Melanin content and MC1R function independently affect UVR-induced DNA damage in cultured human melanocytes

Malignant transformation of melanocytes leads to melanoma, the most fatal form of skin cancer. Ultraviolet radiation (UVR)-induced DNA photoproducts play an important role in melanomagenesis. Cutaneous melanin content represents a major photoprotective mechanism against UVR-induced DNA damage, and generally correlates inversely with the risk of skin cancer, including melanoma. Melanoma risk is also determined by susceptibility genes, one of which is the melanocortin 1 receptor (MC1R) gene. Certain MC1R alleles are strongly associated with melanoma. We hereby present experimental evidence for the role of two melanoma risk factors, constitutive pigmentation, as assessed by total melanin, eumelanin and pheomelanin contents, and MC1R genotype and function, in determining the induction and repair of DNA photoproducts in cultured human melanocytes after irradiation with increasing doses of UVR. We found that total melanin and eumelanin contents (MC and EC) correlated inversely with the extent of UVR-induced growth arrest, apoptosis and induction of cyclobutane pyrimidine dimers (CPD), but not with hydrogen peroxide release in melanocytes expressing functional MC1R. In comparison, melanocytes with loss-of-function MC1R, regardless of their MC or EC, sustained more UVR-induced apoptosis and CPD, and exhibited reduced CPD repair. Therefore, MC, mainly EC, and MC1R function are independent determinants of UVR-induced DNA damage in melanocytes.     

Mouse homologue of skin-specific retroviral-like aspartic protease involved in wrinkle formation

Retroviral proteases are encoded in the retroviral genome and are responsible for maturation and assembly of infectious virus particles. A number of retroviral protease sequences with retroviral elements are integrated in every eukaryotic genome as endogenous retroviruses. Recently, retroviral-like aspartic proteases that were not embedded within endogenous retroviral elements were identified throughout the eukaryotic and prokaryotic genomes. However, the physiological role of this novel protease family, especially in mammals, is not known. During the high throughput in situ hybridization screening of mouse epidermis, as a granular layer-expressing clone, we identified a mouse homologue of SASPase (Skin ASpartic Protease), a recently identified retroviral-like aspartic protease. We detected and purified the endogenous 32-kDa (mSASP32) and 15-kDa (mSASP15) forms of mSASP from mouse stratum corneum extracts and determined their amino acid sequences. Next, we bacterially produced recombinant mSASP15 via autoprocessing of GST-mSASP32. Purified recombinant mSASP15 cleaved a quenched fluorogenic peptide substrate, designed from the autoprocessing site for mSASP32 maximally at pH 5.77, which is close to the pH of the epidermal surface. Finally, we generated mSASP-deficient mice that at 5 weeks of age showed fine wrinkles that ran parallel on the lateral trunk without apparent epidermal differentiation defects. These results indicate that the retroviral-like aspartic protease, SASPase, is involved in prevention of fine wrinkle formation via activation in a weakly acidic stratum corneum environment. This study provides the first evidence that retroviral-like aspartic protease is functionally important in mammalian tissue organization.     

Spawning habitat selection and suitability for Japanese dace, Tribolodon hakonensis

We have investigated the effects of three current velocities and three substrate sizes (gravel to cobble) on the spawning behavior of Japanese dace, Tribolodon hakonensis, in spawning-induction and habitat-selection experiments. In the spawning-induction experiment under laboratory conditions the number of females induced to spawn was significantly fewer at low current velocity (ca. 5 cm s^–1) than at medium (ca. 30 cm s^–1) or high (ca. 50 cm s^–1) current velocity. Females spawned independently of substrate size in the experimental tank, and did not bury their eggs in the substrate bed. In the habitat-selection experiment under field conditions, females selected the substrate microhabitat and spawned more frequently at a site with medium substrate size (very coarse gravel; major axis ca. 40 mm) than at sites with small (fine gravel; ca. 8 mm) or large (cobble; ca. 70 mm) substrate size. We conclude that Japanese dace select spawning sites at least partly on the basis of current velocity and substrate size, which affects the survival rate of eggs.     

Spontaneous elicitation of potent antitumor immunity and eradication of established tumors by administration of DNA encoding soluble transforming growth factor-β II receptor without active antigen-sensitization

Since immunity is generally suppressed by immunoregulatory factors, such as transforming growth factor-beta (TGF-β), interleukin (IL)-10, and vascular endothelial growth factor (VEGF), produced by tumor cells or stromal cells surrounding tumor cells, various kinds of cancer immunotherapy mostly fail to elicit potent antitumor immunity. Herein, we tested whether neutralization of TGF-β can elicit strong antitumor immune responses and tumor regression in tumor-bearing mice. A plasmid DNA, pcDNA-sTGFβR/huIg, encoding a fusion protein consisting of the extracellular domain of TGF-β type II receptor (TGFβRII) and the Fc portion of human IgG heavy chain, was injected through different routes into B6 mice carrying established tumors of E.G7 cells, which consist of the poorly immunogenic tumor cells EL4, transfected with the ovalbumin (OVA) gene. The frequency of OVA-specific cytotoxic T lymphocytes (CTL), in the treated mice. increased resulting in the tumor eradication and relapse-free survival in around 70% of the E.G7-bearing mice. In contrast, administration of mock DNA into E.G7-bearing mice did not elicit tumor-specific immune responses. Therefore, administration of DNA encoding TGFβRII allowed tumor-bearing hosts to elicit sufficiently potent antitumor immune responses without requirement of further active antigen-immunization. This strategy seems to be applicable to clinical therapy against cancer, because it is low-cost, safe, and easy to manipulate.     

Sunitinib induces apoptosis in pheochromocytoma tumor cells by inhibiting VEGFR2/Akt/mTOR/S6K1 pathways through modulation of Bcl-2 and BAD

Sunitinib is an oral multitargeted receptor tyrosine kinase inhibitor with antiangiogenic and antitumor activity that mainly targets vascular endothelial growth factor receptors (VEGFRs). Very recently, sunitinib has been shown to be an active agent for the treatment of malignant pheochromocytomas. However, it is unclear whether sunitinib acts only through an antiangiogenic mechanism or whether it may also directly target tumor cells. Sunitinib markedly induced apoptosis of PC12 cells in a dose-dependent and time-dependent manner. Furthermore, in support of these findings, we found that sunitinib induced a reduction in the expression of the antiapoptotic molecule Bcl-2 as well as dephosphorylation of the proapoptotic molecule BAD, which results in the activation of BAD in these cells. Consistent with these apoptotic effects, our results showed that sunitinib inhibited phosphorylation of Akt and mTOR and was followed by a reduction of S6K1, which is a well-known target of mTOR. Knockdown of VEGFR-2 attenuated the sunitinib-induced effects, including apoptosis and inhibition of signaling pathways such as the phosphorylation of Akt as well as mTOR, and Bcl-2, which confirmed that these effects could be mediated by VEGFR-2. In addition, silencing of S6K1 induced apoptosis accompanied by a decrease in the phosphorylation of BAD and Bcl-2, similar to that observed with sunitinib treatment. Thus, these results together suggest that sunitinib initially exerts its apoptotic effect through the inhibition of VEGFR-2, which, when followed by reduction of its downstream effectors, including Akt/mTOR/S6K1, may lead to inhibition of the antiapoptotic molecule Bcl-2 and activation of the proapoptotic molecule BAD in PC12 cells. However, PC12 cells do not precisely reflect the pathogenesis of malignant cells. Therefore, we confirmed the key findings by replicating these experiments in human neuroblastoma SK-N-SH cells.     

Evaluation of bactericidal effects of low-temperature nitrogen gas plasma towards application to short-time sterilization

To develop a novel low-temperature plasma sterilizer using pure N(2) gas as a plasma source, we evaluated bactericidal ability of a prototype apparatus provided by NGK Insulators. After determination of the sterilizing conditions without the cold spots, the D value of the BI of Geobacillus stearothermophilus endospores on the filter paper was determined as 1.9 min. However, the inactivation efficiency of BI carrying the same endospores on SUS varied to some extent, suggesting that the bactericidal effect might vary by materials of sterilized instruments. Staphylococcus aureus and Escherichia coli were also exposed to the N(2) gas plasma and confirmed to be inactivated within 30 min. Through the evaluation of bactericidal efficiency in a sterilization bag, we concluded that the UV photons in the plasma and the high-voltage pulse to generate the gas plasma were not concerned with the bactericidal effect of the N(2) gas plasma. Bactericidal effect might be exhibited by activated nitrogen atoms or molecular radicals.     

Cysteine catabolism and cysteine desulfhydrase (CdsH/STM0458) in Salmonella enterica serovar typhimurium

Cysteine is potentially toxic and can affect diverse functions such as oxidative stress, antibiotic resistance, and swarming motility. The contribution of cysteine catabolism in modulating responses to cysteine has not been examined, in part because the genes have not been identified and mutants lacking these genes have not been isolated or characterized. We identified the gene for a previously described cysteine desulfhydrase, which we designated cdsH (formerly STM0458). We also identified a divergently transcribed gene that regulates cdsH expression, which we designated cutR (formerly ybaO, or STM0459). CdsH appears to be the major cysteine-degrading and sulfide-producing enzyme aerobically but not anaerobically. Mutants with deletions of cdsH and ybaO exhibited increased sensitivity to cysteine toxicity and altered swarming motility but unaltered cysteine-enhanced antibiotic resistance and survival in macrophages.     

Characterization of bisphenol A metabolites produced by Portulaca oleracea cv. by liquid chromatography coupled with tandem mass spectrometry

The garden plant portulaca (Portulaca oleracea cv.) efficiently removes bisphenol A (BPA), an endocrine-disrupting chemical, from a hydroponic solution, but the molecular mechanisms underlying BPA metabolism by portulaca remain unclear. In this study, BPA metabolites converted by portulaca were analyzed by liquid chromatography coupled with tandem mass spectrometry. We observed the hydroxylation of BPA and the oxidization of it to quinone. Polyphenol oxidases are likely to contribute to BPA degradation by portulaca.     

Purification, characterization, and molecular cloning of lectin from winter buds of Lysichiton camtschatcensis (L.) Schott

A novel lectin was purified to homogeneity from winter buds of Lysichiton camtschatcensis (L.) Schott of the Araceae family. It was a tetramer composed of two non-covalently associated polypeptides with small subunits (11 kDa) and large subunits (12 kDa). Sequencing of both subunits yielded unique N-terminal sequences. A cDNA encoding the lectin was cloned. The isolated cDNA contained an open reading frame that encoded 267 amino acids. It encoded both subunits, indicating that the lectin is synthesized as a single precursor protein that is post-translationally processed into two different subunits with 45% sequence identity. Each subunit contained a mannose-binding motif known to be conserved in monocot mannose-binding lectins, but its activity was not inhibited by monosaccharides, including methyl α-mannoside. Asialofetuin and yeast invertase were potent inhibitors. Lectin activity was detected in the buds formed during the winter season but not in the expanded leaves.     

Modelling the global distribution of fungal species: new insights into microbial cosmopolitanism

Microbes are usually believed to have cosmopolitan distributions. However, for estimating the global distributions of microorganisms, discriminating among cryptic species and eliminating undersampling biases are important challenges. We used a novel approach to address these problems and infer the global distribution of a given fungal ecological guild. We collected mushroom‐forming fungi from Yakushima, Japan. We sequenced the internal transcribed spacer 2 (ITS2) from these samples and queried their sequences against GenBank. After identifying similar sequences, we tracked down the geographical origins of samples that yielded those sequences. We used Bayesian zero‐inflated models to allow for species whose DNA sequences have not yet been deposited in GenBank. Results indicated that the geographical distribution of ectomycorrhizal (ECM) fungi was strongly constrained by host specificity, resulting in the occurrence of these fungi intensively in the neighbouring regions. On the other hand, saprotrophic (SAP) fungi were less constrained by climatic conditions, resulting in a much broader distribution range. We inferred that differences in constraints during colonization between ECM and SAP fungi were responsible for the different geographical distribution ranges. We hypothesize that the degree of host/habitat specificity and the degree of isolation of potentially suitable habitats determine microbial biogeographic patterns.