in soils. Sorption-desorption studies were conducted in five different soils varying in physical and chemical properties. A batch equilibration technique was used with total initial aqueous solution concentrations of DKN at 0.25, 0.75, 2.0, 8.0, 25, 75, 150, and 250 mg l(-1). After the sorption process, two subsequent desorptions were conducted with an equilibration period of 7 days. A high correlation existed between the desorption coefficient, K(Fd) and the organic matter content of soils (r(2)=0.844 for the first desorption and r(2)=0.861 for the second desorption), while the clay content did not greatly influence the desorption of DKN. Although the sorption of DKN was generally reversible, a sorption-desorption hysteresis was apparent in all soils. The site energy distribution curves emphasized the fact that DKN binds tightly to soils with higher organic matter content and greater proportion of DKN was retained by those soils

Journal Article↗
PubMed2000

Epithelial healing rates with topical ciprofloxacin, ofloxacin, and ofloxacin with artificial tears after photorefractive keratectomy.

PURPOSE: To determine whether corneal epithelial healing differs after the use of topical ciprofloxacin alone, topical ofloxacin alone, or topical ofloxacin with artificial tears in patients having photorefractive keratectomy (PRK). SETTING: Department of Ophthalmology and Visual Science, The University of Texas Health Science Center at Houston, Houston, Texas, USA. METHODS: Eighteen patients (6 women, 12 men) with moderate myopia (-1.50 to -6.00 diopters [D]) had standardized PRK. Patient age ranged from 25 to 62 years. The 28 eyes (16 right, 12 left) were randomized into 3 treatment groups: ofloxacin alone, n = 9 eyes; ciprofloxacin, n = 9 eyes; and ofloxacin with Refresh Plus, n = 10 eyes. The drugs were administered immediately after surgery and then every 6 hours. Video recordings of the corneal wounds stained with fluorescein were performed at 8:00 AM and 4:00 PM using a video slitlamp camera with a cobalt-blue light until the wound completely healed. The videotaped images were recorded and analyzed by a computer planimetry program. Wound areas were recorded and compared among the 3 drugs. The square-root transformation was applied to the wound area to obtain a constant healing rate. Statistical comparisons were analyzed using an analysis of variance test. RESULTS: Mean recovery time was 82.67 hours +/- 14.42 (SD) in the ofloxacin eyes, 120.89 +/- 34.05 hours in the ciprofloxacin eyes, and 76.80 +/- 19.30 hours in the ofloxacin with Refresh Plus eyes. Mean healing rate was 0.66 +/- 0.17 hours, 0.54 +/- 0.16 hours, and 0.67 +/- 0.15 hours, respectively. The healing rate was significantly higher in the ofloxacin with Refresh Plus eyes than in the ciprofloxacin eyes (P < .0001). There was no significant difference between the ofloxacin eyes and the ofloxacin with Refresh Plus eyes (P = .42). CONCLUSION: Ofloxacin with Refresh Plus and ofloxacin alone had a more positive effect on epithelial healing than ciprofloxacin. The ciprofloxacin eyes were significantly more prone to impaired or delayed wound healing and to the development of corneal haze.

Administration, Topical↗
PubMed2000

Advantages of rhythmic movements at resonance: minimal active degrees of freedom, minimal noise, and maximal predictability.

Using time delay embedding, the authors applied phase space reconstruction to the time series of rhythmic movements of a hand-held pendulum. Subjects (N = 6) produced the manual oscillations about the wrist at the pendulum's resonant frequency and at a higher and a lower frequency. The number of active degrees of freedom required to capture the dynamics of the rhythmic behavior was 3 for the resonant frequency and 4 for each of the nonresonant frequencies. The residual high-dimensional noise was similarly lowest for the resonant frequency. Whereas 33% and 20%, respectively, of the vectors in the phase spaces of the dynamics higher and lower than resonance were unpredictable, only 12% were unpredictable at resonance. Finally, the predictability of the evolving dynamics extended farther into the future for oscillations at the resonant frequency. At resonance, the prediction horizon was 5 times farther than the prediction horizon for the higher than resonance behavior and 2.5 times farther than that for the lower than resonance behavior. The results suggest that, in pendular oscillations of a limb or limb segment, attunement of the central nervous system to the resonant frequency minimizes the variables to be controlled and maximizes the predictability of the rhythmic movement's chaotic dynamics.

Forecasting↗