Induction of Manganese-Containing Superoxide Dismutase Is Required for Acid Tolerance in Vibrio vulnificus

The manganese-containing superoxide dismutase (MnSOD) of Vibrio vulnificus, normally detected after the onset of the stationary phase, is expressed during the lag that immediately follows the transfer of cells grown exponentially to a fresh medium acidified to pH 5.0, whereas Fe-containing SOD is constitutively expressed. The signal triggering the growth lag and MnSOD induction therein is not low pH but intracellular superoxide accumulated under these conditions, since addition of a superoxide scavenger not only shortened the lag but also abrogated the MnSOD induction. If the lysine decarboxylase reaction proceeds in the presence of sufficient lysine, the broth is rapidly neutralized to abolish the generation of oxidative stress. Accordingly, the acid tolerance response was examined without the addition of lysine. SoxR regulates MnSOD induction. Lack of MnSOD caused by mutations in soxR or sodA resulted in low tolerance to low pH. The fur mutant derepressing MnSOD showed better tolerance than the wild type. Thus, an increase in total cytosolic SOD activity through MnSOD induction is essential for the cell to withstand the acid challenge. The contribution of cuprozinc-containing SOD to acid tolerance is not significant compared with those of cytosolic SODs

Gp78-KO mice spontaneously develop hepatic steatosis with inflammatory infiltrates.

<p>(<b>A</b>) Livers of 1-year-old gp78 KO mice, grown with normal diet were stained with H&E and visualized as indicated magnification. (a) Steatohepatitis shows swelling cytoplasm with lipid droplets (black arrow) and infiltrating cells. (b) Mild lipid droplets and infiltrating cells (Red arrow). (c) Infiltrate cells gather in the absence of lipid droplets. (<b>B</b>) Oil red stain. Arrow indicates lipid droplets. (<b>C</b>) Trichrome stain was preformed to identify blue colored collagen fibers. PV; portal vein, FL; fatty liver area. (<b>D</b>) Percentage of incidence including mild fatty liver, inflammation in 1-year-old KO mice (n = 25 mice per each group).</p

Gp78-KO mice with acute ER stress progresses to severe fatty liver through UPR-driving SREBP-1 activation.

<p>(<b>A</b>) 1mg/kg body weight of tunicamycin (TM) was intraperitoneally injected to 6 months-old gp78-KO mice. Body weight is represented after normalization to the starting weight as the meant ± SE (n = 3 per group). (<b>B</b>) Radish livers are brown colored after TM injection. Liver of gp78-KO at 11 days was not recovered. (<b>C</b>) Acute ER stress potentiates entire fatty liver of gp78-KO. H&E stain at 3 days after TM injection. Ballooned cells (b) are typically bigger size than WT hepatocytes (a). Cytoplasmic lipid droplets (arrow) (40x). (<b>D</b>) Prolonged fatty liver and fibrosis in gp78-KO. H&E, oil red and Trichrome stains at 11 days after TM injection. White ballooned cells (arrows) were maintained on H&E (200x). Frozen tissues were stained with oil-red O. Irregular fibrosis was extended from connective tissue of portal vein surrounding accumulated lipid droplets (white spots) at pinkish gp78-KO liver (100x). (<b>E</b>) Persistent SREBP-1 activation along with UPR up regulation is responsible for fatty liver of gp78-KO. TM-injected mice were scarified respectively (n = 3). Liver extracts at indicated days were subjected to immunoblots. GRP78, Glucose-Regulated Protein; PDI, Protein Disulfide Isomerase, SERBP; Sterol Regulatory Element Binding Transcription Factor; Insig, Insulin Induced Gene. (<b>F</b>) Chop-mediated apoptosis. Cell survival was analyzed with viable counting in gp78-KO mouse embryonic fibroblast (MEF) cells treated with TM (1μg/ml) as indicated times (top). Induction of UPR was analyzed in immunoblots and gp78 expression was visualized after its immunoprecipitation (bottom). Chop; ER stress-mediated apoptosis marker.</p

Aged gp78 knockout mice develop obesity and gp78 is unregulated upon ER stress.

<p>(<b>A</b>) Schematic of gp78-targeting strategy. The gp78 allele disrupted by the insertion of a gene trap vector (OmniBank Vector 76) in the first intron. Genomic DNA isolated from WT, heterozygous and homozygous was genotyped by PCR. Primer set including double reverse primers was designed for internal PCR quality. (<b>B</b>) Total RNA and lysates were prepared in embryo and adult liver and were analyzed in RT-PCR (Top), both lysates were immunoprecipitated with rabbit anti-gp78 antibodies (epitope: 524–537) and then immunoblotted with monoclonal anti-gp78 antibodies (epitope: 451–551) to identify gp78 protein (bottom). (<b>C</b>) Immunohistochemistry (IHC) of liver with monoclonal anti-gp78 antibody. Arrow indicates gp78 positive stain. PV, portal vein. Original image, x400. (<b>D</b>) Upregulation of gp78 in response to ER stress. Immortalized THLE-3 and cancerous HepG2 liver cells were treated with tunicamycin (1ug/ml) for 24 hrs. and lysates were immunoprecipitated with anti-gp78 antibody and immunoblotted. GRP78/BIP, a chaperon of UPR pathways. (<b>F</b>) Photography of abdomen of 1-year-old mice (left). Comparison of body weight of WT and gp78-KO mice at 3 months (n = 25), 6 months (n = 25), 12 months (n = 25) old (right). Asterisk indicates a significance determined by Student’s test (*, p < 0.05).</p