Nuclear omnipotent suppressors of premature termination codons in mitochondrial genes affect the 37S mitoribosomal subunit

Summary nam3 and R705, yeast nuclear omnipotent suppressors of mitochondrial mit ^– mutations, reverse the superimposed spectrum of trans-recessive splicing defects by affecting the protein composition of the small mitoribosomal subunit. Analysis of the suppressor's interaction suggests that suppression results from mutations in the mitoribosomal polypeptides. These data indicate an obligatory connection between mitoribosome function and splicing of introns bI2, bI4 and aI1 in yeast mitochondria.     

Nuclear suppressors of the mitochondrial mutation oxi1-V25 in Saccharomyces cerevisiae

Summary Ten nuclear suppressors (nam mutations) of the mitochondrial oxi1-V25 ochre mutation are characterized. They restore to some extent the functional form of cytochrome oxidase, as judged by the results of growth tests, cytochrome spectra, cytochrome oxidase activities, and electrophoresis of the products of mitochondrial translation. The nam mutants can suppress some mit ^– mutations mapping in four mitochondrial genes. They act on a number of chain-terminating mit ^– mutations. When grown on glycerol medium some double mutants nam _x-V25 show an increased sensitivity to paromomycin, while the growth of others is stimulated by the drug. The nam mutants are probably omnipotent suppressors resulting from mutations in nuclear gene(s) specifying mitoribosomal protein(s).     

Mitochondrial mutagenesis in Saccharomyces cerevisiae

Summary Four types of mit ^– mutations induced with manganese are found in the following relative proportions: oxi3 ^– > cob-box ^– > oxi2 ^– oxi1 ^–1. The frequences of loss of their respective mit ⁺ alleles in manganese-induced rho ^–] primary and secondary clones follow the same order. The possible interdependence between these two sets of data is discussed.     

Recombinational analysis of oxi2 mutants and preliminary analysis of their translation products in S. cerevisiae

Summary Genetic and biochemical studies were performed with mutants allocated to the mitochondrial oxi2 gene.Recombinational analysis of 19 oxi2 mutants was performed using and a mutant strains derived from the same genetic background. The frequencies of wild-type recombinants in oxi2 ^– × oxi2 ^– crosses varied from 0.002 to 17%. The map of oxi2 mutations constructed on the basis of these frequencies shows many internal inconsistencies. In the course of rho ^– deletion mapping five classes of oxi2 mutations were distinguished. The results of deletion analysis are in agreement with those of recombinational mapping.The analysis of mitochondrial translation products by SDS-polyacrylamide electrophoresis of 20 oxi2 mutants shows that 17 of them are connected with conspicuous changes of 22 kd polypeptide band corresponding to subunit III of cytochrome oxidase. At least four of them carried instead of subunit III clearly visible significantly shorter polypeptides (12.8 to 20.1 kd). These were, most likely, shorter fragments of subunit III resulting from chain termination mutations. Colinearity was observed between the lenght of new polypeptides and the positions of the respective mutations on the recombinational map. These data confirm hat oxi2 encodes subunit III of cytochrome oxidase and suggest that translation of the oxi2 gene is in the direction from V303 to V273.     

Molecular screening of patients with nonsyndromic hearing loss from Nanjing city of China

Hearing loss is the most frequent sensory disorder involving a multitude of factors,and at least 50% of cases are due to genetic etiology.To further characterize the molecular etiology of hearing loss in the Chinese population,we recruited a total of 135 unrelated patients with nonsyndromic sensorineural hearing loss (NSHL) for mutational screening of GJB2,GJB3,GJB6,SLC26A4,SLC26A5 IVS2-2A>G and mitochondrial 12SrRNA,tRNA Ser(UCN) by PCR amplification and direct DNA sequencing.The carrier frequencies of deafness-causing mutations in these patients were 35.55% in GJB2,3.70% in GJB6,15.56% in SLC26A4 and 8.14% in mitochondrial 12SrRNA,respectively.The results indicate the necessity of genetic screening for mutations of these causative genes in Chinese population with nonsyndromic hearing loss.     

Sporulation in mitochondrial OXI3 mutants of Saccharomyces cerevisiae

Summary By use of a set of mitochondrial oxi3 mutants (mit ^–, defective in cytochrome oxidase) we have shown that sporulation is possible at a very low level of respiration (below l% of the wild type respiration). A specific role for oxygen in biosynthesis during sporulation is suggested. Correlation of these results with the genetic map of the OXI3 region reveals that one group of mutants, mapping in the central part of the OXI3 region, is capable of sporulation.     

Phenotypic suppression and nuclear accommodation of the mit − oxi1-V25 mutation in isolated yeast mitochondria

Summary Phenotypic suppression by the antibiotic, paromomycin, of the mitochondrial oxi1 ^–-V25 mutation, a mutation which arrests by premature ochre codon the synthesis of the cox 11 subunit, was studied in isolated yeast mitochondria competent in translation. This antibiotic is known to suppress the mutation in vivo (Dujardin et al. 1984) and allowed in vitro, at concentrations of 20–1100 Mg per ml. the synthesis of the cox II subunit. This strongly suggests that phenotypic suppression of mit ^– mutations is due to the direct action of paromomycin on mitochondrial ribosomes. The effect of paromomycin bears a resemblance to the function of the omnipotent nuclear suppressor mutation R705. The nuclear suppression was expressed in isolated mitochondria; suppressor mutation influenced the structure of the mitoribosome. Therefore, it appears that mitoribosomes are indeed the common target in the phenotypical and genetic nuclear suppression of the oxi1-V25 mutation.     

Mitochondrial and nuclear mitoribosomal suppressors that enable misreading of ochre codons in yeast mitochondria

Summary We describe studies on the action spectra of the mitochondrial suppressor mim3-1 and the three alleles of nuclear suppressor nam3. Their specificity of action was tested on 516 mit ^– mutations located in different mitochondrial genes. The degree of suppression was quantified by the extent of cytochrome oxidase and cytochrome b synthesis. We show that the four suppressors are allele-specific gene-nonspecific informational suppressors. They would act by changing the structure of the small mitoribosomal subunit which would decrease fidelity of translation enabling misreading of some but not all ochre codons. The implications of the results on the role of intron encoded maturases are discussed.     

Effects of chronic morphine intake on binding of NAD and GABA-benzodiazepine agonists to synaptic membranes

Nine weeks of compulsory morphine drinking decreased the specific binding of³H-muscimol to GABA receptors and¹⁴C-NAD to rat brain synaptic membranes and increased the synaptosomal uptake of¹⁴C-GABA. These effects of morphine on the GABA-benzodiazepine receptor complex were reversed by excessive doses of vitamin B₃. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 3, pp. 295–301, March, 1999     

Cytopathogenic action ofEscherichia coli cells containing heterologous human type O(H) antigen on human cells in culture

The character of interaction between two enteropathogenic strains ofEscherichia coli of serotype 055K59 with human HeLa cells containing O(H) isoantigen was studied. On the addition of strainE. coli No. 5789, containing heterologous type O(H) antigen to a culture of HeLa cells, a cytopathogenic action was discovered on the third day of interaction in the presence of doses of bacterial cells of 2·10¹⁰, 2·10⁵, and 2·10⁴. A dose of 2·10³ bacterial cells ofE. coli did not give this effect. Strain No. 3827, not containing heterologous antigen of ABO type, had no cytopathogenic action in maximal, average, and small doses of bacterial cells. It is suggested that the cytopathogenic action of strain No. 5789 is connected with the presence of an antigen in this strain which is identical with the group antigen of the human cell culture studied.Research Laboratory of Experimental Immunobiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of medical Sciences of the USSR N. N. Zhukov-Verezhnikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 84, No. 7, pp. 70–72, July, 1977.     

Recombinational analysis of OXI1 mutants and preliminary analysis of their translation products in S. cerevisiae

Summary Recombinational analysis of oxil mutants was performed using a and a mutant strains with the same mitochondrial and nuclear backgrounds, derived from strain 777-3A.In spite of minor inconsistencies the overall map of oxi1 mutations can be constructed on the basis of wild-type recombinant frequencies in the two-point oxi1 ^{– –} x oxi1 ^– crosses. The frequencies of wild-type recombinants varied in a wide range from 0.003% to 16%, reaching the maximal values expected for unlinked mitochondria) markers. No distinct clusters of mutants were observed.The analysis of translation products of oxil mutants showed that all but one of the oxil mutants studied are connected with the conspicuous changes of the polypeptide band corresponding to subunit 11 of cytochrome c oxidase in electrophoresis on polyacrylamide gels. The exceptional G565 mutant showed no conspicuous change in subunit II, but lacked subunit I of cytochrome c oxidase.Various oxi1 mutants seemed to carry premature chain termination mutations. Most of them show a correlation between the length of the putative fragment of subunit II synthesized and the position on the genetic map. The direction of translation is from the V2 to the V60 mutation. The V2 mutation is proximal to cap and V60 proximal to the par locus.     

Effect of pulsed infrared laser radiation on DNA synthesis in tissues of intact rats and during strenuous physical exercise

The motor zone of the rat brain cortex is subjected to pulsed infrared (0.89 μ) laser radiation, which is found to stimulate DNA synthesis both in intact animals and after strenuous physical exercise (swimming). Preliminary laser irradiation exerts a stress-limiting effect on cells of the brain cortex and thymus but does not prevent swimminginduced reduction of³H-thymidine incorporation in nuclear DNA of muscles. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 119, N ^o 6, pp. 625–627, June, 1995 Presented by V. M. Bogolyubov, Member of the Russian Academy of Medical Sciences     

Effect of human defensins on the cytoplasmic Ca2+ content in platelets

The effect of the total fraction of human defensins (HNP-1, HNP-2, and HNP-3) on the cytoplasmic Ca²⁺ content ([Ca²⁺]_i) in the platelets of healthy donors was studied. At concentrations of 0.1–40 μg/ml and an incubation time of 10 min defensins have no effect on [Ca²⁺]_i in platelets labeled with Fura-2AM. However, at higher concentrations (100 μg/ml) they increased platelet [Ca²⁺]_i. In addition, defensins (40 μg/ml) inhibited the Ca²⁺ increase in platelets induced by thrombin, adenosine diphosphate, and the lipopolysaccharide ofS. typhimurium endotoxin. The most pronounced inhibitory effect was observed in a suspension of thrombin-stimulated platelets. It is shown that the effect of human defensins on the functional activity of platelets is due to the alterations in the intracellular Ca²⁺. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 12, pp. 600–603, December, 1994     

The antioxidant Trolox restores mitochondrial membrane potential and Ca2+-stimulated ATP production in human complex I deficiency

Malfunction of mitochondrial complex I caused by nuclear gene mutations causes early-onset neurodegenerative diseases. Previous work using cultured fibroblasts of complex-I-deficient patients revealed elevated levels of reactive oxygen species (ROS) and reductions in both total Ca²⁺ content of the endoplasmic reticulum (ER_Ca) and bradykinin(Bk)-induced increases in cytosolic and mitochondrial free Ca²⁺ ([Ca²⁺]_C; [Ca²⁺]_M) and ATP ([ATP]_C; [ATP]_M) concentration. Here, we determined the mitochondrial membrane potential (Δψ) in patient skin fibroblasts and show significant correlations with cellular ROS levels and ER_Ca, i.e., the less negative Δψ, the higher these levels and the lower ER_Ca. Treatment with 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) normalized Δψ and Bk-induced increases in [Ca²⁺]_M and [ATP]_M. These effects were accompanied by an increase in ER_Ca and Bk-induced increase in [Ca²⁺]_C. Together, these results provide evidence for an integral role of increased ROS levels in complex I deficiency and point to the potential therapeutic value of antioxidant treatment. Felix Distelmaier and Henk-Jan Visch contributed equally to this paper.     

Transport of nitrullin,a new nitrosoalkylurea derivative,into tumor and normal cells

The interaction between the antitumor drug nitrullin and the system providing the transport ofl-lysine into P388 leukemic cells and murine enterocytes is studied. Two types of lysine carriers with low and high affinity for the substrate are identified. Nitrullin competitively inhibits the transport of³H-lysine and shows the same affinity for both carriers. It is similar to that of lysine for the low-affinity carrier and is 80-fold lower than lysine affinity for the high-affinity carrier. Kinetic characteristics of the low-affinity transport of³H-lysine and K_i of nitrullin are similar to those obtained at a reciprocal substrate-inhibitor ratio. Nitrullin does not inhibit active transport of³H-lysine into enterocytes against the background of considerable (70%) passive diffusion. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 122, No. 12, pp. 648–650, December, 1996     

cAMP-dependent inward rectifier current in neurons of the rat suprachiasmatic nucleus

Electrophysiological properties of the inward rectification of neurons in the rat suprachiasmatic nucleus (SCN) were examined by using the single-electrode voltage-clamp method, in vitro. Inward rectifier current (I _H) was produced by hyperpolarizing step command potentials to membrane potentials negative to approximately −60 mV in nominally zero-Ca²⁺ Krebs solution containing tetrodotoxin (1 μM), tetraethylammonium (40 mM), Cd²⁺ (500 μM) and 4-aminopyridine (1 mM).I _H developed during the hyperpolarizing step command potential with a duration of up to 5 s showing no inactivation with time.I _H was selectively blocked by extracellular Cs⁺ (1 mM). The activation of the H-channel conductance (G _H) ranged between −55 and −120 mV. TheG _H was 80–150 pS (n=4) at the half-activation voltage of −84±7 mV (n=4). The reversal potential ofI _H obtained by instantaneous current voltage (I/V) relations was −41±6mV (n=4); it shifted to −51±8mV (n=3) in low-Na⁺ (20 mM) solution and to −24±4 mV (n=4) in high-K⁺ (20 mM) solution. Forskolin (1–10 μM) produced an inward current and increased the amplitude ofI _H. Forskolin did not change the half-activation voltage ofG _H. 8-Bromo-adenosine 3′,5′-cyclic monophosphate (8-Br-cAMP, 0.1–1 mM) and dibutyryl-cAMP (0.1–1 mM) enhancedI _H. 3-Isobutyl-1-methylxanthine (IBMX, 1 mM) also enhancedI _H. The results suggest that the inward rectifier cation current is regulated by the basal activity of adenylate cyclase in neurons of the rat SCN.     

Induced recombination and reversion of theCDC8 gene in relation to the cell cycle of yeast

Summary The induction of mitotic recombination in theCDC8 locus was studied in a diploid strain heteroallelic forcdc8 mutations (cdc8-1/cdc8-3); mitotic reversion was studied in strainscdc8-1/cdc8-1 andcdc8-3/cdc8-3. Conversion and reversion did not occur in those cells blocked at the S stage of the cell cycle by exposure to a nonpermissive temperature. In stationary phase cells irradiated just prior to exposure to temperature stress, the induction of recombinants was rather low and the induction of revertants was minimal. Conversely, a significant induction ofcdc ⁺ occurred in logarithmic phase cells subjected to the same treatment. Irradiation of synchronously dividing cultures revealed that intragenic recombination occurs at all three stages of the cell cycle- G₁, S and G₂. It was also found that UV-induced gene reversion can occur during the S and G₂ stages, but not during the G₁ stage of the cell cycle.     

Chromium resistant mutants of the yeast Saccharomyces cerevisiae

Summary Many strains of Saccharomyces cerevisiae do not grow on YPD agar containing 750 g/ml CrO₃. Mutants able to grow in the presence of 850 g/ml CrO₃ were obtained from such strains after UV mutagenesis. All of the mutants grew even in the presence of 1,000 /ml CrO₃. Chromium resistance was dominant or partial dominant over normal response, therefore it was impossible to determine the number of genetic loci by complementation analysis. However, the segregation of representative mutants strongly indicated that resistance was determined by single mutations. In addition, a limited analysis of recombination suggested that the chromium resistant mutations were located on a certain region of the yeast genome. Although it was determined that the mutants had slightly reduced rates of Cr⁶⁺ uptake, the exact mechanism of resistance was not discovered. According to the studies of interactions between resistant mutations and sensitive mutations, however, we have proposed a preliminary pathway of Cr⁶⁺ detoxification.     

Molecular pathology of well-differentiated thyroid carcinomas

The newly discovered molecular features of well-differentiated thyroid carcinomas derived from follicular cells are reviewed, within the frame of the 2004 WHO classification of thyroid tumours, under the following headings: “Follicular carcinoma”, “Papillary carcinoma”, “Follicular variant of papillary carcinoma” and “Hürthle cell tumours”. A particular emphasis is put on the meaning of PAX8–PPARγ rearrangements, RAS and BRAF mutations, and deletions and mutations of mitochondrial genes and of nuclear genes encoding for mitochondrial enzymes, for thyroid tumorigenesis.     

Functional activity of catecholaminergic system in human fetal midbrain and diencephalon

The development of catecholaminergic system of the midbrain and diencephalon was studied in human embryos and fetuses aged 6, 8, 10, and 12 weeks by specific capture and K⁺-stimulated release of³H-dopaminein vitro. Specific capture of³H-dopamine was first detected in the midbrain of 6-week embryos and in the diencephalon of 8-week fetuses. The time course of the capture points to on-going differentiation of catecholaminergic neurons and fiber growth and the presence of the caudorostral gradient in the development of brain catecholaminergic system. The release of catecholamines was not stimulated in response to membrane depolarization in the midbrain and diencephalon at any of the studied stages of development. The difference in the time of capture and K⁺-stimulated release of catecholamines is related to specific features of differentiation of these neurons in human fetuses. Tranlated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 9, pp. 259–262, September, 1997