Endocytosis of superoxide dismutase is required in order for the enzyme to protect hepatocytes from the cytotoxicity of hydrogen peroxide

Inhibitors of endocytosis have been used to show that internalization of superoxide dismutase is required for the enzyme to protect hepatocytes from the cytotoxicity of hydrogen peroxide. As shown previously (Starke, P. E., and Farber, J. L. (1985) J. Biol. Chem. 260, 10099-10104), superoxide dismutase prevented the killing of cultured hepatocytes by H2O2 generated in the medium by glucose oxidase. Five inhibitors of endocytosis, methylamine, monensin, benzyl alcohol, cytochalasin B, and oligomycin, each abolished the protective effect of superoxide dismutase. Cell-associated superoxide dismutase activity was increased 4-fold in hepatocytes after exposure to superoxide dismutase for 1 h. Each of the inhibitors abolished this increase in the cell-associated superoxide dismutase activity. The uptake of horseradish peroxidase, a measure of fluid phase endocytosis, differed from that of superoxide dismutase in its lower rate, reduced sensitivity to methylamine, and its insensitivity to cytochalasin B. The results of the present study demonstrate that endocytosis of superoxide dismutase is required to protect hepatocytes from the cytotoxicity of hydrogen peroxide. This conclusion may account for some of the conflicting results in the literature with respect to the protective action of superoxide dismutase.     

Ethanol-induced changes in methionine metabolism in rat liver

The administration of alcohol to rats fed a protein-restricted diet results in significant changes in the hepatic content of four enzymes of methionine metabolism. The levels of s-adenosylmethionine synthetase, cystathionine synthase, and betaine-homocysteine methyltransferase increase while the level of methyltetrahydrofolate-homocysteine methyltransferase decreases. These changes represent a reversal of the normal adaptive response to protein-restriction. The resultant impairment in methionine conservation could explain the alcohol-induced increase in the dietary lipotrope requirement.     

A Novel MitoTimer Reporter Gene for Mitochondrial Content,Structure, Stress,and Damage in Vivo

Mitochondrial dysfunction plays important roles in many diseases, but there is no satisfactory method to assess mitochondrial health in vivo. Here, we engineered a MitoTimer reporter gene from the existing Timer reporter gene. MitoTimer encodes a mitochondria-targeted green fluorescent protein when newly synthesized, which shifts irreversibly to red fluorescence when oxidized. Confocal microscopy confirmed targeting of the MitoTimer protein to mitochondria in cultured cells, Caenorhabditis elegans touch receptor neurons, Drosophila melanogaster heart and indirect flight muscle, and mouse skeletal muscle. A ratiometric algorithm revealed that conditions that cause mitochondrial stress led to a significant shift toward red fluorescence as well as accumulation of pure red fluorescent puncta of damaged mitochondria targeted for mitophagy. Long term voluntary exercise resulted in a significant fluorescence shift toward green, in mice and D. melanogaster, as well as significantly improved structure and increased content in mouse FDB muscle. In contrast, high-fat feeding in mice resulted in a significant shift toward red fluorescence and accumulation of pure red puncta in skeletal muscle, which were completely ameliorated by voluntary wheel running. Hence, MitoTimer allows for robust analysis of multiple parameters of mitochondrial health under both physiological and pathological conditions and will be highly useful for future research of mitochondrial health in multiple disciplines in vivo.     

Overexpression of the Lactobacillus plantarum peptidoglycan biosynthesis murA2 gene increases the tolerance of Escherichia coli to alcohols and enhances ethanol production

A major challenge in producing chemicals and biofuels is to increase the tolerance of the host organism to toxic products or byproducts. An Escherichia coli strain with superior ethanol and more generally alcohol tolerance was identified by screening a library constructed by randomly integrating Lactobacillus plantarum genomic DNA fragments into the E. coli chromosome via Cre-lox recombination. Sequencing identified the inserted DNA fragment as the murA2 gene and its upstream intergenic 973-bp sequence, both coded on the negative genomic DNA strand. Overexpression of this murA2 gene and its upstream 973-bp sequence significantly enhanced ethanol tolerance in both E. coli EC100 and wild type E. coli MG1655 strains by 4.1-fold and 2.0-fold compared to control strains, respectively. Tolerance to n-butanol and i-butanol in E. coli MG1655 was increased by 1.85-fold and 1.91-fold, respectively. We show that the intergenic 973-bp sequence contains a native promoter for the murA2 gene along with a long 5′ UTR (286 nt) on the negative strand, while a noncoding, small RNA, named MurA2S, is expressed off the positive strand. MurA2S is expressed in E. coli and may interact with murA2, but it does not affect murA2’s ability to enhance alcohol tolerance in E. coli. Overexpression of murA2 with its upstream region in the ethanologenic E. coli KO11 strain significantly improved ethanol production in cultures that simulate the industrial Melle-Boinot fermentation process.     

The multifunctional role of the pallilysin‐associated Treponema pallidum protein,Tp0750, in promoting fibrinolysis and extracellular matrix component degradation

The mechanisms that facilitate dissemination of the highly invasive spirochaete, Treponema pallidum, are incompletely understood. Previous studies showed the treponemal metalloprotease pallilysin (Tp0751) possesses fibrin clot degradation capability, suggesting a role in treponemal dissemination. In the current study we report characterization of the functionally linked protein Tp0750. Structural modelling predicts Tp0750 contains a von Willebrand factor type A (vWFA) domain, a protein‐protein interaction domain commonly observed in extracellular matrix (ECM)‐binding proteins. We report Tp0750 is a serine protease that degrades the major clot components fibrinogen and fibronectin. We also demonstrate Tp0750 cleaves a matrix metalloprotease (MMP) peptide substrate that is targeted by several MMPs, enzymes central to ECM remodelling. Through proteomic analyses we show Tp0750 binds the endothelial fibrinolytic receptor, annexin A2, in a specific and dose‐dependent manner. These results suggest Tp0750 constitutes a multifunctional protein that is able to (1) degrade infection‐limiting clots by both inhibiting clot formation through degradation of host coagulation cascade proteins and promoting clot dissolution by complexing with host proteins involved in the fibrinolytic cascade and (2) facilitate ECM degradation via MMP‐like proteolysis of host components. We propose that through these activities Tp0750 functions in concert with pallilysin to enable T. pallidum dissemination.     

DNA intermediates of meiotic recombination in synchronous S. pombe at optimal temperature

Crossovers formed by recombination between homologous chromosomes are important for proper homolog segregation during meiosis and for generation of genetic diversity. Optimal molecular analysis of DNA intermediates of recombination requires synchronous cultures. We previously described a mutant, pat1-as2, of the fission yeast Schizosaccharomyces pombe that undergoes synchronous meiosis at 25°C when an ATP analog is added to the culture. Here, we compare recombination intermediates in pat1-as2 at 25°C with those in the widely used pat1-114 temperature-sensitive mutant at 34°C, a temperature higher than optimal. DNA double-strand breaks at most hotspots are similarly abundant in the two conditions but, remarkably, a few hotspots are distinctly deficient at 25°C. In both conditions, Holliday junctions at DNA break hotspots form more frequently between sister chromatids than between homologs, but a novel species, perhaps arising from invasion by only one end of broken DNA, is more readily observed at 25°C. Our results confirm the validity of previous assays of recombination intermediates in S. pombe and provide new information on the mechanism of meiotic recombination.