Effects of Isolation by Continental Islands in the Seto Inland Sea, Japan, on Genetic Diversity of the Large Japanese Field Mouse, Apodemus speciosus (Rodentia: Muridae), Inferred from the Mitochondrial Dloop Region.

To study the effects of post-glacial isolation by islands on population genetic diversity and differentiation of the large Japanese field mouse, Apodemus speciosus, we examined partial nucleotide sequences of the mitochondrial Dloop region (ca. 300 bp) in 231 individuals collected from islands in the Seto Inland Sea and adjacent regions on Honshu and Shikoku Islands in the western part of the Japanese archipelago. Molecular phylogenetic and network analyses showed that haplotypes in each island tended to form monophyletic groups, while those in Honshu and Shikoku (the major Japanese islands) showed scattered relationships and were connected with island haplotypes. These observations suggest that a set of Honshu and Shikoku haplotypes became the ancestral lineages of the island population. No gene flow was detected among island populations, indicating that independent evolution occurred on each island, without the influence of human activities, since the establishment of the islands in the Holocene. Population genetic diversities on each island were lower than those on Honshu and Shikoku. Comparison between genetic diversity and island area size showed positive correlations and supported the suggestion that genetic drift is a major factor that shaped the current haplotype constitution of the islands in the Seto Inland Sea.

Gadolinium causes M1 and M2 microglial apoptosis after intracerebral haemorrhage and exerts acute neuroprotective effects.

OBJECTIVES: Gadolinium (Gd) affects microglial polarization during remyelination. We previously reported that the suppression of proinflammatory microglia was neuroprotective in intracerebral haemorrhage (ICH). The objective of the present study was to investigate the effects of Gd on microglial polarization and neuronal injury after ICH. METHODS: Gadolinium was intraperitoneally administered to ICH mice prepared by an intrastriatal microinjection of collagenase type VII. The polarization of M1, 2a, b and c microglia was evaluated by real-time PCR using the respective markers. Changes in representative mRNAs were also confirmed by immunological methods. Neuroprotective effects were evaluated by counting NeuN-positive cells and a behavioural analysis. KEY FINDINGS: One day after ICH, the mRNA levels of proinflammatory M1 microglial markers, such as inducible nitric oxide synthase (iNOS), and anti-inflammatory M2 microglial markers, such as arginase1 (M2a, c), Ym1 (M2a), and transforming growth factor-ß (M2c), increased, while those of chemokine CCL1 (M2b) only increased after 3 days. Gd decreased the levels of all M1 and M2 markers. Arginase1 and iNOS protein levels also increased, and Gd reduced them due to apoptotic cell death. Gadolinium attenuated oedema, neuron loss, neurological deficits and the mortality rate without affecting haematoma sizes. CONCLUSIONS: Gadolinium induced M1 and M2 microglial apoptosis and exerted acute neuroprotective effects after ICH.

Deciphering and dating the red panda's ancestry and early adaptive radiation of Musteloidea.

Few species have been of more disputed affinities than the red or lesser panda (Ailurus fulgens), an endangered endemic Southeast Asian vegetarian member of the placental mammalian order Carnivora. This peculiar carnivoran has mostly been classified with raccoons (Procyonidae) or bears (Ursidae), grouped with the giant panda (Ailuropoda melanoleuca) in their own family, or considered a separate lineage of equivocal ancestry. Recent molecular studies have indicated a close affinity of the red panda to a clade of procyonids and mustelids (weasels, otters, martens, badgers, and allies), but have failed to unambiguously resolve the position of this species relative to mephitids (skunks and stink badgers). We examined the relationship of the red panda to other extant species of the carnivoran suborder Caniformia using a set of concatenated approximately 5.5-kb sequences from protein-coding exons of five nuclear genes. Bayesian, maximum likelihood, and parsimony phylogenetic analyses strongly supported the red panda as the closest living relative of a clade containing Procyonidae and Mustelidae to the exclusion of Mephitidae. These three families together with the red panda (which is classified here as a single extant species of a distinct family, Ailuridae) compose the superfamily Musteloidea, a clade strongly supported by all our phylogenetic analyses as sister to the monophyletic Pinnipedia (seals, sea lions, walruses). The approximately unbiased, Kishino-Hasegawa, and Templeton topology tests rejected (P<0.05) each of all possible alternative hypotheses about the relationships among the red panda and mephitids, procyonids, and mustelids. We also estimated divergence times for the red panda's lineage and ones of other caniform taxa, as well as the ages of the first appearance datums for the crown and total clades of musteloids and the total clades of the red panda, mephitids, procyonids, and mustelids. Bayesian relaxed molecular-clock analysis using combined information from all sampled genes yielded a approximately 42-Myr timescale to caniform evolution and provided evidence of five periods of increased diversification. The red panda's lineage and those of other extant musteloid families are estimated to have diverged during a 3-Myr interval from the mid-Early Oligocene to near the Early/Late Oligocene boundary. We present fossil evidence that extends the early adaptive radiation of the total clade of musteloids to the Eocene-Oligocene transition and also suggests Asia as a center of this radiation.

Large-scale identification by shotgun proteomics of proteins expressed in porcine liver and salivary gland.

Protein catalogs containing a large number of proteins expressed in a variety of organs can be powerful tools for stem-cell research, because this requires accurate knowledge about how cells differentiate. Salivary gland progenitor (SGP) cells are somatic stem cells isolated from the salivary gland that can differentiate into hepatic or pancreatic cell lineages. Their differentiation state has been assessed by the expression of major protein markers, but to use these cells in regenerative medicine, it will be necessary to establish additional means of quality assessment. We examined the use of shotgun proteomics for porcine salivary gland (a source of SGP cells) and liver (a destination of differentiated SGP cells) for determining the state of SGP cell differentiation. Protein complexes from each organ were digested into peptides and separated by two-dimensional liquid chromatography involving strong cation-exchange chromatography followed by reversed-phase liquid chromatography. The separated peptides were analyzed by on-line electrospray ionization tandem mass spectrometry using a quadrupole-time of flight mass spectrometer (ESI Q-TOF MS/MS), and the spectra obtained were processed to search peptides against a mammalian database for protein identification. Using this method, we identified 117 proteins in porcine salivary gland and 154 proteins in porcine liver. Of these, 72 and 109 were specific to salivary gland and liver, respectively, and some of these were previously shown to be organ specific. The current study can be utilized in the future as a basis to study the pattern of differentiation in protein expression by stem cells.

Further evidence for recombination between mouse hemoglobin beta b1 and b2 genes based on the nucleotide sequences of intron, UTR, and intergenic spacer regions.

Nucleotide sequences of the intron regions and UTRs (Untranslated regions) of the hemoglobin beta adult genes, b1 and b2, and of the intergenic spacer region were determined for mouse strains representing the d, p, and w1 hemoglobin haplotypes defined by protein electrophoretic analyses. The hypothesis of recombination of the b1 and b2 genes between the d and w1 haplotypes previously reported in the cDNA nucleotide sequences was confirmed by neighbor-joining analyses of the intron regions and UTRs within the b1 and b2 genes, suggesting that all of the structures of hemoglobin beta adult genes support the hypothesis that the p haplotype was established by hybridization between d and w1 haplotype mice. The resultant recombinant of the p haplotype was found to have a d-like b1 gene and a w1-like b2 gene. In addition to the possible recombination, a break point was suggested around 2-3 kb downstream of the b1 gene within the intergenic spacer region, despite the absence of clear properties that could stimulate the recombination machinery. Some large insertions or deletions (indels) specific to the p or d haplotypes were located within the intergenic spacer region, in which the 1010-bp indel specific to the p haplotype was shared by all examined strains representing the p haplotype.

Evidence from nuclear DNA sequences sheds light on the phylogenetic relationships of Pinnipedia: single origin with affinity to Musteloidea.

Considerable long-standing controversy and confusion surround the phylogenetic affinities of pinnipeds, the largely marine group of "fin-footed" members of the placental mammalian order Carnivora. Until most recently, the two major competing hypotheses were that the pinnipeds have a single (monophyletic) origin from a bear-like ancestor, or that they have a dual (diphyletic) origin, with sea lions (Otariidae) derived from a bear-like ancestor, and seals (Phocidae) derived from an otter-, mustelid-, or musteloid-like ancestor. We examined phylogenetic relationships among 29 species of arctoid carnivorans using a concatenated sequence of 3228 bp from three nuclear loci (apolipoprotein B, APOB; interphotoreceptor retinoid-binding protein, IRBP; recombination-activating gene 1, RAG1). The species represented Pinnipedia (Otariidae: Callorhinus, Eumetopias; Phocidae: Phoca), bears (Ursidae: Ursus, Melursus), and Musteloidea (Mustelidae: Mustela, Enhydra, Melogale, Martes, Gulo, Meles; Procyonidae: Procyon; Ailuridae: Ailurus; Mephitidae: Mephitis). Maximum parsimony, maximum likelihood, and Bayesian inference phylogenetic analyses of separate and combined datasets produced trees with largely congruent topologies. The analyses of the combined dataset resulted in well-resolved and well-supported phylogeny reconstructions. Evidence from nuclear DNA evolution presented here contradicts the two major hypotheses of pinniped relationships and strongly suggests a single origin of the pinnipeds from an arctoid ancestor shared with Musteloidea to the exclusion of Ursidae.

Plasmacytoid dendritic cells: from specific surface markers to specific cellular functions.

We have recently described a panel of monoclonal antibodies (mAb), that recognize two novel leukocyte surface antigens, BDCA-2 and BDCA-4. BDCA-2 is a novel type II C-type lectin specifically expressed by plasmacytoid dendritic cells (PDCs) that can internalize antigen for presentation to T cells. Furthermore, signaling via BDCA-2 may play a role in switching from interferon (IFN)-alpha/beta-controlled to interleukin (IL)-12-controlled immune response pathways, as triggering of BDCA-2 potently inhibits secretion of IFN-alpha/beta by PDCs and thereby promotes IL-12 p70 production in PDCs and other cells. Viruses may exploit this switch to escape innate antiviral immunity, but it may be beneficial for patients with systemic lupus erythematosus (SLE) if induced, for instance by anti BDCA-2 mAb treatment. BDCA-4 is shown here to be identical to neuropilin-1 (NP-1), a neuronal receptor for the axon guidance factors belonging to the class-3 semaphorin subfamily, and a receptor on endothelial and tumor cells for vascular endothelial growth factor (VEGF-A). In blood and bone marrow, BDCA-4/NP-1 is exclusively expressed on PDCs, but in tonsils also on a few other cells, primarily follicular B helper memory T cells (T(FH)).

Germline-specific Antigens Identified by Monoclonal Antibodies in the Nematode Caenorhabditis elegans1 : (Caenorhabditis elegans/monoclonal antibody/cell-specific antigen/germinal plasm/asymmetry).

Of 27 monoclonal antibodies identified to react, by indirect immunofluorescent antibody staining, with specific cells and tissues of the nematode Caenorhabditis elegans, we report here three monoclonal antibodies pertaining to the gonadal tissues. One antibody defines an antigen that is distributed over the entire embryo at earlier development and later becomes unique to the gonad, including mature oocytes. The antigens recognized by the other two are distributed asymmetrically in the posterior region of the fertilized egg's cytoplasm destined to become the germline precursor cell. Each antigen is successively segregated only to the germline precursor cells of the developing embryo and, postembryonically, is uniquely localized around the germline cell nuclei of the larvae and adults.

Response-guided therapy for patients with chronic hepatitis who have high viral loads of hepatitis C virus genotype 2.

AIM: We evaluated the efficacy of response-guided therapy in patients with hepatitis C virus (HCV) genotype 2. METHODS: We studied 105 patients with an HCV genotype 2 load of higher than 5.0 Log IU/mL who received more than 75% of the target dose of pegylated interferon plus ribavirin. Among patients with rapid viral response (RVR; no HCV RNA detected at week 4), 14 selected 16 weeks of therapy (group A), and 28 selected 24 weeks of therapy (group B). Among non-RVR patients, 40 selected 24 weeks of therapy (group C), and 19 selected 48 weeks of therapy (group D). RESULTS: All patients in group A and B achieved a sustained viral response (SVR). Clinical characteristics did not differ significantly between groups C and D. However, the proportion of patients in whom HCV RNA disappeared at a later week after starting treatment was higher in group D (P = 0.0578). SVR rate was 73% in C, and 79% in D. Among patients in whom HCV RNA disappeared between weeks 5 and 8, SVR was achieved in 28 (82%) of 34 patients in C and 10 (91%) of 11 patients in D. Among patients whose HCV RNA disappeared between weeks 9 and 12, SVR was achieved in one (20%) of five patients in C and five (63%) of eight patients in D (not statistically significant). CONCLUSIONS: 16 weeks of combination therapy could achieve an adequate antiviral effect for RVR patients. Extending therapy could not significantly improve SVR rate in non-RVR patients.

High prevalence of hepatitis C virus infection in Airin district, Osaka, Japan: A hospital-based study of 1162 patients.

AIM: The Airin district, located in Nishinari-ku, Osaka, is known as Japan's largest slum area, and has the largest concentration of day laborers in the country. We conducted a large hospital-based study to determine the prevalence of hepatitis C virus (HCV) infection in the district. METHODS: The subjects were 1162 men (mean age, 57 ± 9 years) admitted to the Osaka Socio-Medical Center Hospital between April 2005 and March 2008. Their case records were retrospectively reviewed. RESULTS: Anti-HCV antibodies were found in 218 (18.8%) patients; in contrast, only 24 (2.1%) patients had hepatitis B surface antigen. The prevalence of anti-HCV antibodies was 59% among the 122 patients admitted for liver diseases and 14% among the 1040 patients with other diseases. Among 927 patients with normal alanine aminotransferase levels (≤40 IU/L), 128 (13.8%) had anti-HCV antibodies. The prevalence of anti-HCV antibodies increased with age significantly (P < 0.001). At least 33 of the 218 (15%) patients with anti-HCV antibodies admitted to having a history of injection drug use. Interferon therapy was initiated in 26 patients (11 with genotype 1, 14 with genotype 2 and one unclassifiable), but only six completed their scheduled regimens. Hepatocellular carcinoma was diagnosed in 20 patients, but only seven had early-stage disease in which curative treatment, such as surgical hepatectomy or percutaneous ablation, was indicated. CONCLUSION: The prevalence of HCV infection in the Airin district is extremely higher than that in the Japanese general population. Patient education and strict action against illegal drug use are indispensable to prevent the spread of HCV infection from the district.

Discovery of a new HBB haplotype w2 in a wild-derived house mouse, Mus musculus.

Genetic characterization of a wild-derived house mouse, Mus musculus, originally collected near Lake Balkhash in the Republic of Kazakhstan, was performed by examining protein polymorphisms and nucleotide sequences for the hemoglobin beta chain (HBB) subunits. Protein electrophoresis, which was performed on a cellulose-acetate plate, showed an independent mobility pattern representing a new, previously undiscovered haplotype. Neighbor-joining analyses of the HBB adult genes, i.e., HBB-T1 and HBB-T2, and the intergenic spacer region showed that the Lake Balkhash mouse possessed genomic components that were mixed from different haplotypes. Compared to the previously determined HBB haplotypes, d, p, and w1, the HBB-T1 gene and ca. 11 kb of the spacer region were most similar to the w1 haplotype; however, the remainder of the spacer region and the HBB-T2 gene were most similar to the d haplotype but may represent a still uncharacterized and divergent haplotype. The recombination event is predicted to have occurred 2.5 kb upstream of the HBB-T2 gene and may have occurred through intersubspecific hybridization between mice of the musculus subspecies group (with the w1 haplotype) and the castaneus subspecies group (with the d-like haplotype). Alternatively, an unknown subspecies group that is equidistantly divergent from each of these subspecies groups may have been involved. Our findings suggest reticulate evolution among the subspecies groups during the evolution of M. musculus.

Cell cycle control by daf-21/Hsp90 at the first meiotic prophase/metaphase boundary during oogenesis in Caenorhabditis elegans.

DAF-21, a Caenorhabditis elegans homologue of Hsp90, is expressed primarily in germline cells. Although mutations in the daf-21 gene affect animal fertility, its cellular roles have remained elusive. To phenocopy daf-21 mutations, we impaired the daf-21 function by RNA interference (RNAi), and found that oocytes skipped the diakinesis arrest and displayed a defective diakinesis arrest, which led to the production of endomitotic oocytes with polyploid chromosomes (Emo phenotype). The same Emo phenotype was also observed with RNAi against wee-1.3. To identify a cause for Emo, we examined the CDK-1 (Cdc2) phosphorylation status in Emo animals, since CDK-1 is a key regulator of the prophase/metaphase transition and is kept inactivated by WEE-1.3 kinase during prophase. We immunostained both daf-21(RNAi) and wee-1.3(RNAi) animals with anti-phosphorylated-CDK-1 antibody and observed no detectable phosphates on CDK-1 in either of the animals. We also examined WEE-1.3 expression in daf-21(RNAi) and found a significant reduction of WEE-1.3. These results indicate that CDK-1 was not phosphorylated in either daf-21(RNAi) or wee-1.3(RNAi) animals, and suggest that daf-21 was necessary for producing functional WEE-1.3. Thus, all together, we propose that DAF-21 indirectly regulates the meiotic prophase/metaphase transition during oocyte development by ensuring the normal function of WEE-1.3.

CD52 is a novel costimulatory molecule for induction of CD4+ regulatory T cells.

We previously reported that 4C8 monoclonal antibody (mAb) provides a costimulatory signal to human CD4+ T cells and consequently induces regulatory T (Treg) cells, which are hypo-responsive and suppress the polyclonal response of bystander CD4+ cells in a contact-dependent manner. In this study, we identified the antigen of 4C8 mAb as CD52. Costimulation with Campath-1H, a humanized anti-CD52 mAb, also induced Treg cells. Anti-CD52-induced Treg cells suppressed the proliferation of both CD4+ and CD8+ T cells provided with polyclonal or allogeneic stimulation. When Treg cells were induced from Staphylococcal enterotoxin B (SEB) treated cells, they suppressed the response to SEB more efficiently than that to another superantigen, SEA. Furthermore, anti-CD52-induced Treg cells could be expanded by culture with IL-2 followed by CD52-costimulation, and co-injection of expanded Treg cells suppressed lethal xenogeneic graft versus host disease (GvHD) reactions in SCID mice caused by human peripheral blood mononuclear cells (PBMCs).

Signal regulatory protein molecules are differentially expressed by CD8- dendritic cells.

A normalized subtracted gene expression library was generated from freshly isolated mouse dendritic cells (DC) of all subtypes, then used to construct cDNA microarrays. The gene expression profiles of the three splenic conventional DC (cDC) subsets were compared by microarray hybridization and two genes encoding signal regulatory protein beta (Sirpbeta1 and Sirpbeta4) molecules were identified as differentially expressed in CD8(-) cDC. Genomic sequence analysis revealed a third Sirpbeta member localized in the same gene cluster. These Sirpbeta genes encode cell surface molecules containing extracellular Ig domains and short intracytoplasmic domains that have a charged amino acid in the transmembrane region which can potentially interact with ITAM-bearing molecules to mediate signaling. Indeed, we demonstrated interactions between Sirpbeta1 and beta2 with the ITAM-bearing signaling molecule Dap12. Real-time PCR analysis showed that all three Sirpbeta genes were expressed by CD8(-) cDC, but not by CD8(+) cDC or plasmacytoid pre-DC. The related Sirpalpha gene showed a similar expression profile on cDC subtypes but was also expressed by plasmacytoid pre-DC. The differential expression of Sirpalpha and Sirpbeta1 molecules on DC was confirmed by staining with mAbs, including a new mAb recognizing Sirpbeta1. Cross-linking of Sirpbeta1 on DC resulted in a reduction in phagocytosis of Leishmania major parasites, but did not affect phagocytosis of latex beads, perhaps indicating that the regulation of phagocytosis by Sirpbeta1 is a ligand-dependent interaction. Thus, we postulate that the differential expression of these molecules may confer the ability to regulate the phagocytosis of particular ligands to CD8(-) cDC.

Plasma membrane-focused proteomics: dramatic changes in surface expression during the maturation of human dendritic cells.

The differential expression of surface molecules on dendritic cells (DC) reflects their functional differences as immature and mature subsets. It is difficult, however, to characterize differences in surface expression by standard proteomic approaches, due mainly to the hydrophobic nature and low abundance of the individual proteins in question. We have established a method for obtaining high-yield plasmalemma preparations which contain surface molecules enriched more than 200-fold by coating cells with beads conjugated with antibody against a cell type-specific cell-surface molecule, followed by nitrogen cavitated disruption, magnetic separation, and density gradient ultracentrifugation. We identified and quantified 339 human monocyte-derived DC transmembrane proteins, including 33 previously uncharacterized molecules. Whereas 106 proteins were selectively expressed in immature cells or down-regulated after maturation, 191 proteins were selectively expressed in mature cells or up-regulated after maturation.

Primordial germ cells originate from the endodermal strand cells in the ascidian Ciona intestinalis.

The origin of germ cells in the ascidian is still unknown. Previously, we cloned a vasa homologue (CiVH) of Ciona intestinalis from the cDNA library of ovarian tissue by polymerase chain reaction and showed that its expression was specific to germ cells in adult and juvenile gonads. In the present study, we prepared a monoclonal antibody against CiVH protein and traced the staining for this antibody from the middle tailbud stage to young adulthood. Results showed that positive cells are present in the endodermal strand in middle tailbud embryos and larvae. When the larval tail was absorbed into the trunk during metamorphosis, the CiVH-positive cells migrated from the debris of the tail into the developing gonad rudiment, and appeared to give rise to a primordial germ cell (PGC) in the young juvenile. The testis rudiment separated from the gonad rudiment, the remainder of which differentiated into the ovary. PGCs of the testis rudiment and the ovary rudiment differentiated into spermatogenic and oogenic cells, respectively. When the larval tail containing the antibody-positive cells was removed, the juveniles did not contain any CiVH-positive cells after metamorphosis, indicating that the PGCs in the juvenile originated from part of the larval tail. However, even in such juveniles, positive cells newly appeared in the gonad rudiment at a later stage. This observation suggests that a compensatory mechanism regulates germline formation in C. intestinalis.

Control characteristics of an automatic matching control system for in vivo EPR spectroscopy.

A method for estimating the characteristics of an automatic matching control (AMC) system for in vivo electron paramagnetic resonance (EPR) spectroscopy is described. AMC is also known as automatic coupling control (ACC) and is used to compensate for perturbation due to movement of the subject animal. The open-loop function of the AMC system is derived and the characteristics (gain, phase margin, steady-state error, system bandwidth, and disturbance rejection) of the system are examined. The proposed method is supported by experimental findings. The frequency response of perturbation due to the movement of an anesthetized rat was measured with a 1.1-GHz continuous-wave EPR spectrometer. Disturbance rejection with the AMC system was also considered for respiratory motion and the heartbeat of the anesthetized rat, and these perturbations were reduced by about 40 dB in the power spectrum of the output of the diode detector in the spectrometer.

Caenorhabditis elegans DAF-21 (HSP90) is characteristically and predominantly expressed in germline cells: spatial and temporal analysis.

Three monoclonal antibodies against antigens that exist in the Caenorhabditis elegans germ line have previously been described. In the present study, a full-length mRNA for one of these antigens was isolated, and by sequencing its corresponding cDNA, it was predicted that the protein would show a high homology with the 90 kDa heat shock protein (HSP90) in other species, and with the protein of daf-21, a previously identified hsp90 homologue. The spatial and temporal distribution of the antigen (DAF-21) was analyzed in C. elegans, and the localization of daf-21 mRNA, as detected by in situ hybridization, agreed with that detected by the monoclonal antibody. Under normal conditions, daf-21 mRNA is characteristically distributed in postembryonic germ cells derived from Z2 and Z3 cells in both hermaphrodites and males. Under heat stress conditions, however, daf-21 mRNA was not only detected in germ cells, but also apparently expressed all over the body. In addition, the DAF-21 protein seemed to be localized in the perinuclear region of somatic cells.