Mark B. Roth

Oxygen deprivation causes suspended animation in the zebrafish embryo

Continuous exposure to oxygen is essential for nearly all vertebrates. We found that embryos of the zebrafish Danio rerio can survive for 24 h in the absence of oxygen (anoxia, 0% O2). In anoxia, zebrafish entered a state of suspended animation where all microscopically observable movement ceased, including cell division, developmental progression, and motility. Animals that had developed a heartbeat before anoxic exposure showed no evidence of a heartbeat until return to terrestrial atmosphere (normoxia, 20.8% O2). In analyzing cell-cycle changes of rapidly dividing blastomeres exposed to anoxia, we found that no cells arrested in mitosis. This is in sharp contrast to similarly staged normoxic embryos that consistently contain more than 15% of cells in mitosis. Flow cytometry analysis revealed that blastomeres arrested during the S and G2 phases of the cell cycle. This work indicates that survival of oxygen deprivation in vertebrates involves the reduction of diverse processes, such as cardiac function and cell-cycle progression, thus allowing energy supply to be matched by energy demands.

A histone-H3-like protein in C. elegans

The segregation of a chromosome during mitosis is mediated by a region of the chromosome known as the centromere, which organizes the kinetochore, to which the spindle microtubules attach. Many organisms have monocentric chromosomes, in which the centromeres map to single loci, whereas others, including the nematode Caenorhabditis elegans, have holocentric chromosomes, in which non-localized kinetochores extend along the length of each chromosome. The centromeres of monocentric chromosomes use specialized nucleosomes containing histone-H3-like proteins (known as CENP-A in mammals and Cse4 in the yeast Saccharomyces cerevisiae). Here we show that a C. elegans histone-H3-like protein is necessary for the proper segregation of chromosomes during mitosis and identifies the centromeres of these holocentric chromosomes, indicating that both holocentric and monocentric chromosomes use centromeric histone-H3-like proteins.

Cell division: A histone-H3-like protein in C. elegans

The segregation of a chromosome during mitosis is mediated by a region of the chromosome known as the centromere, which organizes the kinetochore, to which the spindle microtubules attach. Many organisms have monocentric chromosomes, in which the centromeres map to single loci, whereas others, including the nematode Caenorhabditis elegans, have holocentric chromosomes, in which non-localized kinetochores extend along the length of each chromosome. The centromeres of monocentric chromosomes use specialized nucleosomes containing histone-H3-like proteins (known as CENP-A in mammals and Cse4 in the yeast Saccharomyces cerevisiae). Here we show that a C. elegans histone-H3-like protein is necessary for the proper segregation of chromosomes during mitosis and identifies the centromeres of these holocentric chromosomes, indicating that both holocentric and monocentric chromosomes use centromeric histone-H3-like proteins.

Hydrogen sulfide increases thermotolerance and lifespan in Caenorhabditis elegans

Hydrogen sulfide (H2S) is naturally produced in animal cells. Exogenous H2S has been shown to effect physiological changes that improve the capacity of mammals to survive in otherwise lethal conditions. However, the mechanisms required for such alterations are unknown. We investigated the physiological response of Caenorhabditis elegans to H2S to elucidate the molecular mechanisms of H2S action. Here we show that nematodes exposed to H2S are apparently healthy and do not exhibit phenotypes consistent with metabolic inhibition. Instead, animals exposed to H2S are thermotolerant and long-lived. These phenotypes require SIR-2.1 activity but are genetically independent of the insulin signaling pathway, mitochondrial dysfunction, and caloric restriction. These studies suggest that SIR-2.1 activity may translate environmental change into physiological alterations that improve survival. It is interesting to consider the possibility that the mechanisms by which H2S increases thermotolerance and lifespan in nematodes are conserved and that studies using C. elegans may help explain the beneficial effects observed in mammals exposed to H2S.

Human SR proteins and isolation of a cDNA encoding SRp75

SR proteins are a family of proteins that have a common epitope recognized by a monoclonal antibody (MAb104) that binds active sites of polymerase II transcription. Four of the SR family members have been shown to restore activity to an otherwise splicing-deficient extract (S100 extract). Here we show that two untested SR proteins, SRp20 and SRp75, can also complement the splicing-deficient extract. We isolated a cDNA encoding SRp75 and found that this protein, like other SR proteins, contains an N-terminal RNA recognition motif (RRM), a glycine-rich region, an internal region homologous to the RRM, and a long (315-amino-acid) C-terminal domain composed predominantly of alternating serine and arginine residues. The apparent molecular mass of dephosphorylated SRp75 is 57 kDa, the size predicted from the cDNA clone. We also detected mobility shifts after dephosphorylating SRp55, SRp40, SRp30a, and SRp30b; the sizes of the shifts are proportional to the length of the SR domain, suggesting that serines in this domain are phosphorylated.

SR proteins are autoantigens in patients with systemic lupus erythematosus. Importance of phosphoepitopes.

OBJECTIVE To determine whether members of the highly phosphorylated SR protein family are autoantigens and, if so, to determine the frequency and molecular basis of antigen recognition. METHODS Native human SR proteins were purified to homogeneity from HeLa cells, and an enzyme-linked immunosorbent assay (ELISA) was developed. Further studies employed immunoblotting of both phosphorylated and dephosphorylated SR proteins. RESULTS Anti-SR protein reactivity was frequently detected in the sera of patients with systemic lupus erythematosus (SLE). Sera from 52% of the SLE patients in a group of patients with a variety of autoimmune and other disorders (n = 137) and from 50% of the SLE patients in a separate group (n = 102) were positive in an ELISA. In contrast, sera from patients with other disorders, such as rheumatoid arthritis and primary antiphospholipid syndrome, reacted infrequently. Reactivity with double-stranded DNA (dsDNA), used in the diagnosis of SLE, did not correlate with SR protein reactivity. Anti-SR autoantisera did not bind highly charged unphosphorylated peptides related to the SR domain, which is rich in arginine and phosphoserine residues. Surprisingly, many of the epitopes were influenced by the presence or absence of SR protein phosphorylation. In immunoblots, some patient sera lost reactivity upon SR protein dephosphorylation, while others significantly gained reactivity. CONCLUSION We have identified a novel set of autoantigens in SLE, the SR protein family of non-small nuclear RNP pre-messenger RNA splicing factors. Anti-SR autoantibodies are distinct from those which bind dsDNA. The identification of this new set of autoantigens and the observation that the auto-epitope(s) involves posttranslational modification offer new possibilities for understanding autoimmunity and its development.

Surviving Blood Loss Using Hydrogen Sulfide

BACKGROUND Reduced metabolic activity improves outcome in many clinical and experimental models of injury and diseases that result in insufficient blood supply. Recently, we demonstrated that inhaled hydrogen sulfide gas can be used to reversibly reduce metabolic activity in mice. We hypothesize that hydrogen sulfide will confer benefit in injuries and diseases related to insufficient blood supply. METHODS Sprague-Dawley rats were subjected to controlled hemorrhage to remove 60% of total blood. Hydrogen sulfide was administered to rats either via airway as gas, or intravenous infusion as liquid. Outcome was assayed by survival. RESULTS Using inhaled hydrogen sulfide gas, 75% of treated and 23% of untreated rats survived longer than 24 hours. Using intravenous administered sulfide, 67% of treated and 14% of untreated rats survived longer than 24 hours. Using log-rank analysis, p < 0.001. Surviving rats showed no functional or behavioral abnormalities. Blood chemistry analysis at the end of hemorrhage showed minor but significant differences between treated and control animals. Respirometry results show that hydrogen sulfide stabilized metabolic output during and after hemorrhage. CONCLUSION These data indicate that sulfide can protect rats from lethal hemorrhage. Future studies are needed to analyze the mechanism of benefit as well as whether sulfide is beneficial in other models of human injury and disease.

Hydrogen Sulfide Increases Hypoxia-inducible Factor-1 Activity Independently of von Hippel–Lindau Tumor Suppressor-1 in C. elegans

In C. elegans, hydrogen sulfide (H2S) exposure results in Hif-1 stabilization. hif-1 is required for survival in H2S and constitutive HIF-1 stabilization confers resistance to H2S. H2S-induced HIF-1 reporter activity appears to be independent of VHL-1, whereas VHL-1 is required for hypoxic regulation of HIF-1 reporter activity.

C. Elegans Are Protected from Lethal Hypoxia by an Embryonic Diapause

At least 100 mammalian species exhibit embryonic diapause, where fertilized embryos arrest development in utero until suitable seasonal or nutritional environments are encountered. Delaying maternal investments in producing offspring allows these animals to utilize limited resources to survive while searching for better conditions and ensures that progeny are not produced when they are unlikely to survive. In addition, embryos may be protected from external environmental vicissitudes while in utero. Here we demonstrate embryonic diapause in C. elegans, and show that this diapause protects embryos from otherwise lethal hypoxia. Diapausing embryos in utero require san-1 to survive, indicating that hypoxia-induced embryonic diapause may be mechanistically related to suspended animation. Furthermore, we show that neuronal HIF-1 activity in the adult dictates the O(2) tension at which embryonic diapause is engaged. We suggest that the maternal perception of hypoxia stimulates a response to protect embryos in utero by inducing diapause, a natural form of suspended animation. This response is likely to be an important strategy to improve offspring survival in harsh conditions and allow adults to find environments more suitable for reproductive success.

Adaptive Sugar Provisioning Controls Survival of C. elegans Embryos in Adverse Environments

The ability to adapt to changing environmental conditions is essential to the fitness of organisms. In some cases, adaptation of the parent alters the offsprings phenotype [1-10]. Such parental effects are adaptive for the offspring if the future environment is similar to the current one but can be maladaptive otherwise [11]. One mechanism by which adaptation occurs is altered provisioning of embryos by the parent [12-16]. Here we show that exposing adult Caenorhabditis elegans to hyperosmotic conditions protects their offspring from these conditions but causes sensitivity to anoxia exposure. We show that this alteration of survival is correlated with changes in the sugar content of adults and embryos. In addition, mutations in gene products that alter sugar homeostasis also alter the ability of embryos to survive in hyperosmotic and anoxic conditions and engage in the adaptive parental effect. Our results indicate that there is a physiological trade-off between the presence of glycerol, which protects animals from hyperosmotic conditions, and glycogen, which is consumed during anoxia. These two metabolites play an essential role in the survival of worms in these adverse environments, and the adaptive parental effect we describe is mediated by the provisioning of these metabolites to the embryo.