Disappearance of calcium-induced phase separation in phosphatidylserine-phosphatidylcholine membranes caused by protonation and by electric current

Disappearance of Ca²⁺-induced phase separation in phosphatidylserine-phosphatidylcholine membranes has been studied under several conditions by monitoring electron spin resonance spectrum of spin-labeled phosphatidylcholine. The membranes were prepared in Millipore filters. Electron micrographs of the preparations showed formation of multilayered structures lined on the pore surface. The phase separation was disappeared when the membrane was soaked in non-buffered salt solution (100 ml KCl, pH 5.5). It was markedly contrasting that when the bathing salt solution was buffered no disappearance was observed. Disappearance of the phase separation was also observed when the Ca²⁺-treated membrane was transferred to acidic salt solutions ($\text{?}\text{pH 2.5}$) or to low ionic strength media ($\text{? 10}\text{mM}$) buffered at pH 5.5, and then to the buffered salt solution (100 mM KCl, pH 5.5). These are due to replacement of Ca²⁺ by proton, proton-induced separation, followed by disappearance of the phase separation inthe buffered salt solution. Biological significance of the competition between Ca²⁺ and proton for the phase separation or domain formation in the membranes was emphasized.     

K252a, a new inhibitor of protein kinase C, concomitantly inhibits 40K protein phosphorylation and serotonin secretion in a phorbol ester-stimulated platelets

K252a isolated from microbial origin was found to potently inhibit protein kinase C in vitro (1). This agent inhibits phosphorylation of 40,000 dalton protein (40K protein) induced by 12-0-tetradecanoylphorbol-13-acetate(TPA) in intact rabbit platelets. This indicates that K252a exhibits the inhibition of protein kinase C in intact cells. The serotonin secretion induced by TPA was inhibited by K252a at nearly equal concentrations required to inhibit the phosphorylation of 40K protein. This provides the evidence to support the cause-effect relationship between the protein phosphorylation and the secretion in TPA-stimulated platelets.     

Whole Genome Analyses of Marine Fish Pathogenic Isolate,Mycobacterium sp. 012931

Mycobacterium is a genus within the order Actinomycetales that comprises of a large number of well-characterized species, several of which includes pathogens known to cause serious disease in human and animal. Here, we report the whole genome sequence of Mycobacterium sp. strain 012931 isolated from the marine fish, yellowtail (Seriola quinqueradiata). Mycobacterium sp. 012931 is a fish pathogen causing serious damage to aquaculture farms in Japan. DNA dot plot analysis showed that Mycobacterium sp. 012931 was more closely related to Mycobacterium marinum when compared across several Mycobacterium species. However, little conservation of the gene order was observed between Mycobacterium sp. 012931 and M. marinum genome. The annotated 5,464 genes of Mycobacterium sp. 012931 was classified into 26 subsystems. The insertion/deletion gene analysis shows Mycobacterium sp. 012931 had 643 unique genes that were not found in the M. marinum strains. In the virulence, disease, and defense subsystem, both insertion and deletion genes of Mycobacterium sp. 012931 were associated with the PPE gene cluster of Mycobacteria. Of seven plcB genes in Mycobacterium sp. 012931, plcB_2 and plcB_3 showed low identities with those of M. marinum strains. Therefore, Mycobacterium sp. 012931 has differences on genetic and virulence from M. marinum and may induce different interaction mechanisms between host and pathogen.     

Rapid sexing of bovine preimplantation embryos using loop-mediated isothermal amplification

Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method that amplifies a target sequence specifically under isothermal conditions. The product of LAMP is detected by the turbidity of the reaction mixture without electrophoresis. The objective of this study was to develop a rapid sexing method for bovine preimplantation embryos using LAMP. The first experiment was conducted to optimize the DNA extraction method for LAMP-based embryo sexing. The DNA of single blastomeres was extracted using three methods: heat, NaOH, and proteinase K-Tween 20 (PK-TW) treatments. Sexing was performed with two LAMP reactions, male-specific and male-female common reaction, after DNA extraction. The rates of correct determination of sex were 88.9-94.4%, with no difference among methods. The sensitivity and accuracy of LAMP-based embryo sexing were evaluated in the next experiment. The proportion of samples in which the sex was correctly determined was 75-100% for one to five biopsied cells. Lastly, in vivo-derived embryos were examined to verify the usefulness of LAMP-based embryo sexing, and some of these fresh, sexed embryos were transferred into recipient animals. The time needed for sexing was <1 h. The pregnancy rate was 57.4% and all calves born were of the predicted sex (12 male and 21 female). Therefore, LAMP-based embryo sexing accurately determined gender and is suitable for field application.     

A Novel Prolyl Hydroxylase Inhibitor Protects Against Cell Death After Hypoxia

Hypoxia-inducible factor 1 (HIF-1) is regulated by the oxygen-dependent hydroxylation of proline residues by prolyl hydroxylases (PHDs). We recently developed a novel PHD inhibitor, TM6008, that suppresses the activity of PHDs, inducing continuous HIF-1α activation. In this study, we investigated how TM6008 affects cell survival after hypoxic conditions capable of inducing HIF-1α expression and how TM6008 regulates PHDs and genes downstream of HIF-1α. After SHSY-5Y cells had been subjected to hypoxia, TM6008 was added to the cell culture medium under normoxic conditions. Apoptotic cell death was significantly augmented just after the hypoxic conditions, compared with cell death under normoxic conditions. Notably, when TM6008 was added to the media after the cells had been subjected to hypoxia, the expression level of HIF-1α increased and the number of cell deaths decreased, compared with the results for cells cultured in media without TM6008 after hypoxia, during the 7-day incubation period under normoxic conditions. Moreover, the protein expression levels of heme oxygenase 1, erythropoietin, and glucose transporter-3, which were genes downstream of HIF-1α, were elevated in media to which TM6008 had been added, compared with media without TM6008, during the 7-day incubation period under normoxic conditions. However, the protein expression levels of PHD2 and p53 which suppressed cell proliferation were suppressed in the media to which TM6008 had been added. Thus, TM6008, which suppresses the protein expressions of PHD2 and p53, might play an important role in cell survival after hypoxic conditions, with possible applications as a new compound for treatment after ischemic stroke.     

Photoprotective responses of an ice algal community in Saroma-Ko Lagoon, Hokkaido, Japan

In polar seas, ice algal communities can acclimate to extremely low light conditions. Reduced acclimation to shade in ice algal communities, as a result of shortened ice seasons at the lower latitude limits of sea ice distribution, has been suggested as advantageous for avoiding strong photoinhibition when cells are released into high light levels at the water’s surface. Thermal dissipation of excess energy by xanthophyll cycle pigments in the de-epoxidated state may occur in ice algal communities released from retreating sea ice. A light exposure experiment was conducted on ice algal communities obtained from sea ice at Saroma-Ko Lagoon in Hokkaido, Japan. Photoprotective responses to direct sunlight were examined through non-photochemical quenching (NPQ) of chlorophyll fluorescence and xanthophyll pigments. De-epoxidation of diadinoxanthin (DD) to diatoxanthin (DT) occurred rapidly, and NPQ showed a dynamic response to high light exposure. The linear relationship between the ratio of DT to chlorophyll a and NPQ followed a steeper slope than previously observed for mesophilic diatoms. The steeper slope could be explained by an apparent increase in DT for the mesophilic diatoms and induction of NPQ in response to low temperatures only in the ice algal communities. Enhanced production of DT in mesophilic diatoms could be the result of de-epoxidation of DD plus de novo synthesis, and the enhancement of NPQ might be caused by low temperature stress in the ice algae. Although the response of NPQ might be related to temperature, NPQ independent of DT synthesis should also be studied.     

Serum evaluation of soluble interferon-α/β receptor and high-sensitivity C-reactive protein for diagnosis of the patients with gastrointestinal and hepatobiliary-pancreatic cancer

Serum soluble interferon-α/β receptor (sIFN-α/βR) and high-sensitivity C-reactive protein (hs-CRP) levels were evaluated in the patients with gastrointestinal and hepatobiliary-pancreatic cancer. We compared the sensitivity and specificity of serum sIFN-α/βR with that of serum hs-CRP and evaluated the two diagnostic parameters in combination. Serum sIFN-α/βR levels were measured in 92 patients and 25 healthy individuals by enzyme-linked immunosorbent assay. The diagnoses were 37 cases of hepatocellular carcinoma, 17 cases of pancreatic cancer, 15 cases of colon cancer, 13 cases of biliary tract cancer, and 10 cases of gastric cancer. Serum levels of sIFN-α/βR and hs-CRP were significantly higher in the patients than in healthy individuals (p < 0.05). The optimal cut-off values of sIFN-α/βR and hs-CRP were 3600 pg/ml and 0.5 μg/ml, respectively. The sensitivity and specificity for these thresholds were 94.6% and 88.0%, whereas positive predictive and negative predictive values were 96.7% and 81.5%. These results suggest that a combination of serum sIFN-α/βR and hs-CRP thresholds may be more reliable diagnostic parameter for gastrointestinal and hepatobiliary-pancreatic cancer.     

Binding of FAD to cytochrome b558 is facilitated during activation of the phagocyte NADPH oxidase, leading to superoxide production

The superoxide-producing phagocyte NADPH oxidase can be reconstituted in a cell-free system. The activity of NADPH oxidase is dependent on FAD, but the physiological status of FAD in the oxidase is not fully elucidated. To clarify the role of FAD in NADPH oxidase, FAD-free full-length recombinant p47(phox), p67(phox), p40(phox), and Rac were prepared, and the activity was reconstituted with these proteins and purified cytochrome b(558) (cyt b(558)) with different amounts of FAD. A remarkably high activity, over 100 micromol/s/micromol heme, was obtained in the oxidase with purified cyt b(558), ternary complex (p47-p67-p40(phox)), and Rac. From titration with FAD of the activity of NADPH oxidase reconstituted with purified FAD-devoid cyt b, the dissociation constant K(d) of FAD in cyt b(558) of reconstituted oxidase was estimated as nearly 1 nm. We also examined addition of FAD on the assembly process in reconstituted oxidase. The activity was remarkably enhanced when FAD was present during assembly process, and the efficacy of incorporating FAD into the vacant FAD site in purified cyt b(558) increased, compared when FAD was added after assembly processes. The absorption spectra of reconstituted oxidase under anaerobiosis showed that incorporation of FAD into cyt b(558) recovered electron flow from NADPH to heme. From both K(d) values of FAD and the amount of incorporated FAD in cyt b(558) of reconstituted oxidase, in combination with spectra, we propose the model in which the K(d) values of FAD in cyt b(558) is changeable after activation and FAD binding works as a switch to regulate electron transfer in NADPH oxidase.     

Identification of QTLs for resistance to powdery mildew and SSR markers diagnostic for powdery mildew resistance genes in melon (Cucumis melo L.)

Powdery mildew caused by Podosphaera xanthii is an important foliar disease in melon. To find molecular markers for marker-assisted selection, we constructed a genetic linkage map of melon based on a population of 93 recombinant inbred lines derived from crosses between highly resistant AR 5 and susceptible ‘Earl’s Favourite (Harukei 3)’. The map spans 877 cM and consists of 167 markers, comprising 157 simple sequence repeats (SSRs), 7 sequence characterized amplified region/cleavage amplified polymorphic sequence markers and 3 phenotypic markers segregating into 20 linkage groups. Among them, 37 SSRs and 6 other markers were common to previous maps. Quantitative trait locus (QTL) analysis identified two loci for resistance to powdery mildew. The effects of these QTLs varied depending on strain and plant stage. The percentage of phenotypic variance explained for resistance to the pxA strain was similar between QTLs (R ² = 22–28%). For resistance to pxB strain, the QTL on linkage group (LG) XII was responsible for much more of the variance (41–46%) than that on LG IIA (12–13%). The QTL on LG IIA was located between two SSR markers. Using an independent population, we demonstrated the effectiveness of these markers. This is the first report of universal and effective markers linked to a gene for powdery mildew resistance in melon.     

Ubiquitylation of ε-COP by PIRH2 and regulation of the secretion of PSA

Ubiquitylation appears to be involved in the membrane trafficking system including endocytosis, exocytosis, and ER-to-Golgi transport. We found that PIRH2, which was identified as an interacting protein for androgen receptor or p53, interacts with and ubiquitylates the ε-subunit of coatmer complex, ε-COP. PIRH2 promotes the ubiquitylation of ε-COP in vitro and in vivo and consequently promotes the degradation of ε-COP. The interaction between PIRH2 and ε-COP is affected by the presence of androgen, and PIRH2 in the presence of androgen promotes ubiquitylation of ε-COP in vivo. Furthermore, overexpression of the wild type of PIRH2 in prostate cancer cells causes downregulation of the secretion of prostate-specific antigen (PSA), a secretory protein in prostate epithelial cells and one of diagnostic markers for prostate cancer. Our results indicate that PIRH2 functions as a regulator for COP I complex.