Coordinate Regulation of Ribosome and tRNA Biogenesis Controls Hypoxic Injury and Translation.

The translation machinery is composed of a myriad of proteins and RNAs whose levels must be coordinated to efficiently produce proteins without wasting energy or substrate. However, protein synthesis is clearly not always perfectly tuned to its environment, as disruption of translation machinery components can lengthen lifespan and stress survival. While much has been learned from bacteria and yeast about translational regulation, much less is known in metazoans. In a screen for mutations protecting C. elegans from hypoxic stress, we isolated multiple genes impacting protein synthesis: a ribosomal RNA helicase gene, tRNA biosynthesis genes, and a gene controlling amino acid availability. To define better the mechanisms by which these genes impact protein synthesis, we performed a second screen for suppressors of the conditional developmental arrest phenotype of the RNA helicase mutant and identified genes involved in ribosome biogenesis. Surprisingly, these suppressor mutations restored normal hypoxic sensitivity and protein synthesis to the tRNA biogenesis mutants, but not to the mutant reducing amino acid uptake. Proteomic analysis demonstrated that reduced tRNA biosynthetic activity produces a selective homeostatic reduction in ribosomal subunits, thereby offering a mechanism for the suppression results. Our study uncovers an unrecognized higher-order-translation regulatory mechanism in a metazoan whereby ribosome biogenesis genes communicate with genes controlling tRNA abundance matching the global rate of protein synthesis with available resources.

Ageing and hypoxia cause protein aggregation in mitochondria.

Aggregation of cytosolic proteins is a pathological finding in disease states, including ageing and neurodegenerative diseases. We have previously reported that hypoxia induces protein misfolding in Caenorhabditis elegans mitochondria, and electron micrographs suggested protein aggregates. Here, we seek to determine whether mitochondrial proteins actually aggregate after hypoxia and other cellular stresses. To enrich for mitochondrial proteins that might aggregate, we performed a proteomics analysis on purified C. elegans mitochondria to identify relatively insoluble proteins under normal conditions (110 proteins identified) or after sublethal hypoxia (65 proteins). A GFP-tagged mitochondrial protein (UCR-11 - a complex III electron transport chain protein) in the normally insoluble set was found to form widespread aggregates in mitochondria after hypoxia. Five other GFP-tagged mitochondrial proteins in the normally insoluble set similarly form hypoxia-induced aggregates. Two GFP-tagged mitochondrial proteins from the soluble set as well as a mitochondrial-targeted GFP did not form aggregates. Ageing also resulted in aggregates. The number of hypoxia-induced aggregates was regulated by the mitochondrial unfolded protein response (UPRmt) master transcriptional regulator ATFS-1, which has been shown to be hypoxia protective. An atfs-1(loss-of-function) mutant and RNAi construct reduced the number of aggregates while an atfs-1(gain-of-function) mutant increased aggregates. Our work demonstrates that mitochondrial protein aggregation occurs with hypoxic injury and ageing in C. elegans. The UPRmt regulates aggregation and may protect from hypoxia by promoting aggregation of misfolded proteins.

Divergent mechanisms controlling hypoxic sensitivity and lifespan by the DAF-2/insulin/IGF-receptor pathway.

Organisms and their cells vary greatly in their tolerance of low oxygen environments (hypoxia). A delineation of the determinants of hypoxia tolerance is incomplete, despite intense interest for its implications in diseases such as stroke and myocardial infarction. The insulin/IGF-1 receptor (IGFR) signaling pathway controls survival of Caenorhabditis elegans from a variety of stressors including aging, hyperthermia, and hypoxia. daf-2 encodes a C. elegans IGFR homolog whose primary signaling pathway modulates the activity of the FOXO transcription factor DAF-16. DAF-16 regulates the transcription of a large number of genes, some of which have been shown to control aging. To identify genes that selectively regulate hypoxic sensitivity, we compared the whole-organismal transcriptomes of three daf-2 reduction-of-function alleles, all of which are hypoxia resistant, thermotolerant, and long lived, but differ in their rank of severities for these phenotypes. The transcript levels of 172 genes were increased in the most hypoxia resistant daf-2 allele, e1370, relative to the other alleles whereas transcripts from only 10 genes were decreased in abundance. RNAi knockdown of 6 of the 10 genes produced a significant increase in organismal survival after hypoxic exposure as would be expected if down regulation of these genes by the e1370 mutation was responsible for hypoxia resistance. However, RNAi knockdown of these genes did not prolong lifespan. These genes definitively separate the mechanisms of hypoxic sensitivity and lifespan and identify biological strategies to survive hypoxic injury.

Survival from hypoxia in C. elegans by inactivation of aminoacyl-tRNA synthetases.

Hypoxia is important in a wide range of biological processes, such as animal hibernation and cell survival, and is particularly relevant in many diseases. The sensitivity of cells and organisms to hypoxic injury varies widely, but the molecular basis for this variation is incompletely understood. Using forward genetic screens in Caenorhabditis elegans, we isolated a hypoxia-resistant reduction-of-function mutant of rrt-1 that encodes an arginyl-transfer RNA (tRNA) synthetase, an enzyme essential for protein translation. Knockdown of rrt-1, and of most other genes encoding aminoacyl-tRNA synthetases, rescued animals from hypoxia-induced death, and the level of hypoxia resistance was inversely correlated with translation rate. The unfolded protein response was induced by hypoxia and was required for the hypoxia resistance of the reduction-of-function mutant of rrt-1. Thus, translational suppression produces hypoxia resistance, in part by reducing unfolded protein toxicity.

Systematic identification of gene activities promoting hypoxic death.

The sensitivity of an organism to hypoxic injury varies widely across species and among cell types. However, a systematic description of the determinants of metazoan hypoxic sensitivity is lacking. Toward this end, we screened a whole-genome RNAi library for genes that promote hypoxic sensitivity in Caenorhabditis elegans. RNAi knockdown of 198 genes conferred an invariant hypoxia-resistant phenotype (Hyp-r). Eighty-six per cent of these hyp genes had strong homologs in other organisms, 73 with human reciprocal orthologs. The hyp genes were distributed among multiple functional categories. Transcription factors, chromatin modifying enzymes, and intracellular signaling proteins were highly represented. RNAi knockdown of about half of the genes produced no apparent deleterious phenotypes. The hyp genes had significant overlap with previously identified life span extending genes. Testing of the RNAi's in a mutant background defective in somatic RNAi machinery showed that most genes function in somatic cells to control hypoxic sensitivity. DNA microarray analysis identified a subset of the hyp genes that may be hypoxia regulated. siRNA knockdown of human orthologs of the hyp genes conferred hypoxia resistance to transformed human cells for 40% of the genes tested, indicating extensive evolutionary conservation of the hypoxic regulatory activities. The results of the screen provide the first systematic picture of the genetic determinants of hypoxic sensitivity. The number and diversity of genes indicates a surprisingly nonredundant genetic network promoting hypoxic sensitivity.

Autophagy protects against hypoxic injury in C. elegans.

Macroautophagy has been implicated in a variety of pathological processes. Hypoxic/ischemic cellular injury is one such process in which autophagy has emerged as an important regulator. In general, autophagy is induced after a hypoxic/ischemic insult; however, whether the induction of autophagy promotes cell death or recovery is controversial and appears to be context dependent. We have developed C. elegans as a genetically tractable model for the study of hypoxic cell injury. Both necrosis and apoptosis are mechanisms of cell death following hypoxia in C. elegans. However, the role of autophagy in hypoxic injury in C. elegans has not been examined. Here, we found that RNAi knockdown of the C. elegans homologs of beclin 1/Atg6 (bec-1) and LC3/Atg8 (lgg-1, lgg-2), and mutation of Atg1 (unc-51) decreased animal survival after a severe hypoxic insult. Acute inhibition of autophagy by the type III phosphatidylinositol 3-kinase inhibitors, 3-methyladenine and Wortmannin, also sensitized animals to hypoxic death. Hypoxia-induced neuronal and myocyte injury as well as necrotic cellular morphology were increased by RNAi knockdown of BEC-1. Hypoxia increased the expression of a marker of autophagosomes in a bec-1-dependent manner. Finally, we found that the hypoxia hypersensitive phenotype of bec-1(RNAi) animals could be blocked by loss-of-function mutations in either the apoptosis or necrosis pathway. These results argue that inhibition of autophagy sensitizes C. elegans and its cells to hypoxic injury and that this sensitization is blocked or circumvented when either of the two major cell-death mechanisms is inhibited.

Hypoxic preconditioning requires the apoptosis protein CED-4 in C. elegans.

Hypoxic preconditioning (HP) is a rapid and reversible proadaptive response to mild hypoxic exposure with such a response protecting cells from subsequent hypoxic or ischemic insult. HP mechanisms are of great interest because of their therapeutic potential and insight into metabolic adaptation and cell death. HP has been widely demonstrated in the vertebrate subphylum but not in invertebrates. Here, we report that the nematode Caenorhabditis elegans has a potent HP mechanism that protects the organism as well as its neurons and myocytes from hypoxic injury. The time course of C. elegans HP was consistent with vertebrate-delayed HP, appearing 16 hr after preconditioning and lasting at least 36 hr. The apoptosis pathway has been proposed as either a trigger or target of HP. Testing of mutations in the canonical C. elegans apoptosis pathway showed that in general, genes in this pathway are not required for HP. However, loss-of-function mutations in ced-4, which encodes an Apaf-1 homolog, completely blocked HP. RNAi silencing of ced-4 in adult animals immediately preceding preconditioning blocked HP, indicating that CED-4 is required in adults during or after preconditioning. CED-4/Apaf-1 is essential for HP in C. elegans and acts through a mechanism independent of the classical apoptosis pathway.

Volatile anesthetics bind rat synaptic snare proteins.

BACKGROUND: Volatile general anesthetics (VAs) have a number of synaptic actions, one of which is to inhibit excitatory neurotransmitter release; however, no presynaptic VA binding proteins have been identified. Genetic data in Caenorhabditis elegans have led to the hypothesis that a protein that interacts with the presynaptic protein syntaxin 1A is a VA target. Motivated by this hypothesis, the authors measured the ability of syntaxin 1A and proteins that interact with syntaxin to bind to halothane and isoflurane. METHODS: Recombinant rat syntaxin 1A, SNAP-25B, VAMP2, and the ternary SNARE complex that they form were tested. Binding of VAs to these proteins was detected by F-nuclear magnetic resonance relaxation measurements. Structural alterations in the proteins were examined by circular dichroism and ability to form complexes. RESULTS: Volatile anesthetics did not bind to VAMP2. At concentrations in the clinical range, VAs did bind to SNAP-25B; however, binding was detected only in preparations containing SNAP-25B homomultimers. VAs also bound at clinical concentrations to both syntaxin and the SNARE complex. Addition of an N-terminal His6 tag to syntaxin abolished its ability to bind VAs despite normal secondary structure and ability to form SNARE complexes; thrombin cleavage of the tag restored VA binding. Thus, the VA binding site(s) has structural requirements and is not simply any alpha-helical bundle. VAs at supraclinical concentrations produced an increase in helicity of the SNARE complex; otherwise, VA binding produced no gross alteration in the stability or secondary structure of the SNARE complex. CONCLUSION: SNARE proteins are potential synaptic targets of volatile anesthetics.

Effect of disposable infection control barriers on light output from dental curing lights.

PURPOSE: To prevent contamination of the light guide on a dental curing light, barriers such as disposable plastic wrap or covers may be used. This study compared the effect of 3 disposable barriers on the spectral output and power density from a curing light. The hypothesis was that none of the barriers would have a significant clinical effect on the spectral output or the power density from the curing light. METHODS: Three disposable barriers were tested against a control (no barrier). The spectra and power from the curing light were measured with a spectrometer attached to an integrating sphere. The measurements were repeated on 10 separate occasions in a random sequence for each barrier. RESULTS: Analysis of variance (ANOVA) followed by Fisher's protected least significant difference test showed that the power density was significantly less than control (by 2.4% to 6.1%) when 2 commercially available disposable barriers were used (p < 0.05). There was no significant difference in the power density when general-purpose plastic wrap was used (p > 0.05). The effect of each of the barriers on the power output was small and probably clinically insignificant. ANOVA comparisons of mean peak wavelength values indicated that none of the barriers produced a significant shift in the spectral output relative to the control ( p > 0.05). CONCLUSIONS: Two of the 3 disposable barriers produced a significant reduction in power density from the curing light. This drop in power was small and would probably not adversely affect the curing of composite resin. None of the barriers acted as light filters.

Regulation of hypoxic death in C. elegans by the insulin/IGF receptor homolog DAF-2.

To identify genetic determinants of hypoxic cell death, we screened for hypoxia-resistant (Hyp) mutants in Caenorhabditis elegans and found that specific reduction-of-function (rf) mutants of daf-2, an insulin/insulinlike growth factor (IGF) receptor (INR) homolog gene, were profoundly Hyp. The hypoxia resistance was acutely inducible just before hypoxic exposure and was mediated through an AKT-1/PDK-1/forkhead transcription factor pathway overlapping with but distinct from signaling pathways regulating life-span and stress resistance. Selective neuronal and muscle expression of daf-2(+) restored hypoxic death, and daf-2(rf) prevented hypoxia-induced muscle and neuronal cell death, which demonstrates a potential for INR modulation in prophylaxis against hypoxic injury of neurons and myocytes.

Glutamine metabolism in metabolic acidosis

In chronic metabolic acidosis in the rat, there is increased ammoniagenesis, gluconeogenesis and renal extraction of gluatmine with induction of renal phosphate-dependent glutaninase (PDG). Because the stimulus for these changes is not yet clear and also because acute acidosis is the more common clinical problem, the present study deals mainly with the metabolism of glutamine in acute metabolic acidosis. When acute metabolic acidosis is produced in rats by administration of mineral acid or by causing them to swim, thus inducing a severe lactic acidosis, a factor is found in the plasma which stimulates renal glutamine uptake and ammoniagenesis in vivo as well as in vitro. Acute acidosis dose not induce synthesis of PDG in the kidney but causes a change in enzyme kinetics. The plasma factor not only enhances glutamine entry into cells, but apparently causes a conformational change in PDG, as shown by an increase in V1.0mM/Vmax. Intestinal metabolism of glutamine is also stimulated in vivo and in vitro by the plasma factor of acute acidosis. (AU)

Renal transport and metabolism of glutamine

The experiments described in this thesis were designed to investigate the mechanism of glutamine transport and the effect of acute acidosis on glutamine transport and metabolism in the rat kidney. The experiments done with kidney slices investigated the effect of several amino acids chosen from different structural groups on uptake of glutamine. Proline (an ammino acid) and glycine (at inhibitory concentrations) were found to inhibit ammonia production and glutamine uptake significantly. When a sodium free incubation buffer was used the inhibitory effect of proline and glycine on glutamine uptake disappeared, indicating that the inhibition by proline was sodium dependent. In similar experiments with isolated kidney proximal tubules in a sodium containing buffer, proline also caused a decrease in glutamine uptake and ammonia production. Intracellular/extracellular glutamine distributions ratios in tubules however, showed a marked increase in the presence of proline. The explanation for this is not that proline shares a transport site with glutamine but rather that it is metabolised to glutamate which causes an inhibition of phosphate dependent glutaminase and hence decreased glutamine utilisation. This decreased utilisation leads to an accumulation of glutamine within the renal cell and subsequently increased intracellular/extracellular glutamine distribution ratios. The inhibitory effect of proline on glutamine uptake was much less in experiments with kidney slices from chronically acidotic rats. Isolated tubules from acutely acidotic rats showed increased intracellular/extracellular glutamine distribution ratios, increased ammonia production and glutamine uptake in tubules. When tubules from normal rats were incubated in sera from acutely acidotic rats there was a similar increase in intracellular/extracellular glutamine distribution ratios, ammonia production and glutamine uptake. The effect of the serum however, was not due to the accumulation of glutamate because glutamate levels in tubules were unchanged after incubation in sera from the acutely acidotic rats (AU)