Mammalian mitochondrial mutants selected for resistance to the cytochromeb 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 hada 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-cytochromec oxidoreductase, the respiratory complex containing cytochromeb. As with the previously described mitochondrial antimycinresistant mutant, the initial biochemical and genetic studies indicated that these mutations occur within the mitochondrial cytochromeb 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 apocytochromeb gene. Finally, all four mitochondrial cytochromeb mutants have been analyzed in both cell plating studies and succinate-cytochromec oxidoreductase assays to determine the pattern of cross-resistance to inhibitors of cytochromeb other than the one used for selection.     

Sequence analysis of mouse mitochondrial chloramphenicol-resistant mutants

The nucleotide sequences of the 3 halves of the mitochondrial 16S rRNA genes from four independent mouse chloramphenicol-resistant (CAP-R) mutants were determined. Each contained a different, single base change that encodes the mutational phenotype. The mitochondrial rRNAgene from the SVA31 CAP-R mutant contains a G-to-A transition at nucleotide 2161 of the noncoding strand; the SVIS CAP-R mutant, a G-to-A transition at position 2375; the LA9 CAP-R mutant, an A-to-T transversion at position 2379; and the SVT2 CAP-R mutant, a T-to-C transition at position 2433. Three of these CAP-R mutants appear to be heteroplasmic as the mtDNA populations contain both wild-type and mutant copies of the rRNAgene. The SVIS CAP-R mutation has not been observed in other mammalian CAP-R mutants, although it occurs at a site homologous to one of the yeast mitochondrial CAP-R mutations. Based upon the locations of the mutated sites within the 16S rRNA, and their proximity to previously analyzed sites of mutations conferring increased inhibitor resistance, all these mutations occur within the ribosomal RNA peptidyltransferase domain. These results provide an explanation for the pleiotropic nature of mitochondrial CAP-R mutations in mammalian cells, particularly the observations that some of the mutant lines are partially respiration deficient.     

Temperature-sensitive nuclear mutants of Neurospora crassa deficient in mitochondrial ribosomes

Summary We have isolated two non-allelic nuclear mutants of Neurospora crassa that are temperature-sensitive for the production of cytochromes aa ₃ and b. When grown at 23 °C the mutants are virtually indistinguishable from the parent wild-type strains. When grown at 41 °C the mutants have large amounts of KCN-insensitive respiration and lack cytochromes aa ₃ and b. Further examination of the mutants revealed that they were extremely deficient in their capacity for mitochondrial protein synthesis when grown at 41 °C. This protein synthesis deficiency appears to be related to a virtual absence of both small and large mitochondrial ribosomal subunits following growth at 41 °C. Examination of the mitochondrial RNAs of the mutants suggests that mitochondrial rRNAs are synthesized in greatly reduced amounts or that they are misprocessed when these mutants are grown at the non-permissive temperature.Abbreviations mt mitochondrial - SDS sodium dodecylsulfate - EDTA ethylenediaminetetraacetic acid - CAP chloramphenicol - TCA trichloroacetic acid - SHAM salicylhydroxamic acid     

Alteration of ribonucleotide reductase in aphidicolin-resistant mutants of mouse FM3A cells with associated resistance to arabinosyladenine and arabinosylcytosine

Aphidicolin-resistant mutants of mouse FM3A cells were isolated and characterized. Most of the mutants were of a type showing associated resistance to arabinosyladenine, arabinosylcytosine, deoxyadenosine, and excess thymidine. This phenotype could also be observed in a variant line selected by resistance to a low level of arabinosylcytosine. In cell-cell hybrids, aphidicolin resistance as well as this cross-resistance behaved as codominant traits. The mutants had an increased dATP pool and decreased ability to incorporate labeled deoxycytidine into macromolecules. Genetic and biochemical evidence suggested that the mutation conferring the pleiotropic phenotype resulted from a change in ribonucleotide reductase activity such that the enzyme was desensitized to the allosteric negative effector dATP. This alteration of the enzyme could account for the marked change in deoxynucleotide pools and for the aphidicolin resistance of the mutants.     

Mitochondrial genetics of mammalian cells: A mouse antimycin-resistant mutant with a probable alteration of cytochromeb

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 coxidoreductase 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 bgene and, more specifically, within an antimycin binding domain.     

Two membrane-bound proteins associated with alanine resistance and increased A-system amino acid transport in mutants of CHO-K1

Growth of CHO-K1, a praline auxotroph, is inhibited by amino acids that prevent praline transport. From a hydroxyurea-treated, alanine-resistant, constitutive mutant, ala^r4, we isolated, in a stepwise fashion, mutants, resistant to higher concentrations of alanine, that have increased velocity of amino acid transport through the A system. Two such mutants, ala^r4-H2.1 and ala^r4-H3.9, isolated as resistant to 50 mM and 125 mM alanine, respectively, showed increases in V_max of praline transport through the A system that are directly proportional to their resistance to alanine. Ala^r4-H3.9, as compared to ala^r4 and CHO-K1, has six and 29 times the V_max of praline transport through the A system and two and five times the velocity of transport through the combined ASC and P systems, respectively, and no change in system L. No doubleminute or homologous staining regions were detectable in ala^r4-H3.9. A-system activity of ala^r4-H2.1 and ala^r4-H3.9, when grown under nonselective conditions, was stable for 20 generations and then declined. The phenotype of ala^r4-H3.9 is codominant with that of ala^r4 and partially recessive to that of CHO-K1. Membrane vesicles prepared from ala^r4-H3.9 show increases mainly in A-system transport. In sodium dodecylsulfatepolyacrylamide gel electrophoresis analysis of A-system active membrane vesicles and endoplasmic reticulum, two bands of molecular weight of approximately 62–66 kd and 29 kd are present in higher concentrations in ala^r4-H3.9 than in CHO-K1. These results are compatible with the hypothesis that the phenotype of ala^r4-H3.9 is the result of gene amplification of an A-system transporter structural gene and that the two bands may represent this transporter.     

Protein kinase C inhibitors abolish the increased resistance of diabetic rat heart to ischemia-reperfusion injury

Protein kinase C (PKC) has been implicated in ischemic preconditioning, but whether it plays a role in the cardioprotection observed in the diabetic heart is not known. We assessed the possible role of PKC by investigating whether the inhibition of PKC with staurosporine (Stau, 0.01 microM) or chelerythrine (Chel, 1 microM) can abolish the increased resistance to ischemia (25 min)-reperfusion (30 min) injury in Langendorff perfused hearts from streptozotocin-induced 4-week diabetic rats. In the diabetic heart, pre-ischemic left ventricular developed pressure (LVDP), double product (DP: LVDPxheart rate/1,000), +/- dP/dt(max) and coronary flow rate (CFR) were all reduced compared to the control. The pretreatment with Stau or Chel significantly improved these parameters. The post-ischemic contractile function was recovered to a greater extent in the diabetic heart (116.9 +/- 20.5% of pre-ischemic DP) than in the control (23.3 +/- 2.3% of pre-ischemic DP), indicating the increased resistance of the diabetic heart to ischemia-reperfusion injury. The treatment with Stau or Chel abolished the enhanced recovery in the diabetic heart (36.0 +/- 14.6 and 54.1 +/- 12.8% of pre-ischemic DP, respectively). The reduction in post-ischemic end diastolic pressure (EDP) and lactate dehydrogenase (LDH) release in diabetes (13.5 +/- 2.5 mmHg and 27.2 +/- 6.2 U/g heart) compared to the control (55.8 +/- 2.9 mmHg and 60. 3 +/- 5.7 U/g heart) was significantly (p<0.05) increased by pretreatment with Stau (39.0 +/- 4.9 mmHg and 53.1 +/- 7.6 U/g heart) or Chel (36.2 +/- 3.0 mmHg and 48.8 +/- 4.3 U/g heart). Neither Stau nor Chel had any influence on the post-ischemic values of LVDP, DP, +/- dP/dt(max), EDP and LDH release in the control heart. In the conclusion, the present results suggest that PKC activation may, at least in part, contribute to the increased resistance of the diabetic heart to ischemia-reperfusion injury.     

Properties of cells with increased resistance to some picornaviruses

The resistance to picornaviral infection cells of susceptible lines has similar changes in the phenotype. They have decreased number of nucleoli and increased percentage of euploidy. Also the percentage of euploid cells those were resistant to the picornaviral infection increased in all highly transformed cultures. In resistant cells of all cultures has been found reduction of DNA. RNA amount also decreased both in nucleus and in cytoplasm. All these data correlated with the increased euploidy of the resistant population. The resistant cells had a less transformed phenotype, and decreased proliferative activity. Decreased nucleolar status became apparent by reduction of absolute and relative nucleolar indices. Consequently the reduction of viral titer (viral titters reduction) in resistant cells could be the direct result of diminished activity of the RNA synthesis machinery. It is important to note that the cells lose resistance while another type of virus, even from the same family, infects the culture once.     

High level of aphidicolin resistance with multiple mutations in mouse FM3A cell mutants

Spontaneous mutants of mouse FM3A cells (AC1, AC2, and AC3), highly resistant to aphidicolin (3000-, 2500-, and 300-fold increase in resistance, respectively), were isolated by multistep selection. The DNA synthesizing activity in permeabilized cells of all three mutants was substantially resistant to aphidiolin, like that in intact cells. The DNA polymerase activity in nuclear extracts in AC1 and AC3, but not AC2, was resistant to aphidicolin. Partially purified DNA polymerase from AC3, but not from AC1 or AC2, showed resistance to aphidicolin. The apparentK _i value for aphidicolin of AC3 polymerase was three to four times that of the enzyme from the parent cells, but the apparentK _m values of the enzyme for dCTP and dTTP were normal. All the mutants showed cross-resistance to both arabinofuranosyladenine and arabinofuranosylcytosine. The AC3 mutant had expanded deoxyribonucleoside triphosphate pools. On two-dimensional polyacrylamide gel electrophoresis, AC1 gave a new protein (mol wt 40 kDa). The aphidicolin-resistance trait was reversible in AC2, unlike in AC1 and AC3. These results show that in mammalian cells there are at least two mechanisms of aphidicolin-resistance that involve an altered DNA polymerase that is resistant to aphidicolin and simultaneous expansion of the four DNA-precursor pools.     

Combined expression of tau and the Harlequin mouse mutation leads to increased mitochondrial dysfunction, tau pathology and neurodegeneration

Mitochondrial dysfunction and oxidative stress play an important role in ageing and have been implicated in several age-related neurodegenerative conditions including Alzheimer's disease (AD) and other tauopathies characterized by the presence of intracellular accumulations of the hyperphosphorylated microtubule-associated protein tau. To study the interaction between mitochondrial dysfunction and tau pathology in vivo, we generated a novel mouse model by crossbreeding two existing lines: the Harlequin (Hq) mutant mice which suffer from mitochondrial dysfunction and oxidative stress due to a lack of the mitochondrial apoptosis-inducing factor (AIF), and the P301L tau transgenic mice, a mouse model of human tau pathology.Combined expression of the Hq mouse mutation and the tau transgene in the Tau/Hq double mutant mice led to an increase in tau pathology and apoptotic neurodegeneration when compared to single expression of the two mutations. Neurodegeneration was most prominent in the dentate gyrus and was significantly increased in the cerebellum leading to aggravated motor deficits. Functional activity measurements of the mitochondrial respiratory chain (MRC) in the Tau/Hq mice revealed early decreased activities of multiple MRC complexes and depleted ATP levels which preceded neurodegeneration and elevated oxidative stress markers. These results suggest an age-dependent mutual reinforcement of the tau pathology and mitochondrial dysfunction in vivo, which may contribute to neurodegeneration in patients suffering from AD and other age-related tauopathies.     

Genetic mechanisms of resistance to protease inhibitors and entry inhibitors

Abstract

Resistance to protease inhibitors (PIs) often appears as a result of changes at codons within the protease gene, which differ from one drug to another. When PIs are boosted with low doses of ritonavir, multiple mutations at the protease are required to reduce significantly the antiviral activity of these compounds. Moreover, mutations at gag cleavage sites may restore the loss of viral fitness caused by the selection of specific PI resistance mutations. In respect to entry inhibitors, a new class of compounds already in phase II/III clinical trials, resistance may emerge as a result of mutations outside the pol gene. Loss of susceptibility to agents that block virus attachment or the interaction with co-receptors often select HIV strains with mutations in the gp120 envelope-coding region. In contrast, agents that target gp41-dependent fusion select for HIV variants with mutations in the gp41 envelope gene.     

Genetic mechanisms of resistance to protease inhibitors and entry inhibitors

Resistance to protease inhibitors (PIs) often appears as a result of changes at codons within the protease gene, which differ from one drug to another. When PIs are boosted with low doses of ritonavir, multiple mutations at the protease are required to reduce significantly the antiviral activity of these compounds. Moreover, mutations at gag cleavage sites may restore the loss of viral fitness caused by the selection of specific PI resistance mutations. In respect to entry inhibitors, a new class of compounds already in phase II/III clinical trials, resistance may emerge as a result of mutations outside the pol gene. Loss of susceptibility to agents that block virus attachment or the interaction with co-receptors often select HIV strains with mutations in the gp120 envelope-coding region. In contrast, agents that target gp41-dependent fusion select for HIV variants with mutations in the gp41 envelope gene.     

Characterization of monoclonal antibodies to carcinoembryonic antigen with increased tumor specificity

Nine monoclonal antibodies reacting with carcinoembryonic antigen (CEA) were produced after immunization of mice with either purified CEA or a CEA-producing human cell line. Their specificities were assessed by immunohistochemistry on tissue sections of neoplastic and nonneoplastic lesions. These monoclonal antibodies have different patterns of tissue reactivity. Two of them, D14 and B18, were found to have a high degree of specificity for colonic carcinoma and did not react with formalin-fixed paraffin-embedded sections of normal colon with standardized staining conditions. Most cases of noncolonic adenocarcinomas and normal epithelial structures were not stained by these two monoclonal antibodies. The specificity of the monoclonal antibodies was further investigated immunochemically using intact, reduced, and alkylated or chemically fragmented CEA. Liquid phase radioimmunoassays and antibody competition immunoenzymatic assays confirmed that the antibodies recognize different epitopes of CEA. These data support the concept of CEA heterogeneity and the reactivity of the D14 and B18 monoclonal antibodies with colonic adenocarcinomas indicates that they are useful immunohistochemical probes.     

Characterization of Escherichia coli mutants with altered ploidy

We describe the isolation and characterization of new mutants in the cell cycle of Escherichia coli. The mutants were selected as gain of function mutants that are able to maintain more than the normal number of chromosomes. Our increased ploidy mutants were isolated as resistant to camphor vapours, which selects for cells with more chromosomes than normal. The mutants (called mbr for moth-ball-resistant) map to four chromosomal locations: mbrA at 68 min; mbrB at 88.5 min; mbrC at 89.5 min; and mbrD at 90 min. To investigate the nature of these cell cycle mutants, we have coupled them with defects in recA, to test for induction of the SOS response, and dam, to determine if methylation is required for mbr function. Based on the results of these and other tests, we have made a preliminary placement of the mbr mutants within the context of the cell cycle. mbrA mutations appear to be defective in the coupling of the DNA replication cycle to the cell division cycle, and as such, may define a new link between the two processes. mbrB does not seem to be able to coordinate the cell cycle and the growth rate of the cell. mbrC appears to be defective in partitioning of chromosomes. mbrD, which may be allelic to rpoB (a subunit of RNA polymerase), appears to be defective in either chromosomal partitioning or the later stages of DNA replication.     

Isolation and characterization of three mutants with increased sensitivity to photoactivated 3-carbethoxypsoralen in Saccharomyces cerevisiae

The complementation and genetical analysis of yeast mutants sensitive to photoactivated 3-carbethoxy-psoralen define three novel recessive mutant alleles pso-5-1, pso6-1, and pso7-1. Their cross-sensitivity to UV_254nm, radiomimetic mutagens, and to chemicals enhancing oxidative stress suggest that these mutants are either impaired in metabolic steps protecting from oxidative stress or in mechanisms of the repair of oxygen-dependent DNA lesions. None of the three novel mutant alleles block the induction of reverse mutation by photoactivated mono- and bi-functional psoralens, nitrogen mustards, or UV_254nm.     

Immunological mutants of the mouse

Mutations at more than 30 loci in mice have been shown to cause deleterious effects on the immune system. Immunologic defects caused by certain of these mutations are determined at the level of hematopoietic progenitor cells or at the level of hematopoietic cell-stromal cell interactions. The immunological mutants described in this paper serve as experimental tools with which to increase our understanding of the development and regulation of the mammalian immune system.