Effect of lysophasphatidylcholine on sensitivity of the heart to acetylcholine and binding of quinuclidinyl benzilate to myocardial membranes

Chemical nature of the blood serum factor exerting cholinolytic effect on the animal heart, was determined. The effect is due to the serum lipid component: lysophosphatidylcholine. The latter in concentration 1-25 microM suppresses the sensitivity of the frog perfused heart ventricle to acetylcholine. Phosphatidylcholine was found to protect the heart from the lysophosphatidylcholine effect. Lysophosphatidylcholine also affected the binding of the acetylcholine high affinity antagonist [3H]-quinuclidinylbenzilate to the rabbit atria cell membranes. The mechanism of this effect is related to an increased ability of muscarinic receptors to form oligomeric complexes in presence of lysophosphatidylcholine, the latter manifesting a positive cooperativity on binding to ligand.     

The possibility of peripheral cholinolytic action of psychostimulants

The cholinolytic effect of sydnophen discovered in earlier anesthetized cats was confirmed on unanesthetized fish and frogs: the vagal bradycardia induced by electric stimulation of peripheral vagal end was decreased or even abolished by intravenous injection of sydnophen (0.2-20 mg/kg). The amphetamine (0.2-30 mg/kg) also blocked the vagal bradycardia in anesthetized cats and unanesthetized frogs. The maximum vagolytic action of amphetamine appeared later (in 4-8 min after injection) in compared with sydnophen (1-3 min). The small dose of amphetamine (0.2-0.3 mg/kg) in contrast to sydnophen didn't decrease the vagal bradycardia but even increased it. It was suggested that the cholinolytic effect of sydnophen and amphetamine is due to different mechanisms.     

Characteristics of the psychotropic spectrum of action of mebicar

Under group interaction in cats, a new Soviet tranquilizer mebicar eliminates fear-alarm induced by stimulatin of the emotiogenic zone of the hypothalamus. This action is not associated with myorelaxant or hypnotic action. Mebicar decreases the brain noradrenaline level, exerts no effect on the dopaminergic systems, increases the brain serotonin level, and does not elicit cholinolytic action.     

Study of the mechanism of postcoital contraception in the combination of streoids and the central m-cholinolytic, amizil]

Experiments on female rats showed the blocking of the M-cholinoreactive system with amizyl to significantly contribute to the estrogen/norsteroid contraceptive effect during the postcoital periods. This effect was accompanied by decrease in the gonadotropin level and by the change in the LH/FSH ratio, this creating an unfavourable background for implantation of the fertilized ovicell in the endometrium. There was a change in the transport rate in the tube and a delay in the decidual reaction. Changes in the rate of the ovicell transport were not accompanied by distrubances in the process of fertilization or with the cytotoxic action. Mestranol and ethynylestradiol in combination with norethynodrel (1:20) and with the central cholinolytic amizyl were agents with future prospects for short-term postcoital contraception.     

The functional role of neurospecific protein S-100 in memory processes]

Elaboration of alimentary conditioned reflex in rats is accompanied by an increase of the level of protein S-100 in the left and right cerebral hemispheres. Amnestic factor M-cholinolytic atropine disturbs the elaborated habit and simultaneously decreases the quantity of protein S-100 up to the level of unlearned animals. The elaboration of conditioned reflex of passive avoidance does not change the content of protein S-100 in the rats brain. Intracisternal injection of antiserum to protein S-100 has an expressed amnestic action. Intracisternal injection of protein S-100 against the background of amnestic action of cholinolytic does not lead to restoration of memory. The cholinolytic and antiserum to protein S-100 mutually potentiate the amnestic effect.     

Reactivating and cholinolytic action of trimedoxime bromide at the neuromuscular junction of warm-blooded animals

The reactivating and cholinolytic action of trimedoxime bromide was evaluated during experiments on rat soleus and diaphragm muscles accoridng to the amplitude and time course of miniature end-plate potentials and currents (MEPP and MEPC respectively). This agent reactivates acetylcholinesterase (AChE) phosphorylation. The effects of trimedoxime bromide at concentrations of 5·10^–6–5·10^–4 M following AChE inhibition on the amplitude and duration of MEPP arises from complex interaction between the reactivating and cholinolytic effects. A separate evaluation of the reactivating effect (once the reactivating agent had been removed) revealed that this action increases throughout the entire range of trimedoxime bromide concentration: complete reactivation of AChE phosphorylation was observed under the action of 2–5·10^–4 M trimedoxime bromide. Examination of the cholinolytic effect in isolation (with voltage-clamping at the muscle and intact AChE) showed that blockade of open end-plate ionic channels underlies this effect. Reduction in MEPC amplitude together with retarded (but still exponential) decay of signals were distinguishing features of this blockade, confirming that trimedoxime bromide acts as a very fast blocker.S. V. Kurashov Medical Institute, Ministry of Public Health of the RSFSR, Kazan'. Translated from Neirofiziologiya, Vol. 20, No. 3, pp. 351–357, May–June, 1988.     

X-ray studies in the diagnosis of pyloroduodenal ulcerative stenoses

Combined clinical and x-ray investigation of 100 patients operated on for pyloroduodenal ulcerative stenoses was conducted. Methods of polypositional and polyprojectional x-ray investigation including a double contrast study and a differentiated use of cholinolytic and cholinomimetic drugs were standardized. The x-ray classification of stenosis was improved on the basis of radiological and surgical correlations and quantitative analysis of indices characterizing a degree of stenosis compensation. Criteria for the determination of a degree of stenosis were worked out.     

Ischemic preconditioning inhibits mitochondrial respiration,increases H2O2 release,and enhances K+ transport

Ischemic preconditioning, or the protective effect of short ischemic episodes on a longer, potentially injurious, ischemic period, is prevented by antagonists of mitochondrial ATP-sensitive K+ channels (mitoKATP) and involves changes in mitochondrial energy metabolism and reactive oxygen release after ischemia. However, the effects of ischemic preconditioning itself on mitochondria are still poorly understood. We determined the effects of ischemic preconditioning on isolated heart mitochondria and found that two brief (5 min) ischemic episodes are sufficient to induce a small but significant decrease ( approximately 25%) in mitochondrial NADH-supported respiration. Preconditioning also increased mitochondrial H2O2 release, an effect related to respiratory inhibition, because it is not observed in the presence of succinate plus rotenone and can be mimicked by chemically inhibiting complex I in the presence of NADH-linked substrates. In addition, preconditioned mitochondria presented more substantial ATP-sensitive K+ transport, indicative of higher mitoKATP activity. Thus we directly demonstrate that preconditioning leads to mitochondrial respiratory inhibition in the presence of NADH-linked substrates, increased reactive oxygen release, and activation of mitoKATP.     

A mitochondrial molecular marker, ori-rep-tra, for differentiation of yeast species.

Yeasts exhibit various mechanisms for the inheritance of their mitochondrial genomes. Differences among these mechanisms are based on variations within nuclear as well as mitochondrial genetic elements. Here we report diagnostic differences in the presence of biologically active mitochondrial intergenic sequences, ori-reptra, among related yeasts in the genera Saccharomyces, Arxiozyma, Debaryomyces, Kluyveromyces, Pachytichospora, Torulaspora, and Zygosaccharomyces. A molecular probe containing ori-rep-tra can be employed specifically for the differentiation and identification of isolates belonging to the species complex Saccharomyces sensu stricto.     

Functional and structural role of amino acid residues in the matrix alpha-helices, termini and cytosolic loops of the bovine mitochondrial oxoglutarate carrier

The mitochondrial oxoglutarate carrier belongs to the mitochondrial carrier family and exchanges oxoglutarate for malate and other dicarboxylates across the mitochondrial inner membrane. Here, single-cysteine mutant carriers were engineered for every residue in the amino- and carboxy-terminus, cytoplasmic loops, and matrix alpha-helices and their transport activity was measured in the presence and absence of sulfhydryl reagents. The analysis of the cytoplasmic side of the oxoglutarate carrier showed that the conserved and symmetric residues of the mitochondrial carrier motif [DE]XX[RK] localized at the C-terminal end of the even-numbered transmembrane alpha-helices are important for the function of the carrier, but the non-conserved cytoplasmic loops and termini are not. On the mitochondrial matrix side of the carrier most residues of the three matrix alpha-helices that are in the interface with the transmembrane alpha-helical bundle are important for function. Among these are the residues of the symmetric [ED]G motif present at the C-terminus of the matrix alpha-helices; the tyrosines of the symmetric YK motif at the N-terminus of the matrix alpha-helices; and the hydrophobic residues M147, I171 and I247. The functional role of these residues was assessed in the structural context of the homology model of OGC. Furthermore, in this study no evidence was found for the presence of a specific homo-dimerisation interface on the surface of the carrier consisting of conserved, asymmetric and transport-critical residues.     

The separation of sheep liver cytoplasmic and mitochondrial aldehyde dehydrogenases by isoelectric focusing, and observations on the purity of preparations of the cytoplasmic enzyme, and their sensitivity towards inhibition by disulfiram.

Preparations of sheep liver cytoplasmic aldehyde dehydrogenase obtained by published methods were found by analytical isoelectric focusing in the pH range 5--8 to contain 5--10% by weight of the mitochondrial aldehyde dehydrogenase. Under the conditions used the pI of the cytoplasmic enzyme is 6.2 and that of the mitochondrial enzyme 6.6. The mitochondrial enzyme can be removed from the preparation by selective precipitation of the cytoplasmic enzyme with (NH4)2SO4. Kinetic experiments and inhibition experiments with disulfiram show that the properties of the two sheep liver enzymes are so different that the presence of 10% mitochondrial enzyme in preparations of the cytoplasmic enzyme can introduce serious errors into results. Our results suggest that the presence of 10 microM-disulfiram in assays may completely inactivate the pure cytoplasmic enzyme. This result is in contrast with a previous report [kitson (1978) Biochem. U. 175, 83--90].     

Periodate-oxidized AMP as a substrate, an inhibitor and an affinity label of human placental alkaline phosphatase.

1. Addition of glucose induced an inactivation of mitochondrial enzymes in the yeast Saccharomyces cerevisiae containing normal mitochondrial particles. 2. The glucose-induced inactivation of mitochondrial enzymes was inhibited by the presence of cycloheximide. 3. Pepstatin also inhibited the inactivation, but phenylmethanesulphonyl fluoride accelerated the inactivation. 4. The specific activities of fructose 1,6-bisphosphatase and cytoplasmic malate dehydrogenase were decreased on the exposure to glucose, as well as those of the mitochondrial enzymes. However, the glucose-induced inactivation of cytoplasmic enzymes was not inhibited by the presence of pepstatin. 5. The specific activities of hexokinase and phosphofructokinase, which are cytoplasmic enzymes were increased by the addition of glucose, and this effect was not affected by pepstatin. 6. Addition of glucose resulted in an increase in the synthesis of proteins of the mitochondria and the cytosol, and simultaneously in degradation of these mitochondrial and cytoplasmic proteins.     

Diphenylacetaldehyde-generated excited states promote damage to isolated rat liver mitochondrial DNA, phospholipids, and proteins.

This work studies damage to rat liver mitochondrial protein, lipid, and DNA caused by electronically excited states generated by cytochrome c-catalyzed diphenylacetaldehyde enol oxidation to triplet benzophenone. The extension of lipid peroxidation was estimated by production of thiobarbituric acid-reactive substances and by formation of Schiff bases with membrane proteins, evaluated by SDS-polyacrylamide gel electrophoresis. Concomitant with DPAA-driven mitochondrial permeabilization, extensive mtDNA fragmentation occurred and DNA adducts with aldehydes-products of fatty acid oxidation-were observed. The degree of lipid peroxidation and mtDNA alterations were significantly decreased by butylated hydroxytoluene, a potent peroxidation chain breaker. The lipid peroxidation process was also partially inhibited by the bioflavonoid rutin and urate totally prevented the mitochondrial transmembrane potential collapse. In all cases, the mitochondrial damage was dependent on the presence of phosphate ions, a putative bifunctional catalyst of carbonyl enolization. These data are consistent with the notion that triplet ketones may act like alkoxyl radicals as deleterious reactive oxygen species on biologic structures. Involvement of singlet dioxygen formed by triplet-triplet energy transfer from benzophenone in the model reaction with DPAA/cytochrome c in the presence of DCP liposomes was suggested by quenching of the accompanying chemiluminescence upon addition of histidine and lycopene.     

Central cholinolytic effect of tropane derivatives: structure-activity relationship

The effect of two tropane derivatives on the electric neuronal activity in sensorimotor cortex was studied in rabbits using microiontophoretic method. Unlike atropine they lack aryl and hydroxyl, but possess morpholine and piperazine. The effects of both drugs were opposite to those of acetylcholine. Simultaneous application of tropane derivatives and acetylcholine to one neuron decreased both excitatory and inhibitory neuronal responses to acetylcholine. It is concluded that aryl and hydroxyl are not necessary for tropane derivatives to reveal their central cholinolytic activity.     

Cloning and characterization of FAD1, the structural gene for flavin adenine dinucleotide synthetase of Saccharomyces cerevisiae.

The FAD1 gene of Saccharomyces cerevisiae has been selected from a genomic library on the basis of its ability to partially correct the respiratory defect of pet mutants previously assigned to complementation group G178. Mutants in this group display a reduced level of flavin adenine dinucleotide (FAD) and an increased level of flavin mononucleotide (FMN) in mitochondria. The restoration of respiratory capability by FAD1 is shown to be due to extragenic suppression. FAD1 codes for an essential yeast protein, since disruption of the gene induces a lethal phenotype. The FAD1 product has been inferred to be yeast FAD synthetase, an enzyme that adenylates FMN to FAD. This conclusion is based on the following evidence. S. cerevisiae transformed with FAD1 on a multicopy plasmid displays an increase in FAD synthetase activity. This is also true when the gene is expressed in Escherichia coli. Lastly, the FAD1 product exhibits low but significant primary sequence similarity to sulfate adenyltransferase, which catalyzes a transfer reaction analogous to that of FAD synthetase. The lower mitochondrial concentration of FAD in G178 mutants is proposed to be caused by an inefficient exchange of external FAD for internal FMN. This is supported by the absence of FAD synthetase activity in yeast mitochondria and the presence of both extramitochondrial and mitochondrial riboflavin kinase, the preceding enzyme in the biosynthetic pathway. A lesion in mitochondrial import of FAD would account for the higher concentration of mitochondrial FMN in the mutant if the transport is catalyzed by an exchange carrier. The ability of FAD1 to suppress impaired transport of FAD is explained by mislocalization of the synthetase in cells harboring multiple copies of the gene. This mechanism of suppression is supported by the presence of mitochondrial FAD synthetase activity in S. cerevisiae transformed with FAD1 on a high-copy-number plasmid but not in mitochondrial of a wild-type strain.     

Exposure to arginine analog canavanine induces aberrant mitochondrial translation products,mitoribosome stalling,and instability of the mitochondrial proteome

Impairment of mitochondrial protein homeostasis disrupts mitochondrial function and causes human diseases and aging, but the molecular mechanisms of protein synthesis and quality control in mammalian mitochondria are not fully understood. Here we demonstrate in human cells that misincorporation of an arginine analog, canavanine, during mitochondrial protein synthesis, induced aberrant translation products and destabilized the mtDNA-encoded proteome, leading to loss of mitochondrial respiratory chain complexes. Furthermore, in the presence of a high concentration of canavanine, mitoribosome stalling could be demonstrated. The stalling did not, however, occur at arginine codons, but downstream of those codons. In particular, two adjacent arginines induced the most prominent downstream stalling effect, with the distance between the arginine codons and the stalling peak corresponding roughly to the length of the ribosomal exit tunnel. These results suggest that misincorporated canavanine disrupted the proper folding of the hydrophobic nascent polypeptides within the exit tunnel or while being inserted into the inner mitochondrial membrane. The canavanine treatment provides a model system for studying the consequences of mitoribosome stalling and the responses to misfolded proteins exiting the mitochondrial ribosome.     

Multiple lines of evidence localize signaling, morphology, and lipid biosynthesis machinery to the mitochondrial outer membrane of Arabidopsis

The composition of the mitochondrial outer membrane is notoriously difficult to deduce by orthology to other organisms, and biochemical enrichments are inevitably contaminated with the closely associated inner mitochondrial membrane and endoplasmic reticulum. In order to identify novel proteins of the outer mitochondrial membrane in Arabidopsis (Arabidopsis thaliana), we integrated a quantitative mass spectrometry analysis of highly enriched and prefractionated samples with a number of confirmatory biochemical and cell biology approaches. This approach identified 42 proteins, 27 of which were novel, more than doubling the number of confirmed outer membrane proteins in plant mitochondria and suggesting novel functions for the plant outer mitochondrial membrane. The novel components identified included proteins that affected mitochondrial morphology and/or segregation, a protein that suggests the presence of bacterial type lipid A in the outer membrane, highly stress-inducible proteins, as well as proteins necessary for embryo development and several of unknown function. Additionally, proteins previously inferred via orthology to be present in other compartments, such as an NADH:cytochrome B5 reductase required for hydroxyl fatty acid accumulation in developing seeds, were shown to be located in the outer membrane. These results also revealed novel proteins, which may have evolved to fulfill plant-specific requirements of the mitochondrial outer membrane, and provide a basis for the future functional characterization of these proteins in the context of mitochondrial intracellular interaction.     

Detailing the role of Bax translocation, cytochrome c release, and perinuclear clustering of the mitochondria in the killing of HeLa cells by TNF

Induction of cell death in HeLa cells with TNF and cycloheximide (CHX) required an adequate ATP supply and was accompanied by decrease in intracellular pH, translocation of Bax, perinuclear clustering of the mitochondria, and cytochrome c release. The chloride channel inhibitor furosemide prevented the intracellular acidification, the translocation of Bax and the cell death. Cyclosporin A (CyA) or bongkrekic acid (BK) inhibited the induction of the MPT, the release of cytochrome c and the cell death without affecting the perinuclear clustering of the mitochondria or the translocation of Bax. Energy depletion with the ATP synthase inhibitor oligomycin or the uncoupler FCCP in the presence of 2-deoxy-glucose prevented the perinuclear clustering of the mitochondria and the cell killing. However, mitochondrial translocation of Bax was still observed. By contrast, cytochrome c was released in the oligomycin-treated cells but not in the same cells treated with FCCP. The data demonstrate that apoptosis in HeLa cells is ATP dependent and requires the translocation of Bax. The movement of Bax to the mitochondria occurs before and during the perinuclear clustering of these organelles and does not require the presence of ATP. The release of cytochrome c depends on the induction of the mitochondrial permeability transition but not ATP content.     

Unique uncoupler-stimulation pattern of mitochondrial ATPase activity of tumor cells, brain, and fetal liver

Mitochondria were prepared from various kinds of normal tissues and tumor cells of mice, and their ATPase activities were measured in the presence of an uncoupler. The ATPase activities of all mitochondria were stimulated by the uncoupler when it was added to the mitochondrial suspension just before or after addition of ATP. However, when mitochondria were preincubated with the uncoupler for four minutes or more before the addition of ATP, its stimulating effect on mitochondrial ATPase activities was greatly reduced in all tumor cells tested, but not in normal adult liver. Reduction of the stimulating effect of the uncoupler by preincubation with it was also observed with mitochondrial ATPase of brain and fetal liver. Thus this pattern of change in the effect of uncoupler on preincubation may be common to tumor mitochondria, but it is not specific to tumor mitochondria. The pattern of uncoupler stimulation observed in fetal liver changed rapidly to that of adult liver immediately after birth. Thus the difference between the two uncoupler stimulation patterns is probably not due to a difference in molecular species of mitochondrial ATPase.     

Cloning and characterization of a microsatellite in the mitochondrial control region of the African side-necked turtle,Pelomedusa subrufa

The nucleotide sequence of the African side-necked turtle mitochondrial control region and its flanking tRNA genes was determined. This 73% A+T-rich region is 1194 bp long. Several conserved motifs involved in the regulation of the mitochondrial genome replication process, including one conserved sequence block (CSB1), and three termination-associated sequences were identified. The most remarkable feature found in this control region was the presence of six microsatellite-containing tandem repeats between the CSB1 motif and the tRNA^Phe gene. The potential usefulness of this microsatellite sequence for population-level studies is enhanced by its unique localization in the maternally inherited mitochondrial molecule.