Cloning and Functional Analysis of Skin Host Defense Peptides from Yakushima Tago's Brown Frog () and Development of Serum Endotoxin Detection System.

Amphibian skin is a valuable source of host defense peptides (HDPs). This study aimed to identify HDPs with novel amino acid sequences from the skin of and analyze their functions. cDNAs encoding HDP precursors were cloned and sequenced using RT-PCR and 3'-RACE.

Molecular Cloning, Expression Analyses, and Physiological Roles of Cathelicidins in the Bursa of Fabricius of the Japanese Quail, .

Antimicrobial peptides (AMPs) act directly on pathogens and maintain the anti-inflammatory effects and activation of immunocompetent cells. Therefore, the activation of the immune system in poultry via the elevation of endogenous AMPs has been attempted. In this study, we focused on the host defense mechanisms in the bursa of Fabricius (BF) of Japanese quail, cloned the cDNA of cathelicidin (CATH)-1 to -3, and analyzed their expression sites.

Cell proliferation and neurogenesis in the adult telencephalon of the newt Cynops pyrrhogaster.

Urodele amphibians have the ability to regenerate several organs, including the brain. For this reason, the research on neurogenesis in these species after ablation of some parts of the brain has markedly progressed. However, detailed information on the characteristics and fate of proliferated cells as well as the function of newly generated neurons under normal conditions is still limited.

Dual Roles of Extracellular Histone H3 in Host Defense: Its Differential Regions Responsible for Antimicrobial and Cytotoxic Properties and Their Modes of Action.

Extracellular histones play a dual role-antimicrobial and cytotoxic-in host defense. In this study, we evaluated the antimicrobial and cytotoxic activities of histone H3 and identified the responsible molecular regions for these properties. Broth microdilution assays indicated that histone H3 exhibits growth inhibitory activity against not only Gram-negative and -positive bacteria but also fungi.

Antimicrobial Property and Mode of Action of the Skin Peptides of the Sado Wrinkled Frog, against Animal and Plant Pathogens.

The Sado wrinkled frog has recently been classified as a new frog species endemic to Sado Island, Japan. In this study, we cloned 12 cDNAs encoding the biosynthetic precursors for brevinin-2SSa-2SSd, esculentin-2SSa, ranatuerin-2SSa, brevinin-1SSa-1SSd, granuliberin-SSa, and bradykinin-SSa from the skin of . Among these antimicrobial peptides, we focused on brevinin-2SSb, ranatuerin-2SSa, and granuliberin-SSa, using their synthetic replicates to examine their activities against different reference strains of pathogenic microorganisms that infect animals and plants.

Comparative evaluation of the Geenius HIV 1/2 Confirmatory Assay and the HIV-1 and HIV-2 Western blots in the Japanese population.

Accurate diagnosis of earlier HIV infection is essential for treatment and prevention. Currently, confirmation tests of HIV infection in Japan are performed using Western blot (WB), but WB has several limitations including low sensitivity and cross-reactivity between HIV-1 and HIV-2 antibodies. To address these problems, a new HIV testing algorithm and a more reliable confirmation and HIV-1/2 differentiation assay are required.

Possible involvement of thyrotropin-releasing hormone receptor 3 in the release of prolactin in the metamorphosing bullfrog larvae.

In amphibians, thyrotropin (TSH), corticotropin (ACTH) and prolactin (PRL) are regarded as the major pituitary hormones involved in metamorphosis, their releasing factors being corticotropin-releasing factor (CRF), arginine vasotocin (AVT), and thyrotropin-releasing hormone (TRH), respectively. It is also known that thyrotropes and corticotropes are equipped with CRF type-2 receptor and AVT V1b receptor, respectively. As for PRL cells, information about the type of receptor for TRH (TRHR) through which the action of TRH is mediated to induce the release of PRL is lacking.

Molecular Cloning and Functional Characterization of Antimicrobial Peptides Brevinin-1ULf and Ulmin-1ULa in the Skin of the Newly Classified Ryukyu Brown Frog Rana ulma.

Antimicrobial peptides (AMPs) were previously isolated from the skin of the Ryukyu brown frog Rana okinavana. However, this species has recently been reclassified as two species, i.e.

Distance to retail stores and risk of being homebound among older adults in a city severely affected by the 2011 Great East Japan Earthquake.

Background: after the Great East Japan Earthquake in 2011, inactivity and the homebound status of older victims in affected areas have been a serious public health concern owing to the victims' prolonged existence as evacuees in mountainous areas.

Objective: to evaluate the association between distances to retail stores and risks of being homebound.

Design: secondary analysis of cross-sectional interview survey data with a geographical information analysis.

Bacterial toxin-inducible gene expression of cathelicidin-B1 in the chicken bursal lymphoma-derived cell line DT40: functional characterization of cathelicidin-B1.

Chicken cathelicidin-B1 (chCATH-B1) is a major host defense peptide of the chicken bursa of Fabricius (BF). To investigate the mechanisms of chCATH-B1 gene expression in the BF, we focused on the DT40 cell line derived from chicken bursal lymphoma as a model for analysis. A cDNA encoding chCATH-B1 precursor was cloned from DT40 cells.

Differential mode of antimicrobial actions of arginine-rich and lysine-rich histones against Gram-positive Staphylococcus aureus.

We previously reported the activities and modes of action of arginine (Arg)-rich histones H3 and H4 against Gram-negative bacteria. In the present study, we investigated the properties of the Arg-rich histones against Gram-positive bacteria in comparison with those of lysine (Lys)-rich histone H2B. In a standard microdilution assay, calf thymus histones H2B, H3, and H4 showed growth inhibitory activity against Staphylococcus aureus with minimum effective concentration values of 4.

Molecular cloning and multifunctional characterization of host defense peptides from the bullfrog Harderian gland with special reference to catesbeianalectin.

The Harderian gland (HG) is an orbital gland found in many terrestrial vertebrates that possess a nictitating membrane. Using reverse-transcription polymerase chain reaction (RT-PCR), we cloned five cDNAs encoding antimicrobial peptide (AMP)-homologs, catesbeianalectin, ranacyclin-CBa, ranatuerin-1CBa, ranatuerin-2CBa, and ranatuerin-2CBb, from the bullfrog HG total RNA. Of these, catesbeianalectin has not been thoroughly studied in terms of its biological activities.

Emergence in Japan of an HIV-1 variant associated with transmission among men who have sex with men (MSM) in China: first indication of the International Dissemination of the Chinese MSM lineage.

A survey of HIV-1 strains circulating in the Tokyo-Kanagawa metropolitan area of Japan during 2004 to 2011 (n = 477) identified six Japanese males (patients 1 to 6), who harbored viruses with genome segments derived from a distinct CRF01_AE variant uniquely found among men who have sex with men (MSM) in China (designated CN.MSM.01-1).

Antimicrobial properties of arginine- and lysine-rich histones and involvement of bacterial outer membrane protease T in their differential mode of actions.

There is growing evidence of the antimicrobial properties of histones and histone-derived peptides; however, most of them are specific to lysine (Lys)-rich histones (H1, H2A, and H2B). In the present study, we focused on arginine (Arg)-rich histones (H3 and H4) and investigated their antimicrobial properties in comparison with those of histone H2B. In a standard microdilution assay, calf thymus histones H2B, H3, and H4 showed growth inhibitory activity against the bacterial outer membrane protease T (OmpT) gene-expressing Escherichia coli strain JCM5491 with calculated 50% growth inhibitory concentrations of 3.

Molecular cloning and characterization of cDNAs encoding biosynthetic precursors for the antimicrobial peptides japonicin-1Ja, japonicin-2Ja, and temporin-1Ja in the Japanese brown frog, Rana japonica.

Using a combination of reverse-transcription polymerase chain reaction and the 5'- and/or 3'-rapid amplification of cDNA ends, we cloned, from a Japanese brown frog (Rana japonica) skin total RNA preparation, cDNAs encoding biosynthetic precursors for the antimicrobial peptides (AMPs) japonicin-1Ja (FFPIGVFCKIFKTC), japonicin-2Ja (FGLPMLSILPKALCILLKRKC), and temporin-1Ja (ILPLVGNLLNDLL.NH2). These peptides were previously isolated from an extract of R.

Expression of genes encoding antimicrobial peptides in the Harderian gland of the bullfrog Lithobates catesbeianus.

The Harderian gland is an orbital gland found in many tetrapod species that possess a nictitating membrane. While the main role of the Harderian gland is lubrication of the eyeballs, numerous other functions are attributed to this gland. In amphibians, mast cells have been detected in the Harderian gland, suggesting that the gland is involved in the host's system of innate immunity defending against microbial invasions.

Cloning and expression of genes enocoding antimicrobial peptides and bradykinin from the skin and brain of Oki Tago's brown frog, Rana tagoi okiensis.

Previous studies led to the isolation from skin extracts of Oki Tago's brown frog, Rana tagoi okiensis of five antimicrobial peptides belonging to the brevinin-1 (brevinin-1TOa), temporin (temporin-TOa and -TOb), and ranatuerin-2 (ranatuerin-2TOa and -2TOb) families, and bradykinin (BK) identical to mammalian BK. Using the reverse-transcription polymerase chain reaction (RT-PCR), we have now cloned from skin total RNA preparations cDNAs encoding biosynthetic precursors of brevinin-1TOa and brevinin-1TOb (containing the substitution Gly(1)-->Val), temporin-TOa and -TOb, and ranatuerin-2TOa and -2TOb. In addition, three cDNA clones encoding preprobradykinins were obtained that contained either one, two, or three tandem repeats of the sequence of BK followed by the sequence of [Thr(6)]-BK.

An efficient protocol for DNA amplification of multiple amphibian skin antimicrobial peptide cDNAs.

Antimicrobial peptides (AMPs) play an important role in the host's innate defence system in many organisms. Amphibian skin is expected to be a particularly rich source of novel AMPs. In amphibians, AMPs are produced from precursor proteins via specific cleavage by processing enzymes.

Evidence from the primary structures of dermal antimicrobial peptides that Rana tagoi okiensis and Rana tagoi tagoi (Ranidae) are not conspecific subspecies.

Morphological evidence and data from comparisons of nucleotide sequences of mitochondrial genes demonstrate considerable intraspecies variation among populations of the Japanese brown frog Rana tagoi Okada 1928 (Tago's brown frog). Five peptides with antimicrobial activity were isolated from an extract of the skins of specimens of Rana tagoi okiensis collected on the Oki Islands, Japan. Determination of their primary structures demonstrated that two peptides belong to the ranatuerin-2 family, two peptides to the temporin family, and one peptide to the brevinin-1 family.

Differential expression of genes encoding preprobrevinin-2, prepropalustrin-2, and preproranatuerin-2 in developing larvae and adult tissues of the mountain brown frog Rana ornativentris.

Previous studies led to the isolation of antimicrobial peptides (AMPs) of the brevinin-2, palustrin-2, and ranatuerin-2 families from skin extracts and/or skin secretions of the Japanese mountain brown frog, Rana ornativentris. In the present study, we cloned cDNAs encoding the precursors of brevinin-2Oc, palustrin-2Oa, and ranatuerin-2Ob and -2Oe from skin total RNA preparations from adult R. ornativentris and established a semi-quantitative RT-PCR system to measure the concentrations of these mRNAs.

Developmental and thyroid hormone-induced expression of preprotemporin genes in the skin of Japanese mountain brown frog Rana ornativentris.

Temporins are a group of small, highly hydrophobic, antimicrobial peptides widely distributed in the skin of frogs from the Ranidae family. In order to examine the mechanisms of regulation of temporin gene expression, we measured expression levels of preprotemporin mRNA in the skin of the Japanese mountain brown frog Rana ornativentris, using a semiquantitative RT-PCR system. Preprotemporin mRNAs were not detected in skin prior to the onset of metamorphosis but their levels increased markedly during metamorphosis, reaching a maximum at the stages of metamorphic climax, suggesting a correlation with thyroid hormone concentrations.

Demonstration of the proopiomelanocortin signaling system in the primary immune organ of the quail.

There is growing evidence to suggest that proopiomelanocortin (POMC), a precursor of adrenocorticotropin and alpha-melanocyte-stimulating hormone (alpha-MSH), also exists in extrapituitary organs, including immune organs. We investigated the presence of the POMC signaling system in the avian-specific primary immune organ the bursa of Fabricius (BF) of the adult quail. Immunohistochemical staining revealed the presence of cells showing immunopositive reaction with anti-alpha-MSH antibody in the quail specimens.

Dermal cytolytic peptides and the system of innate immunity in anurans.

Cationic peptides with the propensity to adopt an amphipathic alpha-helical conformation in a membrane-mimetic environment are synthesized in the skin of many species of frogs. These peptides frequently display potent cytolytic activities against a range of pathogenic bacteria and fungi, consistent with the hypothesis that they play a role in host defense. However, the importance of the peptides in the survival strategy of the animal is not clearly understood.

Quantitation of HIV-1 group M proviral DNA using TaqMan MGB real-time PCR.

The level of human immunodeficiency virus type 1 (HIV-1) proviral DNA is likely to be an important marker of the long-term effectiveness of highly active antiretroviral therapy. A new method was developed for quantifying HIV-1 group M proviral DNA using TaqMan real-time PCR, in which degenerate primers and an MGB probe were used to resolve the difference in amplification efficiencies among different subtypes. The present assay provided good linearity and accuracy in the range of 4-5000 copies of proviral DNA in 0.

Different hatching strategies in embryos of two species, pacific herring Clupea pallasii and Japanese anchovy Engraulis japonicus, that belong to the same order Clupeiformes, and their environmental adaptation.

Pacific herring Clupea pallasii and Japanese anchovy Engraulis japonicus, which belong to the same order Clupeiformes, spawn different types of eggs: demersal adherent eggs and pelagic eggs, respectively. We cloned three cDNAs for Pacific herring hatching enzyme and five for Japanese anchovy. Each of them was divided into two groups (group A and B) by phylogenetic analysis.