Immunohistochemical expressions of mGluR5, P2Y2 receptor,PLC-β1, and IP3R-I and -II in Merkel cells in rat sinus hair follicles

We previously found that Merkel cells (MCs) of the rat and monkey show a strong immunoreaction of the -subunit of Gq protein. The Gq-subunit isoform activates isozymes of phospholipase C- (PLC-), which produces inositol-(1,4,5)-triphosphate (IP3) which mobilizes intracellular Ca⁺⁺ from calcium stores via IP3 receptors. Glutamate and adenosine triphosphate (ATP), which are candidates for neurotransmitters in Merkel endings, are known to couple to Gq. Although MCs showed positive immunoreactions of metabotropic glutamate receptor 5 (mGluR5) in our preliminary study, these cells were not reactive to all antibodies to PLC- isozymes. We, therefore, reinvestigated immunohistochemical affinities to MCs of antibodies to PLC- isozymes and mGluRs using frozen sections of rat sinus hair follicles that were briefly postfixed in formaldehyde. We also studied the immunohistochemical expressions of P2Y receptors for ATP and IP3 receptor subtypes using similar sections. Merkel cells showed positive immunoreactions of PLC-1 and mGluR5. It was also found that MCs show positive immunoreactions of P2Y2, IP3R-I, and IP3R-II receptors. These results suggest that the Gq isoform in MCs couples to both the P2Y2 receptor and mGluR5 and regulates the intracellular Ca⁺⁺ concentration via the PLC-–IP3 cascade.     

Assessment of the efficacy of Japanese Bacillus thuringiensis isolates against the cigarette beetle,Lasioderma serricorne (Coleoptera: Anobiidae)

A total of 2,652 Japanese isolates of Bacillus thuringiensis, belonging to at least 54 H serogroups, were examined for assessment of the toxicity against the cigarette beetle, Lasioderma serricorne (Coleoptera: Anobiidae). When tested with spore/parasporal inclusion mixtures, strong larvicidal activities were associated with 28 isolates (1.1%). Serologically, these toxic isolates fell into 4 known H serovars: thuringiensis (9 isolates), kurstaki (2), kenyae (2), and darmstadiensis (15). Purified parasporal inclusions of the 10 selected isolates exhibited no larvicidal activity, while the supernatants of liquid cultures showed larvicidal and/or growth inhibitory effects. The activities were fully retained after heat treatment at 100 degrees C for 10 min. Overall results suggest that beta-exotoxin (or thuringiensin)-related substances are responsible for the toxicity of the present B. thuringiensis isolates against the cigarette beetle.     

Phenotypic characteristics of adipocytes generated from Meckel's chondrocytes in response to chick serum in vitro

When present in the culture medium, chick serum (CKS) modulated the phenotypic change from chondrocytes of Meckel's cartilage to adipocytes in vitro, as revealed by light and electron microscopy, the incorporation of BrdU, and immunocytochemistry. CKS inhibited DNA synthesis in chondrocytes and the proliferation of these cells, while it facilitated the differentiation to adipocytes. CKS contributed to phenotypic changes in undifferentiated chondrocytes, but did not affect the characteristics of differentiated chondrocytes. Electron microscopy revealed that the lipid droplets in adipocytes were enclosed by limiting membranes that fused to yield larger lipid droplets. Immunocytochemical staining of adipocytes with stage-specific antibodies revealed the presence of immunoreactive uncoupling protein (UCP-1) and peroxisome proliferator-activated receptor (PPARgamma) in immature adipocytes, and leptin and glucose transporter (Glut-4) in mature adipocytes. The adipocytes that were formed in the present study were multilocular adipocytes that contained many small lipid droplets, but in many ways they resembled white adipocytes. CKS contains a high level of estrogen, compared with fetal bovine serum, and it is possible that estrogen might have induced the differentiation to adipocytes.     

Molecular cloning of C4 gene and identification of the class III complement region in the shark MHC

To clarify the evolutionary origin of the linkage of the MHC class III complement genes with the MHC class I and II genes, we isolated C4 cDNA from the banded hound shark (Triakis scyllium). Upon phylogenetic tree analysis, shark C4 formed a well-supported cluster with C4 of higher vertebrates, indicating that the C3/C4 gene duplication predated the divergence of cartilaginous fish from the main line of vertebrate evolution. The deduced amino acid sequence predicted the typical C4 three-subunits chain structure, but without the histidine residue catalytic for the thioester bond, suggesting the human C4A-like specificity. The linkage analysis of the complement genes, one C4 and two factor B (Bf) genes, to the shark MHC was performed using 56 siblings from two typing panels of T. scyllium and Ginglymostoma cirratum. The C4 and one of two Bf genes showed a perfect cosegregation with the class I and II genes, whereas two recombinants were identified for the other Bf gene. These results indicate that the linkage between the complement C4 and Bf genes, as well as the linkage between these complement genes and the MHC class I and II genes were established before the emergence of cartilaginous fish >460 million years ago.     

Xenobiotic responsive element in the 5'-upstream region of the human P-450c gene.

The nucleotide sequence of the 5'-upstream region up to about -4.1 kb of the human P-450c gene was determined. Two kinds of repetitive sequences were located; one was the Alu sequence which was inserted at three positions (-3127 to -3038, -3017 to -2770, and -2167 to -1851), and the other was the SINE-R element located just upstream of the most distal Alu sequences. The region other than the two repeated sequences showed an overall similarity of 70% to that of the rat P-450c gene. Survey of XRE or its homologues, responsible for the inducible expression of the rat P-450c gene, revealed eight XRE core sequences in this region of the human P-450c gene. Three of them were carried in the Alu sequences. A fusion gene which was constructed by ligating the upstream region of the human P-450c gene to the chloramphenicol acetyltransferase (CAT) gene expressed the CAT activity in response to the inducer, methylcholanthrene, when transfected into Hepa-1 cells. Stepwise decrease in CAT activity in three regions was observed as the 5'-upstream sequence containing XRE motifs was removed. However, the XRE core sequence in the Alu sequences seemed inactive, because elimination of the three elements in the Alu sequences did not affect the expressed CAT activity. In accordance with this observation, competition experiments using gel mobility shift assay showed that XRE core sequences in the Alu sequences could not compete with the XRE sequence for the inducer-bound receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Environmental parameters and estivation of Rhyacodrilus (Tubificidae, Oligochaeta) in Lake Suwa, Japan

We confirmed that Rhyacodrilus spp of Yasuda and Okino (1987) (Tubificidae, Oligochaeta) migrated entirely downward below 15 cm into the sediment from June to September m Lake Suwa, a shallow eutrophic lake in Japan In October, Rhyacodrilus spp began to return to the upper layer and immediately attained sexual maturity They tended to show reduced body weight and respiratory activity during summer The results indicate that an oligochaete of Rhyacodrilus spp estivated in the deeper layer during summer The estivation ostensibly occurred at 15°C and the species died off at 20°C, so it is evident that they must migrate from the surface to the deeper layer of sediment during summer in order to escape high temperature Furthermore, physiological experiment suggested that this behavior is accelerated by low oxygen concentration with high temperature     

Association of a 66 kDa homolog of Chlamydomonas DC2, a subunit of the outer arm docking complex, with outer arm dynein of sperm flagella in the ascidian Ciona intestinalis

We previously identified a 66 kDa axonemal protein (Ci-Axp66.0) in sperm of the ascidian Ciona intestinalis. Here we found that Ci-Axp66.0 shows sequence similarity to the DC2 subunit of the Chlamydomonas outer arm docking complex. Analysis of secondary structure of Ci-Axp66.0 suggested that the N-terminal two-thirds of the molecule is rich in coiled coil structure, as in Chlamydomonas DC2. Immunogold localization revealed that it is located in the vicinity of outer arm dynein. Ci-Axp66.0 was partly extracted from the axonemes by a high salt solution and co-purified with outer arm dynein. This co-purification was not affected by the absence of Mg(2+) in isolation buffer, indicating that Ci-Axp66.0 is associated with outer arm dynein. These results suggest that Ci-Axp66.0 is a component of the outer arm dynein docking complex in the axonemes of Ciona sperm.     

Anti-V3 humanized antibody KD-247 effectively suppresses ex vivo generation of human immunodeficiency virus type 1 and affords sterile protection of monkeys against a heterologous simian/human immunodeficiency virus infection

In an accompanying report (Y. Eda, M. Takizawa, T. Murakami, H. Maeda, K. Kimachi, H. Yonemura, S. Koyanagi, K. Shiosaki, H. Higuchi, K. Makizumi, T. Nakashima, K. Osatomi, S. Tokiyoshi, S. Matsushita, N. Yamamoto, and M. Honda, J. Virol. 80:5552-5562, 2006), we discuss our production of a high-affinity humanized monoclonal antibody, KD-247, by sequential immunization with V3 peptides derived from human immunodeficiency virus type 1 (HIV-1) clade B primary isolates. Epitope mapping revealed that KD-247 recognized the Pro-Gly-Arg V3 tip sequence conserved in HIV-1 clade B isolates. In this study, we further demonstrate that in vitro, KD-247 efficiently neutralizes CXCR4- and CCR5-tropic primary HIV-1 clade B and clade B' with matching neutralization sequence motifs but does not neutralize sequence-mismatched clade B and clade E isolates. Monkeys were provided sterile protection against heterologous simian/human immunodeficiency virus challenge by the passive transfer of a single high dose (45 mg per kg of body weight) of KD-247 and afforded partial protection by lower antibody doses (30 and 15 mg per kg). Protective neutralization endpoint titers in plasma at the time of virus challenge were 1:160 in animals passively transferred with a high dose of the antibody. The antiviral efficacy of the antibody was further confirmed by its suppression of the ex vivo generation of primary HIV-1 quasispecies in peripheral blood mononuclear cell cultures from HIV-infected individuals. Therefore, KD-247 promises to be a valuable tool not only as a passive immunization antibody for the prevention of HIV infection but also as an immunotherapy for the suppression of HIV in phenotype-matched HIV-infected individuals.