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N Howell

Publications and source records attributed to N Howell.

At least 91 records · Page 5Linked to original sources
PubMed1988

Mutational analysis of the mouse mitochondrial cytochrome b gene.

The protonmotive cytochrome b protein of the mitochondrial bc1 respiratory chain complex contains two reactions centers, designated Qo and Qi, which can be distinguished by the effects of different inhibitors. The nucleotide sequences have been determined of the mitochondrial cytochrome b genes from a series of mouse cell mutants selected for increased inhibitor resistance. Each mutant contains a single nucleotide change which results in an amino acid substitution. When the proximity of the altered amino acid residues to the histidines involved in heme ligation is considered, the results support a model for cytochrome b folding in which there are eight transmembrane domains rather than the nine of the Widger-Saraste model. Replacement of the Gly38 residue by valine results in resistance to the Qi inhibitors antimycin A and funiculosin but not 2-n-heptyl-hydroxyquinoline-N-oxide. Based upon sequence comparisons of mitochondrial and bacterial cytochrome b and chloroplast b6 proteins, the region of the molecule involved in antimycin binding is as highly conserved as those domains involved in heme ligation. It is suggested that the antimycin binding domain of cytochrome b is involved in forming the Qi reaction center. Alterations of the Gly142 and Thr147 residues result in resistance to myxothiazol and stimatellin, respectively. While both inhibitors block the Qo reaction center, the two mutations do not confer cross-resistance to each other. This region of cytochrome b is the most highly conserved during evolution and these inhibitor binding sites probably occur within the protein domain constituting the Qo reaction center. In addition, there is a less conserved region of the protein, defined by the Leu294 residue, which may function in binding the hydrophobic portions of Qo inhibitors.

Animals↗
PubMed1988

Mitochondrial chloramphenicol-resistant mutants can have deficiencies in energy metabolism.

Three pairs of mouse CAP-R PYR-IND OLI-R mitochondrial mutants, and the corresponding CAP-S parental lines, were assayed to determine if cellular expression of these phenotypes was accompanied by changes in cellular energy metabolism: glycolysis, cellular respiration, citric acid cycle activity, and mitochondrial electron transport. Relative to its parental CAP-S line, the SVT2 CAP-R mutant had no significant deficiencies in any of the pathways analyzed. In contrast, the LA9 and SVA31 CAP-R mutants showed significant reductions in cellular respiration. At the biochemical level, respiration deficiency was accompanied by derangements in mitochondrial electron transport. It was also found that the CAP-R mutants had very high levels of glycolysis when the cells were maintained in the presence of chloramphenicol. The possibility is discussed that the sequence changes in the mitochondrial large rRNA gene which determine chloramphenicol resistance can also result, at least in some cases, in reduced levels of mitochondrial biogenesis, leading to respiration deficiency. The PYR-IND and OLI-R phenotypes, which also appear to be encoded by the CAP-R mutations, may result from a compensatory increase in glycolysis-generated ATP or metabolic intermediates.

Animals↗
PubMed1988

Intrarenal effects of [Ala-Pro-Gly-(Ile3-Val5)] angiotensin II in the conscious dog.

Amphibian skin has functional characteristics of epithelium in the mammalian distal nephron and plays an important role in sodium and water metabolism in these animals. A peptide extracted from the skin of the Australian frog Crinia georgiana has been purified, has been determined to have the amino acid sequence of [Ala-Pro-Gly-(Ile3-Val5)]angiotensin II, and recently has been synthesized. We studied the renal effect of synthetic frog skin angiotensin II (FSAII) infused via the renal artery in doses that were confined to the kidney. FSAII was infused intrarenally at 0.2, 2, and 4 pmol/kg.min in uninephrectomized conscious dogs (n = 5) in metabolic balance at a sodium intake of 80 meq/day. FSAII was confined to the kidney, as demonstrated by the absence of any systemic pressor response and/or any increase in plasma aldosterone concentrations during intrarenal FSAII infusion at rats of 0.2 and 2 pmol/kg.min. At 4 pmol/kg.min, FSAII traversed the kidney in amounts sufficient to stimulate aldosterone secretion (P less than 0.05). All three doses of FSAII caused significant antidiuresis and antinatriuresis, and decreased fractional excretion of sodium. There were no changes in the glomerular filtration rate (GFR) or renal plasma flow (RPF) during FSAII infusion at 0.2 and 2 pmol/kg.min. At 4 pmol/kg.min, FSAII engendered a significant decrease in GFR and RPF, while the filtration fraction increased. There were no significant changes in arterial blood pressure at any dose of FSAII. When confined to the kidney, FSAII caused antidiuresis and anti-natriuresis in the absence of a change in GFR and RPF. These results provide evidence that angiotensin acts directly at the renal tubular level to alter renal function.

Angiotensins↗
PubMed1987

The molecular basis of inhibitor resistance in a mammalian mitochondrial cytochrome b mutant.

The mitochondrial gene for the cytochrome b of Complex III has been cloned from a mouse L-cell mutant with increased resistance to 2-n-heptyl-4-hydroxyquinoline-N-oxide and other inhibitors which block reactions at the b562 heme group. Nucleotide sequencing revealed that this gene contained a G:A transition on the coding strand at position 14,830. At the amino acid level, this mutation results in the substitution of an aspartic acid residue for a conserved glycine at position 231 of cytochrome b. Based upon current models for the secondary structure of cytochrome b, the altered amino acid lies in close proximity to one of the invariant histidine residues involved in binding the heme groups. Combining this result with the previous biochemical studies of this mutant, we hypothesize that the insertion of this highly charged side chain alters the conformation around the b562 heme group such that 2-n-heptyl-4-hydroxyquinoline-N-oxide and the other inhibitors of this group have reduced access to the inhibitor binding domain.

Amino Acid Sequence↗
PubMed1987

Characterization of mouse nuclear and mitochondrial mutants with increased resistance to cytochrome b inhibitors.

A series of mouse lines with increased resistance to respiratory inhibitors which block electron transport through the protonmotive cytochrome b of complex III have been isolated in this laboratory. We describe here the isolation of a mutant with increased resistance to HQNO (2-n-heptyl-4-hydroxyquinoline-N-oxide) whose phenotype is due to a nuclear mutation. At the cellular level, there is a severe reduction in respiration with the residual oxygen consumption being resistant to inhibitors of both ubiquinol-cytochrome c oxidoreductase and cytochrome oxidase. At the mitochondrial level, there was a severe derangement in NADH oxidase activity. Electron transport through the succinate oxidase span of the respiratory chain and its coupling to oxidative phosphorylation are also reduced in this nuclear mutant but not to the same extent. It is concluded that the primary defect in the mutant lies within a nuclear gene encoding a component of complex I (NADH-ubiquinol oxidoreductase). In addition, further biochemical characterization of the mitochondrially inherited inhibitor-resistant mutants has demonstrated that they also show significant reductions in the efficiency of energy transduction and in the rate of cytochrome b electron transport.

Animals↗
PubMed1985

Anomalous electrophoretic mobility of mouse mtDNA restriction fragments.

Analysis of the electrophoretic mobilities of mouse mtDNA restriction fragments revealed a high incidence of anomalous migration in polyacrylamide slab gels. Relative to the mobility predicted by the sequence, 6 of 29 200- to 700-bp fragments had deviations of 5-12%. Three of these fragments migrated more slowly than predicted while three were faster. There was little, if any, correlation between electrophoretic mobility and base composition. The anomalous restriction fragments mapped throughout the mitochondrial genome.

Animals↗
PubMed1984

Origin, transmission, and segregation of mitochondrial DNA dimers in mouse hybrid and cybrid cell lines.

Hybrid and cybrid progeny lines were constructed from mouse LA9 cells which contain almost exclusively mtDNA monomers and LDTK cells which contain only unicircular mtDNA dimers. The proportion of mtDNA monomers and dimers in the progeny lines was determined both as a function of the number of population doublings since fusion and of selection for expression of a mutant phenotype encoded on one of the parental mtDNAs. There was no preferential segregation of either parental mtDNA in early-passage progeny lines, irrespective of whether or not selection was applied. In marked contrast, there was an accumulation of mtDNA dimers in late-passage progeny lines maintained in the absence of selection for a drug-resistance marker carried by the parental mtDNA monomers. When such selection was applied, roughly equal mass proportions of both parental mtDNAs were maintained in most lines. However, in several progeny lines, new types of mtDNA dimers carrying the selected resistance marker initially encoded in the monomeric mtDNA were present. In some of these latter lines, the new mtDNA dimers apparently arose from LA9 monomers, possibly by recombination. It is hypothesized that mammalian mitochondria normally have a recombination system which maintains low steady-state levels of mtDNA unicircular oligomers by preferentially resolving dimers into two monomers.

Animals↗
PubMed1984

High-level, unstable adriamycin resistance in a Chinese hamster mutant cell line with double minute chromosomes.

An Adriamycin-resistant Chinese hamster V79 line was isolated previously in this laboratory. There was about a 5-fold increase in Adriamycin resistance in this mutant as determined from survival curve measurements. Using this low-level Adriamycin-resistant mutant, a cell line with a high level of resistance was isolated after a multistep selection process culminating in continuous growth of the cells in medium containing 5.0 micrograms Adriamycin/ml. These cells are about 3000 times more resistant towards the cytotoxic effects of Adriamycin than is the parental V79 line. This high-level resistance phenotype is unstable and lost upon culture in the absence of drug. The highly resistant cells also showed increased cross-resistance to actinomycin D, Colcemid, and vincristine compared to the low-level resistant cells. Cytogenetic studies showed that these mutant cells contained increased numbers of double minute chromosomes and that the number of double minutes decreased proportionately with the reduction of Adriamycin resistance in cultures changed to drug-free medium. Adriamycin uptake assays demonstrated that there was a further decrease in net uptake relative to the low-level resistant mutant.

Animals↗
PubMed1983

Mammalian mitochondrial mutants selected for resistance to the cytochrome b inhibitors HQNO or myxothiazol.

Mouse LA9 cell lines were selected for increased resistance to either HQNO or myxothiazol, inhibitors of electron transport which bind to the mitochondrial cytochrome b protein. Two phenotypically distinguishable HQNO-resistant mutants were recovered while the myxothiazol-resistant isolates had a common phenotype. All three mutant phenotypes were transmitted cytoplasmically in cybrid crosses. Biochemical studies further established that for all three mutant types, resistance at the cellular level was paralleled by an increase in inhibitor resistance of mitochondrial succinate-cytochrome c oxidoreductase, the respiratory complex containing cytochrome b. As with the previously described mitochondrial antimycin-resistant mutant, the initial biochemical and genetic studies indicated that these mutations occur within the mitochondrial cytochrome b gene. This conclusion was strongly supported by the results of mtDNA restriction fragment analyses in which it was found that one HQNO-resistant mutant had undergone a small insertion or duplication in the apocytochrome b gene. Finally, all four mitochondrial cytochrome b mutants have been analyzed in both cell plating studies and succinate-cytochrome c oxidoreductase assays to determine the pattern of cross-resistance to inhibitors of cytochrome b other than the one used for selection.

Animals↗
PubMed1983

Mitochondrial genetics of mammalian cells: a mouse antimycin-resistant mutant with a probable alteration of cytochrome b.

Mouse LA9 antimycin-resistant mutants (ANT-R) were isolated and characterized. Genetic analyses established that this phenotype is encoded within the mtDNA: (1) the ANT-R phenotype showed frequent mitotic segregation and reassortment in hybrid clonal lines; (2) it was transmitted directly in cybrid crosses; and (3) it was cotransmitted in cybrid crosses with the mitochondrial CAP-R marker. Furthermore, the genetic studies suggested that the LA9 CAP-R ANT-R cells were heteroplasmic and contained at least two mtDNA genotypes, cap-r ant-s and cap-s ant-r. Cellular respiration of the ANT-R mutant was markedly more resistant to inhibition by antimycin than that of the parental ANT-S cells. The increased resistance of cellular respiration was entirely accounted for by an increase in the resistance of mitochondrial succinate-cytochrome c oxidoreductase to antimycin inhibition. There was no detectable change in the specific activity of the oxidoreductase in mitochondria of resistant ANT-R cells nor in the sensitivity of the complex to three other specific inhibitors of the complex: TTFA, myxothiazol, and HQNO. Taken together, these studies indicate that the ANT-R phenotype is most likely encoded within the mitochondrial cytochrome b gene and, more specifically, within an antimycin binding domain.

Animals↗
PubMed1983

Origin, cellular expression, and cybrid transmission of mitochondrial CAP-R, PYR-IND, and OLI-R mutant phenotypes.

Chloramphenicol-resistant (CAP-R) mouse and Chinese hamster lines were isolated in a single selection step in drug medium containing pyruvate. Cellular expression of the CAP-R phenotype required pyruvate--or an appropriate substitute--as a nutritional supplement. Subclone lines which were pyruvate independent (PYR-IND) arose in second-step selections at a high frequency. CAP-R PYR-IND Chinese hamster mutants could be directly isolated in single-step selections but at a very low frequency. Subclone lines (OLI-R) which were cross-resistant to oligomycin were isolated in a third selection cycle. The PYR-IND and OLI-R phenotypes were cotransmitted with the CAP-R mtDNA mutation but were expressed at the cellular level only if the number of mutant mitochondrial genomes exceeded a minimum threshold value. Analysis of a mtDNA restriction fragment alteration in one series of mutants supported this model. Threshold limits for cellular expression of mitochondrial mutant phenotypes are likely to be a general phenomenon and will constrain models of the origin and segregation of mtDNA mutations.

Animals↗
PubMed1982

Suppression of transformation and tumorigenicity in interspecies hybrids of human SV40-transformed and mouse 3T3 cell lines.

Somatic cell hybrids, formed by fusion of human SV40-transformed fibroblast lines and mouse 3T3 cells, were isolated and analysed for expression of transformation phenotypes and tumorigenic potential in immunodeficient nude mice. Both tumorigenic and nontumorigenic SV40-transformants were used for these experiments. Regardless of whether or not the parental human transformed parent line was tumorigenic, the hybrid progeny-with rare exception-did not form tumors. The possibility that human SV40-transformed lines and the derived hybrid progeny were immunologically rejected in nude mice was unlikely since fusion of these human lines to a tumorigenic 3T3 variant produced hybrids which were highly tumorigenic. In addition, in these hybrid lines, there was not a coordinate expression of SV40 T-antigen and transformation traits. The phenotypic separation in somatic cell hybrids of SV40 T-antigen expression and transformation phenotypes and tumorigenicity is evidence that host genetic changes, occurring during or subsequent to viral integration, also contribute to the expression of those traits in SV40-transformed cells. The general inability of human SV40-transformations to form tumors in nude mice would result from their inherent nontumorigenicity and reflect the relative rarity, compared to SV40-transformed mouse cells, of those host genetic changes determining tumorigenicity. The postulated role of an interaction between viral and cellular genetic changes was supported by other results hybrid lines formed by fusion of LNSV cells and the tumorigenic 3T3 derivative were markedly more anchorage independent than either parental line or LNSV/3T3 hybrid cells.

Animals↗
PubMed1981

Genomic rearrangements in a mouse cell line containing integrated SV40 DNA.

In the SV40-transformed mouse embryo fibroblast cell line SVT2/S, genomic rearrangements involving the SV40 DNA and flanking host sequences were identified by Southern blot hybridization using viral DNA as probe. No rearrangements of SV40 DNA integrated into nonpermissive mouse cells have been previously described. The standard arrangement found in the majority of subclones was mapped with 20 restriction enzymes, 10 of which cleave sites within the SV40 DNA. A single copy of a defective integrated viral genome is present, in which the late region is missing from about nucleotide 200 clockwise to about nucleotide 1750. The rest of the viral genome including the origin of replication and T antigen binding region is present and colinear with SV40 DNA, except for an internal repeat of about 1750 bp located between nucleotides 2750 and 4500. Rearrangements were found in 4 out of 20 random subclones of the parental SVT2/S cell line and 3 of the 4 continued to rearrange. The thioguanine-resistant cell line 281-1-4, derived from SVT2/S, remained stable on subculture but a chloramphenicol-resistant mutant, 107-6-4, derived from 281-1-4, was highly unstable. In 107-6-4, unique rearrangements were found in 6 of 31 subclones of a population that had undergone abut 25 doublings from a single-cell isolate. The high rate of rearrangement and the sporadic expression of rearrangement potential are characteristic of the transposable controlling elements discovered by McClintock.

Animals↗