Pythium polare, a new heterothallic oomycete causing brown discolouration of Sanionia uncinata in the Arctic and Antarctic

Pythium polare sp. nov. is a new heterothallic oomycete species isolated from fresh water and moss from various locations in both the Arctic and Antarctic. This water mould is able to infect stems and leaves of Sanionia moss (Sanionia uncinata). Pythium polare causes brown discolouration in in vitro inoculation tests at 5 °C after 5 weeks of inoculation. It is characterized by globose sporangia with various lengths of discharge tubes releasing zoospores and aplerotic oospores with usually one to five antheridia. The sexual structures are only produced in a dual culture of antheridial and oogonial isolates. Phylogenetic analysis, based on ITS sequencing, places all isolated strains of P. polare in a unique new clade, hence it is considered a novel species. Pythium canariense and Pythium violae are the most closely related species of P. polare based both on morphology and the phylogenetic analysis.     

Neuronal subtype specification in the cerebellum and dorsal hindbrain

In the nervous system, there are hundreds to thousands of neuronal cell types that have morphologically, physiologically, and histochemically different characteristics and this diversity may enable us to elicit higher brain function. A better understanding of the molecular machinery by which neuron subtype specification occurs is thus one of the most important issues in brain science. The dorsal hindbrain, including the cerebellum, is a good model system to study this issue because a variety of types of neurons are produced from this region. Recently developed genetic lineage-tracing methods in addition to gene-transfer technologies have clarified a fate map of neurons produced from the dorsal hindbrain and accelerated our understanding of the molecular machinery of neuronal subtype specification in the nervous system.     

Inflammatory changes in adipose tissue enhance expression of GPR84, a medium-chain fatty acid receptor: TNFα enhances GPR84 expression in adipocytes

In this study we aimed to identify the physiological roles of G protein-coupled receptor 84 (GPR84) in adipose tissue, together with medium-chain fatty acids (MCFAs), the specific ligands for GPR84. In mice, high-fat diet up-regulated GPR84 expression in fat pads. In 3T3-L1 adipocytes, co-culture with a macrophage cell line, RAW264, or TNFα remarkably enhanced GPR84 expression. In the presence of TNFα, MCFAs down-regulated adiponectin mRNA expression in 3T3-L1 adipocytes. Taken together, our results suggest that GPR84 emerges in adipocytes in response to TNFα from infiltrating macrophages and exacerbates the vicious cycle between adiposity and diabesity.     

Differential Role for CD80 and CD86 in the Regulation of the Innate Immune Response in Murine Polymicrobial Sepsis

Background

Inflammation in the early stages of sepsis is governed by the innate immune response. Costimulatory molecules are a receptor/ligand class of molecules capable of regulation of inflammation in innate immunity via macrophage/neutrophil contact. We recently described that CD80/86 ligation is required for maximal macrophage activation and CD80/86^−/− mice display reduced mortality and inflammatory cytokine production after cecal ligation and puncture (CLP). However, these data also demonstrate differential regulation of CD80 and CD86 expression in sepsis, suggesting a divergent role for these receptors. Therefore, the goal of this study was to determine the individual contribution of CD80/86 family members in regulating inflammation in sepsis.

Methodology/Principal Findings

CD80^−/− mice had improved survival after CLP when compared to WT or CD86^−/− mice. This was associated with preferential attenuation of inflammatory cytokine production in CD80^−/− mice. Results were confirmed with pharmacologic blockade, with anti-CD80 mAb rescuing mice when administered before or after CLP. In vitro, activation of macrophages with neutrophil lipid rafts caused selective disassociation of IRAK-M, a negative regulator of NF-κB signaling from CD80; providing a mechanism for preferential regulation of cytokine production by CD80. Finally, in humans, upregulation of CD80 and loss of constitutive CD86 expression on monocytes was associated with higher severity of illness and inflammation confirming the findings in our mouse model.

Conclusions

In conclusion, our data describe a differential role for CD80 and CD86 in regulation of inflammation in the innate immune response to sepsis. Future therapeutic strategies for blockade of the CD80/86 system in sepsis should focus on direct inhibition of CD80.     

Efficient generation of recessive traits in diploid sake yeast by targeted gene disruption and loss of heterozygosity

Sake yeast, a diploid Saccharomyces cerevisiae strain, is useful for industry but difficult to genetically engineer because it hardly sporulates. Until now, only a few recessive mutants of sake yeast have been obtained. To solve this problem, we developed the high-efficiency loss of heterozygosity (HELOH) method, which applies a two-step gene disruption. First, a heterozygous disruptant was constructed by gene replacement with URA3, followed by marker recycling on medium containing 5-fluoroorotic acid (5-FOA). Subsequently, spontaneous loss of heterozygosity (LOH) yielding a homozygous disruptant was selected for in a second round of gene integration. During this step, the wild-type allele of the heterozygous disruptant was marked by URA3 integration, and the resulting transformants were cultivated in non-selective medium to induce recombination and then grown on medium with 5-FOA to enrich for mutants that had undergone LOH. Although the frequency with which LOH occurs is extremely low, many homozygous disruptants were obtained with the HELOH method. Thus, we were able to efficiently construct homozygous disruptants of diploid sake yeast without sporulation, and sake yeast strains with multiple auxotrophies and a protease deficiency could be constructed. The HELOH method, therefore, facilitated the utilization of diploid sake yeast for genetic engineering purposes.     

Chemical and immunochemical identification of propanoyllysine derived from oxidized n-3 polyunsaturated fatty acid

It is known that n-3 polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid and eicosapentaenoic acid, are rapidly oxidized in vitro. N?-(propanoyl)lysine (propionyllysine, or PRL) is formed from the reaction of the oxidized products of n-3 PUFAs and lysine. To evaluate the oxidized n-3 PUFA-derived protein modifications in vivo, we have developed detection methods using a novel monoclonal antibody against PRL as well as liquid chromatography–mass spectrometry (LC/MS/MS). The antibody obtained specifically recognized PRL. A strong positive staining in atherosclerotic lesions of hypercholesterolemic rabbits was observed. We have also simultaneously identified and quantified both urinary PRL and urinary N?-(hexanoyl)lysine, using LC/MS/MS using isotope dilution methods. The level of urinary PRL (21.6 ± 10.6 μmol/mol of creatinine) significantly correlated with the other oxidative stress markers, 8-oxo-deoxyguanosine, dityrosine, and isoprostanes. The increase in the excretion of amide adducts into the urine of diabetic patients was also confirmed compared to healthy subjects. These results suggest that PRL may be good marker for n-3 PUFA-derived oxidative stress in vivo.     

Bioorganic synthesis of end-capped anti-HIV peptides by simultaneous cyanocysteine-mediated cleavages of recombinant proteins

Bioorganic synthesis of N- and C-terminal end-capped peptides by two simultaneous S-cyanocysteine-mediated cleavages of recombinant proteins is described. This approach is demonstrated in the preparation of anti-HIV fusion inhibitory peptides.     

A Novel RNA-Binding Protein,Ossa/C9orf10, Regulates Activity of Src Kinases To Protect Cells from Oxidative Stress-Induced Apoptosis

During the process of tumor progression and clinical treatments, tumor cells are exposed to oxidative stress. Tumor cells are frequently resistant to such stress by producing antiapoptotic signaling, including activation of Src family kinases (SFKs), although the molecular mechanism is not clear. In an attempt to identify the SFK-binding proteins selectively phosphorylated in gastric scirrhous carcinoma, we identified an uncharacterized protein, C9orf10. Here we report that C9orf10 (designated Ossa for oxidative stress-associated Src activator) is a novel RNA-binding protein that guards cancer cells from oxidative stress-induced apoptosis by activation of SFKs. Exposure to oxidative stress such as UV irradiation induces the association of Ossa/C9orf10 with regulatory domains of SFKs, which activates these kinases and causes marked tyrosine phosphorylation of C9orf10 in turn. Tyrosine-phosphorylated Ossa recruits p85 subunits of phosphatidylinositol 3-kinase (PI3-kinase) and behaves as a scaffolding protein for PI3-kinase and SFKs, which activates the Akt-mediated antiapoptotic pathway. On the other hand, the carboxyl terminus of Ossa has a distinct function that directly binds RNAs such as insulin-like growth factor II (IGF-II) mRNA and promotes the extracellular secretion of IGF-II. Our findings indicate that Ossa is a dual-functional protein and might be a novel therapeutic target which modulates the sensitivity of tumors to oxidative stress.Tumor cells are exposed to oxidative stress in various situations in vivo. Reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (H₂O₂) are generated by exposure of cancer cells to hypoxia, followed by reperfusion; radiotherapy; photodynamic therapy; and some chemotherapeutic agents such as cisplatin (6, 25). This production of ROS generally induces apoptosis, whereas some tumor cells become resistant to this kind of apoptosis by some mechanism such as elevated expression of antioxidant thiols in the cells (17, 27).Src family kinases (SFKs) play important roles in various cell functions such as cell proliferation, cell adhesion, and cell migration (26), and the activities of SFKs often correlate with the malignant potential of cancer and a poor prognosis (37). Activation of c-Src is observed after the cells are exposed to oxidative stress (1, 9, 11, 34), and the activation of SFKs contributes to the resistance to apoptosis induced upon cellular stress. For instance, treatment of cells with oxidative stress such as UV irradiation or H₂O₂ causes apoptotic cell death, which is rescued by expression of v-Src (28). In our attempt to identify the key molecules that promote the expansion of gastric scirrhous carcinoma in vivo by mediating signals from activated SFKs, we identified an uncharacterized protein, C9orf10.C9orf10 (Homo sapiens chromosome 9 open reading frame 10) was originally found by the human genome sequence project as an annotated protein, and the gene was mapped to chromosome 9q22.31 (12). C9orf10 was recently detected within the Purα-containing mRNA-protein complex in the brain, although no functional information about this protein is available (14). We show that C9orf10 protects cells from apoptosis through activation of SFKs in response to oxidative stress. The kinase activity of SFKs is regulated by two intramolecular interactions. The inactive form is achieved by interaction of the SH2 domain with the phosphorylated C-terminal tail and association of the SH3 domain with a polyproline type II helix formed by the linker region between the SH2 domain and the catalytic domain (30). C9orf10 functions as a novel activator of SFKs that unfolds the inactive form of SFKs by association with both the SH2 and SH3 domains of SFKs. Tyrosine phosphorylation of C9orf10 is induced by the activated SFKs in turn, producing scaffolds to recruit phosphatidylinositol 3-kinase (PI3-kinase) and activate PI3-kinase-Akt signaling, which plays a key role in protecting cancer cells from oxidative stress-induced apoptosis. Therefore, we named C9orf10 Ossa (oxidative stress-associated Src activator).We also showed that the carboxyl terminus of Ossa directly binds to RNA, suggesting a distinct role for Ossa as an RNA-binding protein. As one of the target RNAs, Ossa directly binds to insulin-like growth factor II (IGF-II) mRNA, which subsequently enhances the extracellular secretion of IGF-II. Because an increase in IGF-II promotes cell proliferation, the RNA-binding function of Ossa may also contribute to the survival of cancer cells in vivo.Scirrhous gastric carcinoma diffusely infiltrates a broad region of the stomach and is frequently associated with metastasis to lymph nodes and peritoneal dissemination and therefore has the worst prognosis among the various types of gastric cancer (35). Blocking of the survival signaling mediated by Ossa, which sensitizes the cancer cells to stress-induced apoptosis, may be a novel therapeutic approach for gastric scirrhous carcinoma cells.     

The evolutionary pattern of early life history in water currents

Explanation of the characteristics of the early developmental stage of organisms is an important problem in evolutionary biology. In studies to date, evolutionary biologists have proposed some theories that successfully explain egg size variation. Mesoscale water movements may transport early life stage organisms in the aquatic biosphere. We propose a novel biological view to explain the duration of the retention period at the spawning ground and egg size variations in aquatic organisms with a planktonic stage at least during the early part of their life history. We develop a life history model of the early life stage of such aquatic organisms that takes into account their adaptations to water currents and biotic environmental gradients in the currents. We hypothesize that the distance from the spawning grounds to the nursery grounds and the biological richness of the currents affect the adaptive life history design of these aquatic organisms, including adaptive retention time at the spawning ground and egg size. Various studies of fish biology describe in passing phenomena that suggest the validity of our deductions, but explicit empirical attempts to evaluate our predictions in the field of evolutional biology are needed.