Biomedical subjects

G Krishna

Publications and source records attributed to G Krishna.

At least 55 records · Page 3Linked to original sources

PubMed1995

Immunofluorescent and confocal laser cytometric analyses of centromeres in V79 cells.

Previously, a modified anticentromere antibody (ACA) technique was established in the V79 Chinese hamster lung cells to simultaneously analyze chromosome damage and aneuploidy induced by various agents. Using this method, cyclophosphamide (CP) was evaluated further in the presence and absence of S9 activation for micronucleus/aneuploidy induction. The specific binding nature of ACA to the centromeric region was also analyzed using a confocal scanning laser cytometry. The results indicated that CP was primarily a clastogen and S9 activation was required for its activity. Vinblastine, the positive control for aneuploidy, produced predominantly centromere containing micronuclei and the addition of S9 was not required for its activity. X-radiation, the positive control for clastogenicity, predominantly produced centromere negative micronuclei confirming its clastogenicity. An evaluation of centromeric region under the standard fluorescence microscope indicated that ACA generally binds to most centromeric regions in a cell. However, by confocal imaging it was found that ACA binds to the central core proteins of the centromere region and not to the peripheral proteins.

Aneuploidy↗

PubMed1995

Simultaneous evaluation of dexamethasone-induced apoptosis and micronuclei in rat primary spleen cell cultures.

Apoptosis or programmed cell death is a biological event that is biochemically and morphologically distinct from cellular necrosis. Nonetheless, its relationship has not been studied in terms of a cytogenetic endpoint such as micronucleus formation. In the present study, based on cytological observations, the incidence of dexamethasone-induced apoptotic cells was related to the frequency of micronucleated cells in vitro. Rat primary spleen cells were grown in 6-well plates with RPMI 1640 media using concanavalin A and lipopolysaccharide as mitogens. At culture initiation, the test agent dexamethasone (10, 20 or 40 microM) and a cytokinesis inhibitor cytochalasin B (3 micrograms/ml) were added. Cultures were harvested 18 h and 40 h later. Slides were prepared and stained with Diff-Quik stain. Frequencies of apoptotic cells and micronucleated binucleate cells were enumerated cytologically based on 500 cells per treatment from the same slides. The results showed a dose-dependent increase in the number of apoptotic cells in rat spleen cultures treated with dexamethasone. At 18 h, the percentages of apoptotic cells were 0.8, 1.6, 3.4 and 4.4 with 0, 10, 20 and 40 microM dexamethasone, respectively. The corresponding percentages of apoptotic cells at 40 h were: 2.8, 2.6, 5.6 and 10.4. However, at the same concentrations of dexamethasone, the micronucleus frequency in binucleate cells remained relatively unchanged. The phenomenon of apoptosis induced by dexamethasone was confirmed biochemically based on a characteristic DNA 'ladder' pattern by gel electrophoresis. These data suggest that dexamethasone at the concentrations which induced apoptosis did not produce cytogenetic damage. Also, these findings indicate that micronucleus formation and nuclear changes leading to apoptosis are separate events and these endpoints may not be closely correlated for dexamethasone.

Animals↗

PubMed1995

Use of cyclophosphamide as a positive control in dominant lethal and micronucleus assays.

Analyses of dominant lethal (DL) mutations and micronuclei (MN) are 2 important and widely used genotoxicity assays to measure drug-induced chromosome damage in germ cells and somatic cells, respectively. Cyclophosphamide (CP) has been widely used as a positive control in the single-dose mouse MN assay; however, its utility as a positive control for the DL assay has not been fully studied. In the present study, CP was tested in both assays under similar experimental conditions and MN seen in somatic tissue (bone marrow) were correlated with DL mutations seen in germinal tissue. In a dose-range finding study, groups of 5 male mice were dosed i.p. daily for 5 days at 0, 30 or 40 mg/kg CP and bone marrow was harvested 24 h later for MN assay. CP induced a dose-related increase (7- and 11-fold over control at 30 and 40 mg/kg) in micronucleated polychromatic erythrocytes (MNPCEs) and decreased %PCEs (to 60% and 54% of controls at 30 and 40 mg/kg, respectively). Based on this, a definitive DL and MN study was conducted using separate groups of 30 male mice at 0 and 40 mg/kg CP with a daily times 5 dosing regimen. For the MN assay, bone marrow was collected 24 h after the last dose from 5 animals and evaluated for MNPCEs and %PCEs. For the DL assay, each male was caged with 2 untreated females per week for 8 weeks to cover the postmeiotic germ cell stages. On day 17 after the initiation of breeding, the females were evaluated for the number of implantation sites and live, dead and resorbed implants. The results indicated that CP induced about a 17-fold increase in MNPCEs and a 46% decrease in PCEs in relation to controls. In the DL assay, CP produced a slight (13%) but statistically significant reduction in fertility index at week 7 of mating. Also, the total number of implants was significantly lower during weeks 1, 2, 3, 6 and 7 and the numbers of dead implants and postimplantation loss (PIL) were increased for weeks 1, 2 and 3 (55%, 71% and 34% PIL, respectively) over controls. These data clearly show that CP produced clastogenicity and some toxicity in both somatic tissue and germinal tissue. It was concluded that a dose of 40 mg/kg CP can be used as a positive control compound in the DL assay and in the multiple-dose marrow MN assay.

Animals↗

PubMed1995

An in vivo/in vitro method for assessing micronucleus and chromosome aberration induction in rat bone marrow and spleen. 1. Studies with cyclophosphamide.

The mouse micronucleus assay has long been used as an indicator of in vivo genotoxicity. Recently, it was shown that no single protocol is adequate to detect all clastogens. As a first step in developing a potentially more sensitive assay, micronucleus induction by cyclophosphamide (CP) was assessed in an in vivo/in vitro system using rat bone marrow and spleen cells. In each of two independent experiments, two rats/dose were treated i.p. with 0, 20, or 40 mg CP/kg and killed 6 h later. Cultures were then established in the presence of growth stimulants (interleukin-3 and granulocyte-macrophage colony stimulating factor for bone marrow; lipopolysaccharide and concanavalin A for spleen) and cytochalasin B, a cytokinesis inhibitor. Bone marrow cells were harvested and slides prepared 24 h after initiation, while spleen cells were harvested at 48 h. One thousand cells/tissue/group were scored for cell cycle kinetics and 1000 binucleate (BN) cells were scored for micronuclei. In addition, spleen cells were concurrently assayed for chromosome aberrations. A dose-related cell cycle delay was observed in both tissues in both experiments. Bone marrow showed a 6% average background frequency of micronucleated BN cells, while the low dose induced an average of 20%, and the high dose 31%. For spleen, the average control frequency of micronucleated BN cells was 3%, the low dose induced a 40% average frequency, and the high dose 65%. Also in splenocytes, a dose-dependent increase in chromosome aberrations was observed, with an almost 40-fold increase observed over the control value at the high dose. Thus, the in vivo/in vitro approach described here shows great potential in detecting drug induced genotoxicity. Also, spleen appears more sensitive than bone marrow to CP.

Animals↗

PubMed1995

An in vivo/in vitro method for assessing micronucleus and chromosome aberration induction in rat bone marrow and spleen. 2. Studies with chlorambucil and mitomycin C.

An in vivo/in vitro system using rat bone marrow cells and spleen cells to assess micronucleus (MN) and structural chromosome aberrations (SCA) simultaneously (Moore et al., 1995) was further developed. In two separate experiments, two rats/dose/experiment were treated i.p. with 0, 5, 10 and 15 mg chlorambucil (CA)/kg or with mitomycin C (MMC) at 0, 1, 2, 4 mg/kg (experiment 1) or 0, 4, 6, and 8 mg/kg (experiment 2) and killed 6 h later. Cultures were then established in the presence of growth stimulants (interleukin-3 and granulocyte-macrophage colony stimulating factor for bone marrow; lipopolysaccharide and concanavalin A for spleen) and cytochalasin B, a cytokinesis inhibitor. Bone marrow cells were harvested 24 h after establishment of cultures, while spleen cells were harvested at 48 h. In addition, spleen cells were concurrently assayed for chromosome aberrations. With the MN endpoint, spleen cells appeared more sensitive than bone marrow cells to the effects of CA due both to a lower background and an increased response. For MMC, bone marrow cells exhibited both a higher background of MN and a greater numerical response than did spleen cells. However, on the basis of a fold-increase over control values, spleen cells were more sensitive than bone marrow cells. In general, the MN endpoint appeared more sensitive than the SCA in spleen cells after treatment with CA or MMC. Thus, the approach described here shows greater potential in detecting genotoxicity.

Animals↗

PubMed1995

Expression of mRNA for atrial natriuretic peptide receptor-A in human liver: detection using RT-PCR.

cDNA preparations obtained from human liver poly A+ RNA by reverse transcription were subjected to polymerase chain reaction. Primers used in the reaction were designed from the reported cDNA sequence for human placental atrial natriuretic peptide receptor-A (ANPRA). Sequence analysis of the amplified product (approximately 700 bp) showed 100% identity to the 2476-3189 bp region of the reported cDNA sequence of human placental ANPRA. Northern blot analysis of human liver poly A+ RNA fractions showed a hybridization signal at approximately 4.4 kb, identical to the signal obtained from the poly A+ RNA fractions of human placenta. These results indicate the possible mRNA expression for atrial natriuretic peptide receptor-A in human liver.

Atrial Natriuretic Factor↗

PubMed1994

Human retina expresses both constitutive and inducible isoforms of nitric oxide synthase mRNA.

The present study provided evidence for the presence of two forms of nitric oxide synthase(NOS) gene in the human retina. Expression of retinal constitutive type(rbNOS) and inducible type(riNOS) of NOS was detected in human retinal poly A+RNA by reverse transcriptase polymerase chain reaction (RT-PCR) method. The deduced amino acid sequence of the human retinal rbNOS showed more than 99% homology with human brain bNOS and that of riNOS was identical to the chondrocytes inducible iNOS with the exception for one amino acid. These differences in amino acid sequences of rbNOS and riNOS, with their counterparts in human brain and human chondrocytes sequences, were only in the non-cofactor binding sites. Northern blot analysis of the human retinal poly A+RNA and total RNA, using the PCR-amplified riNOS probe revealed the existence of riNOS message with the appearance of the band with the expected size of 4.4kb, while the message for rbNOS was not detectable. This was the first report of the deduced nucleotide sequence identification of two NOS genes from a human tissue, while there had been earlier reports from culture cells.

Adolescent↗

PubMed1994

In vivo generation of hydroxyl radicals and MPTP-induced dopaminergic toxicity in the basal ganglia.

The in vivo generation of .OH free radicals in specific brain regions can be measured by intracerebral microdialysis perfusion of salicylate, avoiding many of the pitfalls inherent in systemic administration of salicylate. Direct infusion of salicylate into the brain can minimize the hepatic hydroxylation of salicylate and its contribution to brain levels of 2,5-DHBA. Levels of 2,5-DHBA detected in the brain dialysate may reflect the .OH adduct plus some enzymatic hydroxylation of salicylate in the brain. After minimizing the contribution of enzyme and/or blood-borne 2,5-DHBA, the present data demonstrate the validity of the use of 2,3-DHBA and apparently 2,5-DHBA as indices of .OH formation in the brain. Therefore, intracranial microdialysis of salicylic acid and measurement of 2,3-DHBA appears to be a useful .OH trapping procedure for monitoring the time course of .OH generation in the extracellular fluid of the brain. These results indicate that nonenzymatic and/or enzymatic oxidation of the dopamine released by MPTP analogues in the extracellular fluid may play a key role in the generation of .OH free radicals in the iron-rich basal ganglia. Moreover, a site-specific generation of cytotoxic .OH free radicals and quinone/semiquinone radicals in the striatum may cause the observed lipid peroxidation, calcium overload, and retrograde degeneration of nigrostriatal neurons. This free-radical-induced nigral injury can be suppressed by antioxidants (i.e., U-78517F, DMSO, and deprenyl) and possibly hypothermia as well. In the future, this in vivo detection of .OH generation may be useful in answering some of the fundamental questions concerning the relevance of oxidants and antioxidants in neurodegenerative disorders during aging. It could also pave the way for the research and development of novel neuroprotective antioxidants and strategies for the early or preventive treatment of neurodegenerative disorders, such as Parkinson's disease (Wu et al., this issue), amyotrophic lateral sclerosis, head trauma, and possibly Alzheimer's cognitive dysfunction as well. In conclusion, this in vivo free-radical trapping procedure provides evidence to support a current working hypothesis that a site-specific formation of cytotoxic .OH free radicals in the basal ganglia may be one of the neurotoxic mechanisms underlying nigrostriatal degeneration and Parkinsonism caused by the dopaminergic neurotoxin MPTP. Addendum added in proof: The controversy concerning possible neurotoxic and/or neuroprotective roles of NO. in cell cultures was discussed and debated at the symposium (Wink et al., this issue; Dawson et al., this issue; Lipton et al., this issue).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1994

Expression of mRNA for atrial natriuretic peptide receptor guanylate cyclase (ANPRA) in human retina.

1. Guanylate cyclase plays an important role in the visual cycle. Here we report the mRNA expression for the atrial natriuretic peptide receptor type A form of guanylate cyclase (ANPRA) in human retina. 2. Polymerase chain reaction using two sets of primers on the cDNAs reverse-transcribed from human retinal poly(A)+ RNA amplified two products under two different reaction conditions. The primers used in the reaction were designed from the reported sequence of human placental ANPRA cDNA. 3. Sequencing of the amplified products showed 100% sequence homology to the human placental ANPRA gene. Northern blot analysis indicated the presence of a 4.4-kb ANPRA mRNA in human retina, similar to that present in human brain.

Base Sequence↗

PubMed1994

Comparison of the pharmacokinetics of an ondansetron solution (8 mg) when administered intravenously, orally, to the colon, and to the rectum.

Ondansetron, an antagonist of the serotonin type 3 (5-HT3) receptor, is indicated for the treatment of chemotherapy-induced emesis. This study compares the pharmacokinetics, especially the bioavailability, of an ondansetron 8-mg solution when administered intravenously, orally, to the colon via nasogastric intubation, and to the rectum using a retention enema. Six healthy, male volunteers received ondansetron infused into the colon during the first treatment period. These subjects then received the remaining three treatments in random order, with a minimum 1-week washout period between treatments. Serial plasma samples were obtained for up to 24 hr after dosing in each treatment period. Absolute bioavailability after the oral dosing, colonic infusion, and rectal administration averaged 71 +/- 14, 74 +/- 26, and 58 +/- 18%, respectively. These values were not significantly different (P > 0.05). Values of Tmax and Cmax were also not significantly different among the nonparenteral routes. Mean absorption half-lives were 0.66, 1.1, and 0.75 hr after the oral, colonic, and rectal administrations, respectively. These results indicate that ondansetron is well absorbed in the intestinal segments studied including the upper small intestine, the colon, and the rectum and that sustained-release and suppository formulations of ondansetron are feasible.

Administration, Oral↗

PubMed1994

Cloning and sequencing of heat shock factor (HSF1) cDNA from human retina.

Transcription of heat shock genes takes place in response to the exposure of cells to elevated temperatures or other stress conditions. Heat shock transcription factor (HSF) plays an important role in the activation of such transcription. We report here the identification of human heat shock factor from human retina by RT-PCR and cloning studies. Human retinal poly A+ RNA was reverse transcribed and the cDNAs thus obtained were subjected to PCR using a set of primers designed from the reported cDNA sequence for human HSF1. The amplified product showed 100% sequence identity with the appropriate region of HSF1. A human retinal cDNA library was screened to isolate the full length cDNA for heat shock factor, which showed 100% sequence identity to that reported for human HSF1. Northern blot analysis showed the size of human retinal HSF to be approximately 2.4 kb.

Amino Acid Sequence↗

PubMed1994

Anaesthetic considerations for magnetic resonance imaging.

Magnetic resonance imaging (MRI) has become an increasingly popular non-invasive radiological diagnostic procedure, with several distinct advantages over computerised tomography (CT). The images are produced using a strong (1.5-Tesla) magnetic field and radiofrequency (RF) pulses. Due to the effects of the strong magnetic field, certain groups of patients with implanted ferromagnetic objects and women in their first trimester of pregnancy are precluded from undergoing MRI. While most of the patients undergo MRI awake or with light sedation, few need heavy sedation or general anaesthesia. The problems related to anaesthesia in MRI include the constant presence of a strong magnetic field, the RF pulses and their effect on the anaesthesia machine, monitoring devices, magnetically coded material, and loose ferromagnetic objects. In this article, the current availability of MRI-compatible anaesthesia machine, various monitoring devices, and safe conduct of anaesthesia during MRI for patients of all ages are discussed. In addition, the implications of the strong magnetic field on patient resuscitation inside the MRI suite and the recommended procedure for a successful outcome are outlined.

Adult↗

PubMed1993

Studies on the interaction of the dye, stains-all, with individual calcium-binding domains of calmodulin.

We show that the calcium-mimic dye, Stains-all, is a convenient probe to study the structural features of the individual calcium-binding sites of calmodulin (CaM) and related calcium-binding proteins (CaBP). These peptides bind the dye in their calcium-binding sites, and induce a circular dichroism (CD) band in the bound dye in the 620 nm (J band) region, which is abolished upon the addition of calcium. Replacement of Asp by Asn in the + x position of the weaker calcium-binding site (site I of CaM) abolishes the dye binding, while the same change in the higher affinity site IV attenuates the binding of the dye and does not abolish it. Replacement of Tyr in site IV with Trp does not distort the geometry, although it increases the dye binding affinity.

Amino Acid Sequence↗

PubMed1992

Expression of guanylate cyclase-A mRNA in the rat retina: detection using polymerase chain reaction.

A technique based on RNA-PCR was successfully employed for the detection of guanylate cyclase-A (GC-A) mRNA in the rat retina. Three sets of primers designed from the published cDNA sequence of rat brain guanylate cyclase-A (GC-A) produced amplification products of expected sizes from the retina as well as brain. Analysis of retinal PCR products yielded a 970 bp sequence, which showed 100% homology to the cDNA sequence of GC-A (2343-3312 bp region). Northern blot analysis was not very sensitive for the detection of GC-A mRNA in the retina. The results indicate that the mRNA for GC-A (or a closely related form) is probably expressed in the retina, but at a lower level than that found in the brain.

Animals↗

PubMed1992

Genotoxicity assessment of pirmenol, a new antiarrhythmic drug.

The genotoxicity of pirmenol was tested in the E. coli and S. typhimurium mutagenesis assay, an in vitro mammalian cell chromosome-aberration assay and an in vivo mouse micronucleus assay. The E. coli tester strain WP2s was exposed to concentrations of pirmenol as high as 10,000 micrograms/plate both in the absence (S9-) and presence (S9+) of metabolic activation. Five strains of S. typhimurium (TA98, TA100, TA1535, TA1537, TA1538) were exposed to concentrations of pirmenol as high as 5000 micrograms/plate in the absence and presence of S9. Pirmenol was not mutagenic toward either E. coli or S. typhimurium. Chinese hamster lung V79 cell cultures were exposed to pirmenol at concentrations of 500-2500 micrograms/ml (S9-) and 500-3000 micrograms/ml (S9+). Pirmenol increased the frequency of structural chromosome aberrations (SCAs). The minimum clastogenic concentration was 1500 micrograms/ml (both S9- and S9+) with a peak clastogenic response of 6% (S9-) and 34% (S9+) cells with aberrations. Although there were statistically significant results in the S9- experiment, the percent cells with aberration values for treated groups were within the historical control range (0-6%) of this laboratory. The observed effects in both the absence and presence of S9 appear at high concentrations compared to human circulating plasma levels of 1-3 micrograms/ml and the clastogenicity was confined to chromosome gaps and breaks. Consequently, this in vitro effect would not be expected to be reflected by either in vivo clastogenic or carcinogenic activity. This was supported by findings in the mouse micronucleus study of pirmenol in which single oral doses administered to male CD-1 mice at 5, 55, or 115 mg/kg (80% LD50) produced no statistically significant increases in the frequency of micronucleated polychromatic erythrocytes in bone marrow at 24, 48 or 72 h postdosing. Additionally, no evidence of carcinogenicity was seen in a mouse or rat bioassay.

Animals↗

PubMed1992

Simultaneous analysis of chromosome damage and aneuploidy in cytokinesis-blocked V79 Chinese hamster lung cells using an antikinetochore antibody.

A modified antikinetochore antibody technique was established in the V79 Chinese hamster lung cells to simultaneously analyze chromosome damage and aneuploidy induced by various agents. The method involved sequential treatment of slides with crest serum, fluoresceinated goat-antihuman and swine-antigoat antibodies, and propidium iodide. In this method, cytoplasm (green), nuclei or micronuclei (red), and kinetochores (yellow), are identified using the same filter setting under blue excitation (440-490 nm) with a barrier filter at 520 nm. Using this method, three agents, vinblastine (VB), X-rays, and methyl methanesulfonate (MMS) were tested for micronucleus/aneuploidy induction. An aneugen, VB and a clastogen, X-rays, induced predominantly kinetochore positive (K+) and negative (K-) micronucleated binucleate (MNBN) cells, respectively, in a dose-dependent fashion. An alkylating agent, MMS, produced both K+ and K- MNBN cells. These results are comparable with the results reported in the literature on these compounds using various methods and thus demonstrate the usefulness of this assay in distinguishing clastogenicity from aneugenicity.

Aneuploidy↗

PubMed1992

Simultaneous evaluation of clastogenicity, aneugenicity and toxicity in the mouse micronucleus assay using immunofluorescence.

An improved antikinetochore antibody technique was established in the mouse micronucleus assay to simultaneously evaluate toxicity, clastogenicity and aneugenicity induced by various test agents. The procedure involved the use of cellulose column fractionated cytospun slides for analysis. The staining method consisted of sequential treatment of slides with crest serum, fluorosceinated goat-antihuman and swine-antigoat antibodies, and propidium iodide. In this method, polychromatic erythrocytes (PCEs, dark red), normochromatic erythrocytes (NCEs, green), chromosome(s)/fragments/micronuclei (orange), and kinetochores (yellow), are identified using the same filter setting under blue excitation (440-490 nm) with a barrier filter at 520 nm. Using this method, three agents, cyclophosphamide, X-rays and vincristine were tested for micronucleus/aneuploidy induction and bone marrow toxicity. The aneugen, vincristine, and clastogens, X-rays and cyclophosphamide, induced predominantly kinetochore positive (K+) and negative (K-) micronucleated PCEs, respectively. At the doses tested, cyclophosphamide caused a slight but statistically significant decrease in PCEs in females, and other agents did not produce any severe bone-marrow toxicity in either male or female mice. These results are comparable with the results reported in the literature on these compounds with various methods and thus demonstrate the usefulness of this assay in distinguishing clastogenicity from aneugenicity and in evaluating toxicity.

Aneuploidy↗

PubMed1992

Intracranial microdialysis of salicylic acid to detect hydroxyl radical generation through dopamine autooxidation in the caudate nucleus: effects of MPP+.

Ringer's solution containing salicylic acid (5 nmol/microliters/min) was infused directly through an intracranial microdialysis probe to detect the generation of hydroxyl radicals (.OH) reflected by the formation of dihydroxybenzoic acids (DHBA) in the caudate nucleus of anesthetized rats. Brain dialysate was assayed for dopamine, 2,3-, and 2,5-DHBA by a high-pressure liquid chromatography-electrochemical (HPLC-EC) procedure. 1-Methyl-4-phenylpyridinium ions (MPP+, 0 to 150 nmol) increased dose-dependently the release of dopamine and the formation of DHBA. A positive linear correlation between the release of dopamine and the formation of 2,3- or 2,5-DHBA was observed (R2 = .98). The present results demonstrate the validity of the use of not only 2,3-DHBA but also 2,5-DHBA as an in vivo index of oxidative damage generated by reactive .OH radicals. In conclusion, the present study demonstrates a novel use of intracranial microdialysis of salicylic acid to assess the oxidative damage elicited by .OH in living brain.

1-Methyl-4-phenylpyridinium↗