The RNase P associated with HeLa cell mitochondria contains an essential RNA component identical in sequence to that of the nuclear RNase P.

The mitochondrion-associated RNase P activity (mtRNase P) was extensively purified from HeLa cells and shown to reside in particles with a sedimentation constant ( approximately 17S) very similar to that of the nuclear enzyme (nuRNase P). Furthermore, mtRNase P, like nuRNase P, was found to process a mitochondrial tRNA(Ser(UCN)) precursor [ptRNA(Ser(UCN))] at the correct site. Treatment with micrococcal nuclease of highly purified mtRNase P confirmed earlier observations indicating the presence of an essential RNA component. Furthermore, electrophoretic analysis of 3'-end-labeled nucleic acids extracted from the peak of glycerol gradient-fractionated mtRNase P revealed the presence of a 340-nucleotide RNA component, and the full-length cDNA of this RNA was found to be identical in sequence to the H1 RNA of nuRNase P. The proportions of the cellular H1 RNA recovered in the mitochondrial fractions from HeLa cells purified by different treatments were quantified by Northern blots, corrected on the basis of the yield in the same fractions of four mitochondrial nucleic acid markers, and shown to be 2 orders of magnitude higher than the proportions of contaminating nuclear U2 and U3 RNAs. In particular, these experiments revealed that a small fraction of the cell H1 RNA (of the order of 0.1 to 0.5%), calculated to correspond to approximately 33 to approximately 175 intact molecules per cell, is intrinsically associated with mitochondria and can be removed only by treatments which destroy the integrity of the organelles. In the same experiments, the use of a probe specific for the RNA component of RNase MRP showed the presence in mitochondria of 6 to 15 molecules of this RNA per cell. The available evidence indicates that the levels of mtRNase P detected in HeLa cells should be fully adequate to satisfy the mitochondrial tRNA synthesis requirements of these cells.

Autoimmunity to munc-18 in Rasmussen's encephalitis.

Rasmussen's encephalitis (RE) is a rare disease of the central nervous system characterized by severe epileptic seizures, progressive degeneration of a single cerebral hemisphere, and autoimmunity directed against glutamate receptor subunit, GluR3. We report here the identification of high-titer autoantibodies directed against munc-18 in the serum of a single patient with RE previously shown to have anti-GluR3 antibodies. Munc-18 is an intracellular protein residing in presynaptic terminals, which is required for secretion of neurotransmitters. These findings are consistent with the possibility of intermolecular epitope spreading between GluR3, a postsynaptic cell surface protein, and munc-18, a presynaptic intracellular protein. Immune attack on these two proteins, which participate at distinct steps of synaptic transmission, could act in an additive or synergistic manner to impair synaptic function and lead to seizures and neuronal death.

Seizure disorders in mutant mice: relevance to human epilepsies.

The rate at which mutant genes producing an epileptic phenotype in mice have been identified over the past few years has been astounding. Manipulating the genome of mice has led to identification of a diversity of genes whose absence or modification either causes epileptic seizures or, conversely, limits epileptogenesis. In addition, positional cloning of genes in which spontaneously arising mutations cause epilepsy in mice has led to the identification of genes encoding voltage- and ligand-gated ion channels. Finally, engineering a mutation that mimics a rare form of human epilepsy has led to a mouse line with a phenotype similar to that of the human disease. Taken together, these discoveries promise to shed light on the mechanisms underlying genetic control of neuronal excitability, suggest candidate genes underlying genetic forms of human epilepsy, and provide a valuable model with which to elucidate how the genotype produces the phenotype of a rare form of human epilepsy.

Evaluation of the alpha(2A)-adrenergic receptor gene in a heritable form of temporal lobe epilepsy.

An autosomal dominant form of human temporal lobe epilepsy (TLE) has been mapped to a region of chromosome 10q that contains the intronless alpha(2A)-adrenergic receptor (alpha(2A)AR) gene. Because mutation of the alpha(2A)AR gene in the mouse fosters epileptogenesis, we developed methods for analysis of the alpha(2A)AR coding region applicable to any pathophysiologic state in which the alpha(2A)AR could be implicated in the disease mechanism. This study rules out mutations in the alpha(2A)AR coding region as causal for this form of autosomal dominant TLE.

The deafness-associated mitochondrial DNA mutation at position 7445, which affects tRNASer(UCN) precursor processing, has long-range effects on NADH dehydrogenase subunit ND6 gene expression.

The pathogenetic mechanism of the deafness-associated mitochondrial DNA (mtDNA) T7445C mutation has been investigated in several lymphoblastoid cell lines from members of a New Zealand pedigree exhibiting the mutation in homoplasmic form and from control individuals. We show here that the mutation flanks the 3' end of the tRNASer(UCN) gene sequence and affects the rate but not the sites of processing of the tRNA precursor. This causes an average reduction of approximately 70% in the tRNASer(UCN) level and a decrease of approximately 45% in protein synthesis rate in the cell lines analyzed. The data show a sharp threshold in the capacity of tRNASer(UCN) to support the wild-type protein synthesis rate, which corresponds to approximately 40% of the control level of this tRNA. Strikingly, a 7445 mutation-associated marked reduction has been observed in the level of the mRNA for the NADH dehydrogenase (complex I) ND6 subunit gene, which is located approximately 7 kbp upstream and is cotranscribed with the tRNASer(UCN) gene, with strong evidence pointing to a mechanistic link with the tRNA precursor processing defect. Such reduction significantly affects the rate of synthesis of the ND6 subunit and plays a determinant role in the deafness-associated respiratory phenotype of the mutant cell lines. In particular, it accounts for their specific, very significant decrease in glutamate- or malate-dependent O2 consumption. Furthermore, several homoplasmic mtDNA mutations affecting subunits of NADH dehydrogenase may play a synergistic role in the establishment of the respiratory phenotype of the mutant cells.

Epilepsy genetics: an abundance of riches for biologists.

Twenty-five genes have been identified in which mutations cause epileptic seizures in mice. The gene for a Na+/H+ exchanger has recently been found to underlie the spontaneous mutant slow wave epilepsy. Studies of such mutants should help elucidate the mechanisms that control neuronal excitability.

Nucleotide sequences of the RNA subunit of RNase P from several mammals.

Sequences of the RNA subunit of RNase P from five primate and two rodent species have been determined. The extent of the differences among these sequences and the corresponding RNA from human tissue correlates to known phylogenetic relationships. All the sequences can be drawn in a secondary structure with common features.

Chromosomal localization of gene for human glutamate receptor subunit-7.

We isolated a human glutamate receptor subunit 7 (GluR-7) cosmid after high stringency screening of a human genomic placental library using a rat GluR-7 cDNA as a probe. A 614-bp fragment of the GluR-7 cosmid was sequenced, and an exon that encodes 53 amino acids was found between two introns. The exon exhibited 89% and 96% identity at the nucleotide and amino acid levels, respectively, with the corresponding region of rat GluR-7. The human GluR-7 was classified as a kainate subtype glutamate receptor based on its homology to rat GluR-7. Using somatic cell hybrid analysis, human GluR-7 was localized to chromosome 1. Fine mapping analysis using FISH localized the gene to 1p33-34. Since glutamate receptors of the kainate subtype have been implicated in neurodegenerative disorders, establishing the precise map position of human GluR-7 subunit is an important step towards evaluating this locus as a candidate for mutations in neurodegenerative disorders.

Cloning and expression of a novel Na(+)-dependent neutral amino acid transporter structurally related to mammalian Na+/glutamate cotransporters.

A cDNA has been isolated from human hippocampus that appears to encode a novel Na(+)-dependent, Cl(-)-independent, neutral amino acid transporter. The putative protein, designated SATT, is 529 amino acids long and exhibits significant amino acid sequence identity (39-44%) with mammalian L-glutamate transporters. Expression of SATT cDNA in HeLa cells induced stereospecific uptake of L-serine, L-alanine, and L-threonine that was not inhibited by excess (3 mM) 2-(methylamino)-isobutyric acid, a specific substrate for the System A amino acid transporter. SATT expression in HeLa cells did not induce the transport of radiolabeled L-cysteine, L-glutamate, or related dicarboxylates. Northern blot hybridization revealed high levels of SATT mRNA in human skeletal muscle, pancreas, and brain, intermediate levels in heart, and low levels in liver, placenta, lung, and kidney. SATT transport characteristics are similar to the Na(+)-dependent neutral amino acid transport activity designated System ASC, but important differences are noted. These include: 1) SATT's apparent low expression in ASC-containing tissues such as liver or placenta; 2) the lack of mutual inhibition between serine and cysteine; and 3) the lack of trans-stimulation. SATT may represent one of multiple activities that exhibit System ASC-like transport characteristics in diverse tissues and cell lines.

The gene encoding the glutamate receptor subunit GluR5 is located on human chromosome 21q21.1-22.1 in the vicinity of the gene for familial amyotrophic lateral sclerosis.

Genomic clones of the human non-N-methyl-D-aspartate (non-NMDA) glutamate receptor subunit GluR5 were isolated by high-stringency screening of a cosmid library using the rat cDNA as a probe. The chromosomal localization of the human GluR5 gene has been established. Southern hybridization of DNA isolated from mapping panels of Chinese hamster-human hybrid cell lines and high-resolution in situ suppression hybridization localize the GluR5 gene to chromosome 21q21.1-22.1. This coincides with the localization of a mutant gene causing familial amyotrophic lateral sclerosis (ALS), as Siddique et al. established by linkage analyses [Siddique, T., Figlewicz, D. A., Pericak-Vance, M. A., Haines, J. L., Rouleau, G., Jeffers, A. J., Sapp, P., Hung, W. Y., Bebout, J., McKenna-Yasek, D., Deng, G., Horvitz, H. R., Gusella, J. F., Brown, R. H. & Roses, A. D. (1991) N. Engl. J. Med. 324, 1381-1384]. Convergent evidence from other investigators suggests that chronic pathologic activation of motor neurons via non-NMDA glutamate receptors might induce excitotoxic injury of motor neurons, culminating in ALS. Together with the demonstration that GluR5 transcripts are expressed in the ventral horn of the spinal cord, the region in which susceptible motor neurons reside, the chromosomal localization suggests that a mutated GluR5 gene may be responsible for the familial form of ALS.

Functions of cytochrome c in regulation of electron transfer and protein folding.

Cytochrome c, a "mobile electron carrier" of the mitochondrial respiratory chain, also occurs in detectable amounts in the cytosol, and can receive electrons from cytochromes present in endoplasmic reticulum and plasma membranes as well as from superoxide and ascorbate. The pigment was found to dissociate from mitochondrial membranes in liver and kidney when rats were subjected to heat exposure and starvation, respectively. Treating cytochrome c with hydroxylamine gives a partially deaminated product with altered redox properties; decreased stimulation of respiration by deficient mitochondria, increased reduction by superoxide, and complete loss of reducibility by plasma membranes. Mitochondria isolated from brown adipose tissue of cold-exposed rats are found to be sub-saturated with cytochrome c. The ability of cytochrome c to reactivate reduced ribonuclease is now reinterpreted as a molecular chaperone role for the hemoprotein.

Oxidative activities in mitochondria-like particles from Setaria digitata, a filarial parasite.

The oxidative metabolic potential of Setaria digitata, a filarial parasite found in the intraperitoneal cavity of cattle, was investigated. These worms showed active wriggling movements which were not affected by respiratory poisons such as cyanide, rotenone and malonate. They also possessed cyanide-insensitive and glucose-independent oxygen consumption pathways. By differential centrifugation of sucrose homogenates, a fraction containing mitochondria-like particles was obtained in which the activity of the marker enzyme, succinate dehydrogenase, was recovered. This fraction catalysed succinate- and NADH-dependent reduction of both cytochrome c and dyes. Oxygen uptake found with succinate, NADH and ascorbate as substrates was not sensitive to cyanide. Cytochromes could not be detected in either this fraction or homogenates of the worms. H2O2 generation with a number of substrates and lipid peroxidation by measuring malondialdehyde formed as well as by accompanying oxygen uptake were demonstrated in the mitochondria-like particles. A lipid quinone, possibly with a short side chain and related to ubiquinone, was detected in the worms. The results suggested the existence of two cyanide-insensitive oxygen-consuming reactions in Setaria: one respiratory substrate-independent lipid peroxidation, and a second substrate-dependent reaction that requires an auto-oxidizable quinone but not a cytochrome system.

Oxidations in kidney mitochondria of heat-exposed rats: regulation by cytochrome c.

Exposure of rats to higher environmental temperature (36-37 degrees C) decreased the capacity of their kidney mitochondria to oxidize succinate. The decrease was corrected on the addition of exogenous cytochrome c. Kidney mitochondria of heat-exposed animals showed decreased rates of H2O2 generation when alpha-glycerophosphate, but not succinate, was used as electron donor. These mitochondria also showed decreased activity of alpha-glycerophosphate dehydrogenase but not of succinate dehydrogenase. The content of cytochrome c in kidney mitochondria of heat-exposed animals was low even though the concentration of the pigment in the whole tissue did not decrease. Starvation as well as administration of an antithyroid agent like propylthiouracil simulated some of the effects of heat exposure on kidney mitochondria, but the cytochrome c-dependent reversal of inhibition of oxidation was obtained only in heat exposure.

Noradrenaline treatment of rats stimulates H2O2 generation in liver mitochondria.

Treatment of rats with noradrenaline stimulated H2O2 generation in liver mitochondria using succinate, choline or glycerol 1-phosphate as substrate. The dehydrogenase activity with either succinate or choline as substrate showed no change, whereas that with glycerol 1-phosphate increased. The effect was obtained with noradrenaline, but not with dihydroxyphenylserine. Phenoxybenzamine and yohimbine, but not propranolol, prevented the response to noradrenaline treatment. Phenylephrine could stimulate H2O2 generation, whereas isoprenaline had only a marginal effect. Theophylline treatment slightly decreased the generation of H2O2 in liver mitochondria, but treatment with pargyline, Ro4-1284 and dibutyryl cyclic AMP had little effect. These studies showed that noradrenaline might possibly be acting through the alpha 2-adrenergic system.