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Biomedical subjects

M T Smith

Publications and source records attributed to M T Smith.

At least 199 records · Page 11Linked to original sources
PubMed1989

Expression of the rtp gene of Bacillus subtilis is required for replication fork arrest at the chromosome terminus.

It was earlier proposed that clockwise replication fork arrest at the chromosome terminus in Bacillus subtilis is dependent upon expression of the rtp gene adjacent to the site of arrest, terC [Smith and Wake, J. Bacteriol. 170 (1988) 4083-4090]. A merodiploid strain of B. subtilis, in which rtp was placed under the control of the IPTG-inducible spac-1 promoter, was constructed. Replication fork arrest at terC, as monitored by the level of a forked DNA molecule of predicted dimensions, was shown to be dependent upon IPTG-induced expression of rtp in this strain. The very low concentration of IPTG needed to induce a substantial level of fork arrest suggests that relatively little RTP, the protein product of rtp, is needed for fork arrest at terC.

Bacillus subtilis↗
PubMed1989

Ca2+ oscillations induced by hormonal stimulation of individual fura-2-loaded hepatocytes.

Isolated rat hepatocytes were loaded with the Ca2+ indicator fura-2 to measure cytosolic free Ca2+ concentrations ([Ca2+]i) in individual cells by digital ratio imaging microscopy. Stimulation with 0.1 nM vasopressin, 0.5 microM phenylephrine, or 0.5 microM ATP caused repetitive spikes of high [Ca2+]i in a high percentage of cells, in agreement with Woods et al. (Woods, N. M., Cuthbertson, K. S. R., and Cobbold, P. H. (1986) Nature 319, 600-602), but unlike the results of Monck et al. (Monck, J. R., Reynolds, E. E., Thomas, A. P., and Williamson, J. R. (1988) J. Biol. Chem. 263, 4569-4575). Reduction in extracellular [Ca2+] decreased the frequency but not the amplitude of the spikes, suggesting that the spikes result from dumping of intracellular stores and that the entry of extracellular Ca2+ affects only the rate of replenishment of those stores. Membrane depolarization failed to elevate [Ca2+]i and had an effect similar to removal of extracellular Ca2+ in decreasing the frequency of agonist-evoked [Ca2+]i oscillations or inhibiting them altogether, arguing against any significant role for voltage-operated Ca2+ channels.

Adenosine Triphosphate↗
PubMed1989

Denitrosation of 1,3-bis(2-chloroethyl)-1-nitrosourea by class mu glutathione transferases and its role in cellular resistance in rat brain tumor cells.

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) is known to be detoxified by a denitrosation reaction catalyzed by glutathione-dependent enzymes in rat liver cytosol (R. E. Talcott and V. A. Levin, Drug Metab. Dispos., 11:175-176, 1983). Using a modification of their procedure, we have measured the ability of different purified rat glutathione transferase isoenzymes to denitrosate BCNU. The catalytic efficiencies of the isoenzymes for the denitrosation reaction expressed as the ratio of Vmax to Km were as follows (isoenzyme, Vmax/Km): 1-2, 2.3; 3-3, 12.2; 3-4, 29.2; and 4-4, 26.1. Thus, the class mu isoenzymes containing subunit 4 are by far the best catalysts of the BCNU denitrosation reaction. The class pi transferase 7-7 and class alpha transferases 1-1 and 1-2 demonstrated very weak catalytic activity with BCNU. Determination of the glutathione transferase isoenzyme profiles of 9L rat brain tumor cells and the BCNU-resistant 9L-2 subline by immunoblotting revealed that although the resistant 9L-2 cells contain lower total glutathione transferase activity than 9L cells, they have elevated levels of the class mu transferases. Also, the class pi transferases were found to be down-regulated in 9L-2 as compared with 9L cells. Thus, the increased resistance of 9L-2 cells to BCNU may, in part, be explained by up-regulation of class mu transferase expression with consequent increased capacity for BCNU detoxication. Further support for this hypothesis comes from the fact that pretreatment of 9L-2 cells with the glutathione transferase inhibitors ethacrynic acid or triphenyltin chloride enhanced the cytotoxic effects of BCNU. These results suggest that the class mu transferases play a role in the resistance of brain tumor cells to BCNU.

Animals↗
PubMed1989

Species delineation in the genus Nadsonia Sydow.

The genus Nadsonia Sydow is revised on the basis of morphology, physiology, amino acid and fatty acid composition, electrophoretic patterns of some enzymes and DNA relatedness. Two species, N. commutata (type CBS 6640) and N. fulvescens, with two varieties, N. fulvescens var. fulvescens (type CBS 2596) and N. fulvescens var. elongata (type CBS 2594) nov. comb. are recognized. A modified diagnosis of the genus and a key are given.

Amino Acids↗
PubMed1989

A protein involved in termination of chromosome replication in Bacillus subtilis binds specifically to the terC site.

The small basic protein encoded by the open reading frame adjacent to the terC site in the Bacillus subtilis chromosome and previously implicated in termination of the replication process was purified. Band retardation assays established that this protein (now called the replication terminator protein, encoded by the rtp gene) binds specifically to a 209-base-pair fragment of DNA within which terC is located.

Bacillus subtilis↗
PubMed1989

Peroxidase-dependent metabolism of benzene's phenolic metabolites and its potential role in benzene toxicity and carcinogenicity.

The metabolism of two of benzene's phenolic metabolites, phenol and hydroquinone, by peroxidase enzymes has been studied in detail. Studies employing horseradish peroxidase and human myeloperoxidase have shown that in the presence of hydrogen peroxide phenol is converted to 4,4'-diphenoquinone and other covalent binding metabolites, whereas hydroquinone is converted solely to 1,4-benzoquinone. Surprisingly, phenol stimulates the latter conversion rather than inhibiting it, an effect that may play a role in the in vivo myelotoxicity of benzene. Indeed, repeated coadministration of phenol and hydroquinone to B6C3F1 mice results in a dramatic and significant decrease in bone marrow cellularity similar to that observed following benzene exposure. A mechanism of benzene-induced myelotoxicity is therefore proposed in which the accumulation and interaction of phenol and hydroquinone in the bone marrow and the peroxidase-dependent formation of 1,4-benzoquinone are important components. This mechanism may also be responsible, at least in part, for benzene's genotoxic effects, as 1,4-benzoquinone has been shown to damage DNA and is shown here to induce multiple micronuclei in human lymphocytes. Secondary activation of benzene's phenol metabolites in the bone marrow may therefore play an important role in benzene's myelotoxic and carcinogenic effects.

Animals↗
PubMed1988

Fructose prevents 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced ATP depletion and toxicity in isolated hepatocytes.

The loss of viability of isolated rat hepatocytes exposed to either 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or its toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) was prevented by addition of fructose to the incubation medium. This protection was dependent on fructose concentration, being complete at 10 mM. Addition of fructose dramatically delayed MPTP- and MPP+-induced depletion of ATP and was accompanied by a significant accumulation of lactate, indicating the occurrence of enhanced glycolytic production of ATP. Glucose was much less effective against MPTP and MPP+ toxicity, probably because it is a relatively poor substrate for glycolysis in liver cells. We conclude that depletion of ATP is a critical event in MPTP cytotoxicity in our in vitro model system, and that the use of alternative sources of ATP production may represent an important protective device against the effects of this toxic agent.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1988

Relationships between intracellular vitamin E, lipid peroxidation, and chemical toxicity in hepatocytes.

The cellular content of vitamin E was measured in isolated rat hepatocytes exposed to various types of chemical injury. Vitamin E was determined as alpha-tocopherol by HPLC with in-line uv and electrochemical detection. The cytotoxicity of diquat, a redox cycling compound, was accompanied by a decrease in cellular alpha-tocopherol and a stimulation of lipid peroxidation. Both the loss of alpha-tocopherol and the accumulation of lipid peroxidation products could be prevented by addition of either the antioxidant N,N'-diphenyl-p-phenylenediamine (DPPD) or the reducing agent dithiothreitol (DTT). DTT also prevented the oxidation of soluble and protein thiols and completely protected against cytotoxicity, while DPPD addition only delayed the onset of hepatocyte death. Cytotoxic doses of the naphthoquinone, menadione, and the pyridine compounds 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 1-methyl-4-phenyl-pyridinium ion did not deplete alpha-tocopherol levels, nor did they result in significant lipid peroxidation. On the other hand, a peroxidizing, but noncytotoxic dose of ADP-Fe3+ rapidly decreased cellular alpha-tocopherol levels. These data demonstrate that cellular alpha-tocopherol loss is neither a prerequisite for, nor a necessary consequence of toxicity. Moreover, a substantial depletion (ca. 50%) of alpha-tocopherol does not necessarily result in cell death. Although alpha-tocopherol protects against the oxidation of cellular lipids, the maintenance of hepatocyte alpha-tocopherol content does not prevent the oxidation of soluble and protein thiols. These other targets of oxidative damage seem to play a more critical role in hepatocyte toxicity.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1988

DNA sequence requirements for replication fork arrest at terC in Bacillus subtilis.

The replication terminus, terC, of Bacillus subtilis is the chromosomal site at which movement of the clockwise replication fork is blocked. The effect of deletion or modification of DNA sequences on either side of terC (defined by the sequence location of the arrested clockwise fork junction) has been investigated. Deletion of sequences ahead of terC to within 250 base pairs (bp) had no effect on fork arrest, whereas removal of a further 130 bp abolished it. The 250-bp segment immediately ahead of terC encompassed the previously identified inverted repeat region as well as potential promoters for the transcription of an adjoining open reading frame (ORF). Deletion of DNA from the other side of terC up to 80 bp from it also abolished fork arrest. This deletion removed the bulk of the ORF. Disruption of this ORF by the insertion of 4 bp also abolished fork arrest. A model for clockwise fork arrest at terC, implicating both the inverted repeat region and the protein product of the ORF, is proposed.

Amino Acid Sequence↗
PubMed1987

Cytotoxicity of the redox cycling compound diquat in isolated hepatocytes: involvement of hydrogen peroxide and transition metals.

Diquat is a hepatotoxin whose toxicity in vivo and in vitro is mediated by redox cycling and greatly enhanced by pretreatment with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase. The mechanism by which redox cycling mediates diquat cytotoxicity is unclear, however. Here, we have attempted to examine the roles of three potential products of redox cycling, namely superoxide anion radical (O2-.), hydrogen peroxide (H2O2), and hydroxyl radical (.OH), in the toxicity of diquat to BCNU-treated isolated hepatocytes. Addition of high concentrations of catalase, but not superoxide dismutase, to the incubations provided some protection against the toxic effect of diquat, but much better protection was observed when catalase was added in combination with the iron chelator desferrioxamine. Addition of desferrioxamine alone also provided considerable protection, whereas the addition of copper ions enhanced diquat cytotoxicity. Taken together, these results indicate that both H2O2 and the transition metals iron and copper could play major roles in the cytotoxicity of diquat. The role of O2-. remains less clear, however, but studies with diethylenetriaminepentaacetic acid indicate that O2-. is unlikely to significantly contribute to the reduction of Fe3+ to Fe2+. The hydroxyl radical or a related species seems the most likely ultimate toxic product of the H2O2/Fe2+ interaction, but hydroxyl radical scavengers afforded only minimal protection.

Animals↗
PubMed1987

Sequence features of the replication terminus of the Bacillus subtilis chromosome.

The sequence of 1267 nucleotides spanning the replication terminus, terC, of the Bacillus subtilis 168 chromosome has been determined. The site of arrest of the clockwise fork, which defines terC, has been localized to a 30-nucleotide portion (approximately) within this sequence. The arrest site occurs in an A + T-rich region between two open reading frames and very close to one of two imperfect inverted repeats (47-48 nucleotides each) which are separated by 59 nucleotides. The closeness of approach of the arrested clockwise fork to the first imperfect inverted repeat encountered in this region raises the possibility of a role for the inverted repeats in the mechanism of fork arrest.

Amino Acid Sequence↗
PubMed1987

Increased efflux rather than oxidation is the mechanism of glutathione depletion by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).

Incubation of isolated hepatocytes in the presence of either the parkinsonian-inducing compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or its putative toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) led to a depletion of intracellular reduced glutathione (GSH), which was mostly recovered as glutathione disulfide (GSSG). However, both MPTP- and MPP+-induced glutathione perturbances were relatively unaffected by the prior inhibition of glutathione reductase with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), suggesting that intracellular oxidation was not the major mechanism involved in the GSH loss. Inclusion of cystine in the incubation mixtures revealed a time-dependent formation of cysteinyl glutathione (CySSG), indicating that an increased efflux was mostly responsible for the MPTP- and MPP+-induced GSH depletion. Therefore, the measurement of GSSG, which is apparently formed extracellularly, was not associated with oxidative stress.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗