Accurate delimitation of Phanerochaete chrysosporium and Phanerochaete sordida by specific PCR primers and cultural approach

White rot fungi, Phanerochaete chrysosporium and Phanerochaete sordida, have been mostly studied in a variety of industrial processes like biopulping and pulp bleaching as well as in bioremediation. Whereas P. sordida is widely distributed in the North Temperate Zone, P. chrysosporium is reported in the restricted area and hundreds of reports have been described from a few strains of P. chrysosporium, which are deposited at various fungal collections in the world. The isolates of two species are not easily discriminated because of their morphological and molecular similarity. Through the ITS sequence analyses, a region containing substantial genetic variation between the two species was identified. PCR amplification using two specific primers was successfully used to differentiate P. chrysosporium from P. sordida. These results were supported by cultural studies. The growth rates at 37 degrees C on PDA, MEA, and Cza and the microscopic features of conidia on PDA and YMA were also very useful to differentiate those two species.     

A RAP1/TRF2 complex inhibits nonhomologous end-joining at human telomeric DNA ends

The mechanisms by which telomeres are distinguished from DNA double-strand breaks are poorly understood. Here we have defined the minimal requirements for the protection of telomeric DNA ends from nonhomologous end-joining (NHEJ). Neither long, single-stranded overhangs nor t loop formation is essential to prevent NHEJ-mediated ligation of telomeric ends in vitro. Instead, a tandem array of 12 telomeric repeats is sufficient to impede illegitimate repair in a highly directional manner at nearby DNA ends. The polarity of end protection is consistent with the orientation of naturally occurring telomeres and is well suited to minimize interference between chromosome capping and the repair of DNA double-strand breaks in subtelomeric sequences. Biochemical fractionation and reconstitution revealed that telomere protection is mediated by a RAP1/TRF2 complex, providing evidence for a direct role for human RAP1 in the protection of telomeric DNA from NHEJ.     

Ras-related proteins (Rab) are key proteins related to male fertility following a unique activation mechanism

Ras-related protein Rab (Rab) proteins, member of Ras superfamily of monomeric G proteins, are well known key regulators of intracellular vesicular transport. Recently, it has been reported that Rab 2A and 3A are related to acrosomal exocytosis in spermatozoa and Rab 2A can be used to fertility-related biomarker in male. However, the role and mechanism of Rab proteins in spermatozoa has not been fully understood yet. Therefore, the study to analyze the expression and location of various Rab proteins in spermatozoa is required to understand the role and mechanism of Rab proteins in spermatozoa. In present study, to analyze the expression level and location of various Rab proteins (Rab 2A, Rab3A, Rab4, Rab5, Rab8A, Rab9, Rab11, Rab14, Rab25, Rab27A, and Rab34) and Rab protein regulators (RabGAP, RabGDI, RabGEF) in spermatozoa following capacitation, immunofluorescence and western blot analysis were performed. All of 11 Rab proteins were expressed in acrosomal region and tail of spermatozoa. Furthermore, all Rab proteins and Rab protein regulators, except RabGAP, have decreased expression patterns after capacitation. Taken together, Rab proteins were located in sperm head and tail. In addition, expression patterns of Rab proteins in spermatozoa were altered following capacitation. Therefore, our results suggested that Rab proteins may be key proteins related with capacitation as well as playing important role with uniquely activation pathway for male fertility.     

Taxonomic review and distribution of giant water bugs (Hemiptera: Belostomatidae: Lethocerinae) in the Palearctic,Oriental, and Australian regions

The aim of the present study was to analyze the morphology and COI sequences of giant water bugs (Belostomatidae: Lethocerinae) that inhabit the Palearctic, Oriental, and Australian regions (i.e., three Lethocerus and a single Kirkaldyia species). Analysis revealed that both L. patruelis and L. indicus share two light stripes on the pronotum, but L. patruelis possesses narrower stripes and L. insulanus possesses an additional longitudinal narrow dark median band, whereas K. deyrolli was characterized by not having such markings. With regards to genetic analysis, the average intraspecific genetic distances of L. patruelis and L. indicus (from Palearctic and Oriental regions are 0.3 and 0.8%, respectively), but K. deyrolli which was collected from Northeast Asia, showed much higher intraspecific genetic distance (3.7%). The genetic distance between Kirkaldyia and Lethocerus (16.4–16.8%) is similar to that found between the genera of other hemipteran taxa. This study also newly reported the extended easternmost distribution of L. patruelis up to Surat Thani Province in southern Thailand. Along with K. deyrolli, which is considered threatened in South Korea and Japan, Lethocerus species have been facing threats in Southeast Asia, where they are heavily harvested and commonly sold in local markets as food. Therefore, future conservation efforts should be directed toward Lethocerus species in tropical Southeast Asia, especially the rarely found L. patruelis.     

Halorubrum halophilum sp. nov., an extremely halophilic archaeon isolated from a salt-fermented seafood

A novel, red-pigmented, pleomorphic and short rod-shaped haloarchaeon, designated B8^T, was isolated from a salt-fermented seafood. Strain B8^T was found to be able to grow at 20–45 °C, in the presence of 15–30 % (w/v) NaCl and at pH 7.0–9.0. The optimum requirements were found to be a temperature range of 35–40 °C, pH 8.0 and the presence of 25 % NaCl. The cells of strain B8^T were observed to be Gram-staining negative and lysed in distilled water. Anaerobic growth did not occur in the presence of nitrate, l-arginine, dimethyl sulfoxide or trimethylamine N-oxide. The catalase and oxidase activities were found to be positive and nitrate was reduced in aerobic conditions. Tween 20, 40 and 80 were found to be hydrolyzed, whereas casein, gelatin and starch were not hydrolyzed. Indole or H₂S was not formed and urease activity was not detected. A phylogenetic analysis based on the 16S rRNA gene sequences indicated that strain B8^T is most closely related to members of the genus Halorubrum in the family Halobacteriaceae. Strain B8^T was found to have three 16S rRNA genes, rrnA, rrnB and rrnC; similarities between the 16S rRNA gene sequences are 99.0–99.8 %. Strain B8^T shared 99.0 % 16S rRNA gene sequence similarity with Halorubrum (Hrr.) lipolyticum JCM 13559^T and Hrr. saccharovorum DSM 1137^T, 98.8 % with Hrr. kocurii JCM 14978^T, 98.3 % with Hrr. lacusprofundi DSM 5036^T, 98.0 % with Hrr. arcis JCM 13916^T, 97.7 % with Hrr. aidingense JCM 13560^T and 97.0 % with Hrr. aquaticum JCM 14031^T, as well as 93.7–96.5 % with other type strains in the genus Halorubrum. The RNA polymerase subunit B′ gene sequence similarity of strain B8^T with Hrr. kocurii JCM 14978^T is 97.2 % and lower with other members of the genus Halorubrum. DNA–DNA hybridization experiments showed that strain B8^T shared equal or lower than 50 % relatedness with reference species in the genus Halorubrum. The genomic DNA G+C content of strain B8^T was determined to be 64.6 mol%. The major isoprenoid quinone of strain B8^T was identified as menaquinone-8 and the major polar lipids as phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, sulfated mannosyl glucosyl diether and an unidentified phospholipid. Based on this polyphasic taxonomic study, strain B8^T is considered to represent a new species in the genus Halorubrum, for which the name Hrr. halophilum sp. nov. is proposed. The type strain is B8^T (=JCM 18963^T = CECT 8278^T).     

Protein phosphatase 1 dephosphorylates Orc2

Phosphorylation of Thr¹¹⁶ and Thr²²⁶ on Orc2, one of the six subunits of the origin recognition complex (ORC), by cyclin A/CDK2 during S phase leads to the dissociation of Orc2, Orc3, Orc4, and Orc5 subunits (Orc2-5) from human chromatin and replication origins. The phosphorylated Orc2 becomes dephosphorylated in the late M phase of the cell cycle. Here we show that protein phosphatase 1 (PP1) dephosphorylates Orc2. Dephosphorylation of Orc2 was accompanied by associating the dissociated Orc subunits with chromatin. Inhibitors of PP1 preferentially inhibited the dephosphorylation of Orc2. The overexpression of the α, β and γ PP1 isoforms decreased the amount of phosphorylated Orc2, and the depletion of these isoforms by RNA interference increased the amount of phosphorylated Orc2. These results suggest that PP1 dephosphorylates Orc2 to promote the binding of ORC to chromatin.     

Testosterone Stimulates Duox1 Activity through GPRC6A in Skin Keratinocytes

Testosterone is an endocrine hormone with functions in reproductive organs, anabolic events, and skin homeostasis. We report here that GPRC6A serves as a sensor and mediator of the rapid action of testosterone in epidermal keratinocytes. The silencing of GPRC6A inhibited testosterone-induced intracellular calcium ([Ca²⁺]_i) mobilization and H₂O₂ generation. These results indicated that a testosterone-GPRC6A complex is required for activation of G_q protein, IP₃ generation, and [Ca²⁺]_i mobilization, leading to Duox1 activation. H₂O₂ generation by testosterone stimulated the apoptosis of keratinocytes through the activation of caspase-3. The application of testosterone into three-dimensional skin equivalents increased the apoptosis of keratinocytes between the granular and stratified corneum layers. These results support an understanding of the molecular mechanism of testosterone-dependent apoptosis in which testosterone stimulates H₂O₂ generation through the activation of Duox1.     

A novel delivery platform for therapeutic peptides

Although many peptides have therapeutic effects against diverse disease, their short half-lives in vivo hurdle their application as drug candidates. To extend the short elimination half-lives of therapeutic peptides, we developed a novel delivery platform for therapeutic peptides using an anti-hapten antibody and its corresponding hapten. We selected cotinine because it is non-toxic, has a well-studied metabolism, and is physiologically absent. We conjugated WKYMVm-NH₂, an anti-sepsis therapeutic peptide, to cotinine and showed that the conjugated peptide in complex with an anti-cotinine antibody has a significantly improved in vivo half-life while retaining its therapeutic efficacy. We suggest that this novel delivery platform for therapeutic peptides will be very useful to develop effective peptide therapeutics.     

Low immunogenicity of allogeneic human umbilical cord blood-derived mesenchymal stem cells in vitro and in vivo

Evaluation of the immunogenicity of human mesenchymal stem cells (MSCs) in an allogeneic setting during therapy has been hampered by lack of suitable models due to technical and ethical limitations. Here, we show that allogeneic human umbilical cord blood derived-MSCs (hUCB-MSCs) maintained low immunogenicity even after immune challenge in vitro. To confirm these properties in vivo, a humanized mouse model was established by injecting isolated hUCB-derived CD34+ cells intravenously into immunocompromised NOD/SCID IL2γnull (NSG) mice. After repeated intravenous injection of human peripheral blood mononuclear cells (hPBMCs) or MRC5 cells into these mice, immunological alterations including T cell proliferation and increased IFN-γ, TNF-α, and human IgG levels, were observed. In contrast, hUCB-MSC injection did not elicit these responses. While lymphocyte infiltration in the lung and small intestine and reduced survival rates were observed after hPBMC or MRC5 transplantation, no adverse events were observed following hUCB-MSC introduction. In conclusion, our data suggest that allogeneic hUCB-MSCs have low immunogenicity in vitro and in vivo, and are therefore “immunologically safe” for use in allogeneic clinical applications.