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V I Grishko

Publications and source records attributed to V I Grishko.

13 recordsLinked to original sources
PubMed1999

Nitric oxide-induced damage to mtDNA and its subsequent repair.

Mutations in mitochondrial DNA (mtDNA) have recently been associated with a variety of human diseases. One potential DNA-damaging agent to which cells are continually exposed that could be responsible for some of these mutations is nitric oxide (NO). To date, little information has been forthcoming concerning the damage caused by this gas to mtDNA. Therefore, this study was designed to investigate damage to mtDNA induced by NO and to evaluate its subsequent repair. Normal human fibroblasts were exposed to NO produced by the rapid decomposition of 1-propanamine, 3-(2-hydroxy-2-nitroso-1-propylhydrazino) (PAPA NONOate) and the resultant damage to mtDNA was determined by quantitative Southern blot analysis. This gas was found to cause damage to mtDNA that was alkali-sensitive. Treatment of the DNA with uracil-DNA glycosylase or 3-methyladenine DNA glycosylase failed to reveal additional damage, indicating that most of the lesions produced were caused by the deamination of guanine to xanthine. Studies using ligation-mediated PCR supported this finding. When a 200 bp sequence of mtDNA from cells exposed to NO was analyzed, guanine was found to be the predominantly damaged base. However, there also was damage to specific adenines. No lesions were observed at pyrimidine sites. The nucleotide pattern of damage induced by NO was different from that produced by either a reactive oxygen species generator or the methylating chemical, methylnitrosourea. Most of the lesions produced by NO were repaired rapidly. However, there appeared to be a subset of lesions which were repaired either slowly or not at all by the mitochondria.

Base Sequence↗
PubMed1999

Molecular laser biotechnology.

Laser technology has developed to the point where it is possible to utilize lasers as a sophisticated but accessible tool in understanding and manipulating gene functioning. This review emphasizes some of the systems that employ lasers in the new and growing field of molecular laser biotechnology. Here the main emphasis is on the manipulation and understanding of bacterial and plant systems.

Journal Article↗
PubMed1998

Glial cell-specific differences in response to alkylation damage.

Oligodendrocytes are preferentially sensitive to the toxic, carcinogenic, and teratogenic effects of methylnitrosourea (MNU). The mechanisms responsible for this enhanced sensitivity have not been fully elucidated. One of the most vulnerable cellular targets for this chemical is mitochondrial DNA (mtDNA). To determine if differences in mtDNA damage and repair capacity exist among the different CNS glial cell types, the effects of MNU exposure on oligodendroglia, astroglia, and microglia cultured separately from neonatal rat brain were compared. Quantitative determinations of mtDNA initial break frequencies and repair efficiencies showed that whereas no cell type-specific differences in initial mtDNA damage were detected, mtDNA repair in oligodendrocytes, oligodendrocyte progenitors, and microglia was significantly reduced compared to that of astrocytes. In astrocytes, and all other cell types previously evaluated in our laboratory, >60% of N-methylpurines were removed from the mtDNA by 24 hr. In contrast, only 35% of lesions were removed from mtDNA of oligodendrocytes, oligodendrocyte progenitors, and microglia during the same time period. Mitochondrial perturbations by a variety of xenobiotics have been linked to apoptosis. In the present study, apoptosis, as determined by DNA laddering and ultrastructural analysis, was clearly induced by MNU treatment of cultured oligodendrocyte progenitors and microglia, but not in astroglia. These data demonstrate a correlation between diminished mtDNA repair capacity and the induction of apoptosis. However, further experimentation is necessary to determine if a causal relationship exists and contributes to the vulnerability of oligodendroglia following exposure to N-nitroso compounds in the environment or in chemotherapeutic regimen.

Alkylating Agents↗
PubMed1997

Repair of oxidative damage in nuclear DNA sequences with different transcriptional activities.

This study was designed to investigate the repair of oxidative damage in nuclear DNA sequences with different transcriptional activities. Chinese hamster ovary (CHO) cells were treated with the oxygen radical generator hypoxanthine/xanthine oxidase (Hyp/XO). Damage and repair were evaluated in 14-kb restriction fragments containing either the DHFR gene, a 3'-non-transcribed flanking region, or the c-fos gene using a quantitative Southern blot technique. Damage to the sugar-phosphate backbone and abasic sites were detected by measuring their lability in alkali conditions. Lesions in DNA bases were identified using the bacterial repair enzyme endonuclease III, which predominantly recognizes damage to thymines and cytosines, and formamidopyrimidine-DNA glycosylase, which recognizes 8-oxoguanine and purines with fractured imidazole rings. The results showed that similar amounts of all types of oxidative damage were produced in both the transcribed and non-transcribed sequences following a 1-h exposure to the radical generator. Repair in all sequences was rapid, with approximately 60% removal of lesions observed by 1 h. Therefore, within these sequences, the repair of oxidative lesions is much faster than that of other types of damage, such as those induced by alkylating toxins and UV irradiation, and the repair is not affected appreciably by transcriptional status.

Animals↗
PubMed1996

Catalytic activity of poly(ADP-ribose) polymerase is necessary for repair of N-methylpurines in nontranscribed, but not in transcribed, nuclear DNA sequences.

The role of poly(ADP-ribose) polymerase (PADPRP) in nuclear DNA repair and other nuclear processes has been intensely studied and debated for decades. Recent studies have begun to shed new light on these arguments with firm experimental data for its role, primarily, as a remodeler of chromatin structure. Those studies imply that PADPRP plays an indirect role in DNA repair, serving to expose DNA to repair enzymes through chromatin remodeling. Only DNA that is tightly packaged would require PADPRP activity for its repair; while DNA in an open conformation would be accessible to DNA repair enzymes and not require PADPRP activity. The purpose of the current studies was to address the above hypothesis directly. Using quantitative Southern blot analysis, we studied repair in transcribed and nontranscribed nuclear DNA sequences in ADPRT 351 cells 95% deficient in PADPRP activity. Cells were exposed to methylnitrosourea (MNU) for 1 h and allowed to repair for 8 or 24 h. Densitometric scans of autoradiographs revealed that, when compared to their parental V79 cell line, ADPRT 351 cells 95% deficient in PADPRP activity were equally as efficient in repair of N-methylpurines in the transcribed sequence containing the dihydrofolate reductase gene. However, the ADPRT 351 cells were deficient in the ability to repair these lesions in the nontranscribed sequence containing the IgE gene compared to repair of the same sequence in the parental V79 cells. Nucleoid sedimentation assays demonstrated that the ADPRT 351 cells are deficient in repair across the entire genome when compared to the parental V79 cells. These studies indicate that PADPRP activity is not required for repair of N-methylpurines in transcribed nuclear DNA sequences but is necessary for the repair of these lesions in nontranscribed nuclear DNA sequences as well as across the entire genome since the DNA in a given cell is predominantly nontranscribed.

Alkylation↗
PubMed1996

Defective repair of oxidative damage in the mitochondrial DNA of a xeroderma pigmentosum group A cell line.

Recent evidence has linked mitochondrial DNA (mtDNA) damage to several disease processes,including cancer and aging. An important source of such damage is reactive oxygen species. These molecules can be generated endogenously via the electron transport system or may arise from a host of exogenous sources. It has been reported that extracts from cells of individuals with xeroderma pigmentosum group A (XP-A) do not repair some types of oxidative DNA damage. The current experiments were designed to determine whether there is a correlation between the inadequate repair of oxidatively damaged nuclear DNA in XP-A cells and the capacity of such cells to repair similar damage to their mtDNA. The ability of karyotypically normal human fibroblasts (WI-38) and XP-A fibroblasts to repair alloxan-generated oxidative damage to nuclear and mtDNA was assessed using a quantitative Southern blot method in conjunction with the repair enzymes endonuclease III and formamidopyrimidine DNA glycosylase. The data indicate that both nuclear and mtDNA repair of each damage type investigated is more efficient in the WI-38 cells. These findings suggest a similarity between the process(es) used to repair oxidative damage to nuclear and mtDNA in that both are inhibited by the defect in XP-A.

Alloxan↗
PubMed1994

Universal spectropolarimeter based on overtone circular dichroism measurements in the near-infrared region.

A novel and highly sensitive circular dichroism spectropolarimeter for the near-infrared region has been developed. In this instrument, a solid state titanium-sapphire laser that can be spectrally tuned from 670 to 1030 nm was used as the light source. The laser beam was converted into linearly polarized light by a polarizer and into left circularly polarized light and right circularly polarized light at 42 kHz by a photoelastic modulator (PEM). A limit of detection of 1.1 x 10(-6) AU was achieved by this instrument for (+)-Co(en)3(3+) at 765 nm. Further improvement including employing double modulation (at 42 kHz by the PEM and at 85 Hz by a chopper just before the laser beam was converted to CPL), demodulating and amplifying the signal with high-performance lock-in amplifiers, was made to enable the instrument to have the required sensitivity for the measurements of the circular dichroism of overtones and combination transitions of saturated chiral compounds, e.g., (R)- and (S)-camphor, (R)- and (S)-2-octanol, and (R)- and (S)-2-amino-1-octanol. Because the measured CD spectra originate from the overtones and combination transitions of the C-H and O-H groups, the spectropolarimeter can be used to detect virtually any compounds that have O-H and/or C-H groups.

Aluminum Oxide↗
PubMed1994

Chiral detection in high-performance liquid chromatography by vibrational circular dichroism.

A novel chiral detector for high-performance liquid chromatography has been developed. This detector is based on the measurement of circular dichroism of chiral effluents in the infrared region, i.e., vibrational circular dichroism (VCD). In this instrument, a solid-state spectral tunable (from 2.4 to 3.5 microns) F-center laser was used as the light source. The linearly polarized laser beam was converted into left circularly polarized light (LCPL) and right circularly polarized light (RCPL) at 42 kHz by means of a photoelastic modulator. The intensity of the LCPL and RCPL transmitted through the sample was measured by a liquid nitrogen cooled indium antimonide detector. Double modulation was employed to reduce the noise associated with the laser beam. Specifically, the linearly polarized laser beam, prior to being converted to CPL, was modulated at 85 Hz by a mechanical chopper. Demodulation and amplification were accomplished with the use of two lock-in amplifiers. In its present configuration, the instrument can be used to measure the VCD of O-H groups. Its sensitivity is so high that it was able, for the first time, to detect chirally (with limits of detection of micrograms) (R)- and (S)-2,2,2-trifluoro-1-(9- anthryl)ethanol and (R)- and (S)-benzoin when these compounds were chromatographically separated from the corresponding racemic mixtures by a Chiralcel-OD column. The main advantage of this chiral detector is, however, its universality; i.e., it can be used to virtually detect any chiral compounds which has O-H group (e.g, aliphatic alcohols such as 2-octanol).

Chromatography, High Pressure Liquid↗
PubMed1994

Thermal lens technique for sensitive and nonintrusive determination of isotopic purity.

A new technique in which absorption in the infrared region is sensitively monitored in the visible has been developed. This was accomplished using the visible laser to probe the thermal lens effect induced in a sample as a consequence of its absorption of radiation in the infrared. The sensitivity of the technique is much higher than that of conventional transmission measurements because, in addition to its inherent ultrasensitivity, infrared absorption is monitored in the visible region, which is relatively less noisy, and is detected by a phase-lock-detecting method. The technique is so sensitive that it can be used to determine the isotopic purity of methanol and its 13C and deuterated samples by measuring, not directly the absorption of the C-H group but rather indirectly the absorption of the O-H group. With use of the multivariate calibration method to analyze the data, this technique can be utilized for the nondestructive, noninvasive, and sensitive determination of isotopic impurity in methanol, 13CH3OH, CD3OH, CD2HOH, and CDH2OH at concentrations as low as 10(-3)% (w/w).

Carbon Isotopes↗
PubMedDate not supplied

[The restriction fragment length polymorphism of the DNA loci of human chromosome 7].

The results of comparative RFLP analysis in some DNA loci of chromosome 7 in the populations of different Ukrainian regions are presented. Significant differences in RFLP-genotype distributions among regional populations are found. The role of different genetical processes which take place in the populations of different regions of the Ukraine is under discussion.

Adult↗
PubMedDate not supplied

[The polymorphism of the tandem repeats in the dystrophin gene in the Ukrainian population].

The genetic heterogeneity of populations of different regions of Ukraine was studied using four polymorphic LNA sequences (GTAA-tandem repeats from the 3'-untranslated region of the dystrophin gene, the polymorphic systems pERT87-15--BamHI, pERT8718--TaqI, and pERT87-15--XmnI of the pERT locus of human chromosome X). We analyzed 500 DNA samples of healthy donors from the Kiev, Lvov, Poltava, and Lugansk regions. The above-mentioned polymorphic DNA sequences were used as markers to determine genetic distances between the regional populations of Ukraine. The data obtained indicate the existence of significant genetic differences among the regional populations of Ukraine. DNA polymorphism in the pERT87-8--TaqI system contributes the most significantly to these differences.

Alleles↗
PubMedDate not supplied

[The distribution of deletions in the dystrophin gene in patients with Duchenne muscular dystrophy in Ukraine].

DNA analysis of deletions within 14 exons and promoter region of dystrophin gene was performed in 34 Ukrainian families with high risk of muscular Duchenne dystrophy. The presence of dystrophin gene deletions in different exons was found clustering near central part of dystrophin gene. The necessity of DNA analysis of mutations in dystrophin gene and its use for screening and prenatal diagnosis of muscular Duchenne dystrophy is under discussion.

Dystrophin↗