Changes in responses of UVB irradiated skin of brownish guinea pigs with aging

It is known that skin often shows irregular pigmentation during aging, which is frequently associated with hyperpigmentation. Many studies have utilized brownish A1 guinea pigs to investigate the pathogenesis of ultraviolet B (UVB)-induced skin pigmentation, however, responses associated with aging following UVB irradiation have not been elucidated. To characterize those responses, dorsal skin of A1 guinea pigs from 14-weeks to 5-yr old were investigated. The minimal erythema dose was found to increase with aging. Further, in pigmentation induced by UVB radiation, skin brightness (DeltaL*-value) decreased equally in both the 14-week old (young) group and in the 3-yr old (old) group of guinea pigs. The DeltaL*-value recovered in the young group from 21 d after UVB irradiation, whereas no such recovery was seen in the old group. In addition, the amount of melanin and the number of melanocytes returned near pre-irradiation levels in the young group, while they remained high in the old group. Our results therefore demonstrate for the first time that skin responses following UVB irradiation change with aging in A1 guinea pigs.     

GObar: A Gene Ontology based analysis and visualization tool for gene sets

Background  

Microarray experiments, as well as other genomic analyses, often result in large gene sets containing up to several hundred genes. The biological significance of such sets of genes is, usually, not readily apparent.     

Osteoclast‐specific Dicer gene deficiency suppresses osteoclastic bone resorption

Osteoclasts are unique cells that resorb bone, and are involved in not only bone remodeling but also pathological bone loss such as osteoporosis and rheumatoid arthritis. The regulation of osteoclasts is based on a number of molecules but full details of these molecules have not yet been understood. MicroRNAs are produced by Dicer cleavage an emerging regulatory system for cell and tissue function. Here, we examine the effects of Dicer deficiency in osteoclasts on osteoclastic activity and bone mass in vivo. We specifically knocked out Dicer in osteoclasts by crossing Dicer flox mice with cathepsin K‐Cre knock‐in mice. Dicer deficiency in osteoclasts decreased the number of osteoclasts (N.Oc/BS) and osteoclast surface (Oc.S/BS) in vivo. Intrinsically, Dicer deficiency in osteoclasts suppressed the levels of TRAP positive multinucleated cell development in culture and also reduced NFATc1 and TRAP gene expression. MicroRNA analysis indicated that expression of miR‐155 was suppressed by RANKL treatment in Dicer deficient cells. Dicer deficiency in osteoclasts suppressed osteoblastic activity in vivo including mineral apposition rate (MAR) and bone formation rate (BFR) and also suppressed expression of genes encoding type I collagen, osteocalcin, Runx2, and Efnb2 in vivo. Dicer deficiency in osteoclasts increased the levels of bone mass indicating that the Dicer deficiency‐induced osteoclastic suppression was dominant over Dicer deficiency‐induced osteoblastic suppression. On the other hand, conditional Dicer deletion in osteoblasts by using 2.3 kb type I collagen‐Cre did not affect bone mass. These results indicate that Dicer in osteoclasts controls activity of bone resorption in vivo. J. Cell. Biochem. 109: 866–875, 2010. © 2009 Wiley‐Liss, Inc.     

Isolation of protoplast from aerial mycelia and fruit-body formation from regenerated mycelia in Pleurotus cornucopiae

We have developed a simple and efficient method for protoplast isolation from Pleurotus cornucopiae. Protoplasts were isolated from aerial mycelia cultured on potato dextrose agar medium without time-consuming propagation in liquid culture. Protoplast yield was significantly increased by means of a decompressing pretreatment of mycelia in enzyme solution and a subsequent enzyme reaction with vibrational mixing. The isolated protoplasts regenerated mycelia and these mycelia formed fruit-bodies without any morphological abnormalities.     

Allatotropin-like peptide in Heliothis virescens: tissue localization and quantification

The mating-induced increase in juvenile hormone (JH) biosynthesis in Heliothis virescens females may be stimulated by production and/or release of stimulatory neuropeptides such as allatotropins (AT). Although there is evidence that H. virescens allatotropin may be structurally related to Manduca sexta allatotropin (Manse-AT), little is known of its occurrence and distribution in H. virescens. An enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody against Manse-AT was used to quantify concentrations of Manse-AT immunoreactivity in tissue extracts of H. virescens. In mated females, the highest concentrations of Manse-AT-like material occurred in the brain. The ventral nervous system and the accessory glands also contained considerable amounts of Manse-AT-like material, whereas concentrations were very low in ovaries, fat body, and flight muscle. The Manse-AT antibody was used for whole-mount immunocytochemistry to localize Manse-AT-immunoreactivity in the central nervous system. Several groups of Manse-AT-immunoreactive cells were discovered in the brain, subesophageal ganglion, and thoracic and abdominal ganglia of H. virescens females and males. Strong immunoreactivity was detected in axons going through the corpora cardiaca and branching out over the surface of the corpora allata. The presence of Manse-AT-like material in various locations in the central nervous system suggests that these peptides may have other as yet unknown functions. At the posterior margin of the terminal ganglion of males, a group of large immunoreactive cells was observed that was not present in females. Other than that, there were no obvious differences between virgin and mated females or males. The lack of differences in AT distribution in mated and virgin females suggests that mating-induced differences in female JH biosynthesis rates may be caused by changes in cellular response to AT at the level of the CA, rather than by changes in the amounts of AT acting on the CA.     

Protruding disordered loop of gC1qR is specifically exposed and related to antiapoptotic property in germ cell lineage

We established a monoclonal antibody (MAb), 5G9, with the use of a fixed seminoma tissue from an archival paraffin-embedded specimen, as an immunogen. Without antigen retrieval, positive 5G9-immunohistochemical staining was confined mostly to primordial germ cells, spermatogonia and various germ cell tumors. 5G9 recognized a mitochondrial 32-kD protein with an isoelectric point of pH 4.2, identified as a multifunctional ubiquitous protein, receptor for globular head of C1q (gC1qR), whose epitope was mapped in a disordered loop connecting the β3 and the β4 strands. Reflecting the ubiquitous distribution of gC1qR, with antigen retrieval, 5G9 was found reactive to a wide range of normal and tumor tissues. Since several co-precipitated and phosphorylated bands were observed in various human cell lines but not in germ cell tumor cell lines by in vitro phosphorylation assay, we speculate that the epitope of gC1qR is specifically unmasked in the germ cell lineage. By reducing gC1qR by siRNA, a significant increase was observed in the number of apoptotic cells in ITO-II and TCam-2 cell lines, but to a lesser extent in the Colo201 colon cancer cell line, showing an antiapoptotic property of gC1qR in the germ cells. Since protein–protein interaction is partially preserved by fixation, archival paraffin-embedded specimens can be a valuable source of immunogens for generating monoclonal antibodies (MAbs) that recognize tissue-specific protein conformation.     

Role of VAMP-2, VAMP-7, and VAMP-8 in constitutive exocytosis from HSY cells

We evaluated the role of VAMP-2/synaptobrevin, VAMP-7/TI-VAMP, and VAMP-8/endobrevin in exocytic pathways of HSY cells, a human parotid epithelial cell line, by coexpressing these VAMP proteins tagged with green fluorescent protein (GFP) and human growth hormone (hGH) as a secretory cargo. Exocytosis of hGH was constitutive and the fluorescent signal of hGH–GFP was observed in the Golgi area and small vesicles quickly moving throughout the cytoplasm. The cytoplasmic vesicles containing hGH overlapped well with VAMP-7-GFP, but did so scarcely with VAMP-2-GFP or VAMP-8-GFP. However, when the vesicle transport from the trans-Golgi network to the plasma membrane was arrested by incubation at 20°C for 2 h and then released by warming up to 37°C; VAMP-2-GFP and hGH were clearly colocalized together in small cytoplasmic vesicles. Neither VAMP-7-GFP nor hGH–GFP was colocalized with LAMP-1, a marker for lysosomes and late endosomes. These results suggest that (1) VAMP-2 can be one of the v-SNAREs for constitutive exocytosis; (2) VAMP-7 is involved in the constitutive exocytosis as a slow, minor v-SNARE, but not in the lysosomal transport; and (3) VAMP-8 is unlikely to be a v-SNARE for constitutive exocytosis in HSY cells.     

Differential inhibitory effects of mu-opioids on substance P- and capsaicin-induced nociceptive behavior in mice

The antinociceptive mechanisms of the selective mu-opioid receptor agonists [D-Ala2,NMePhe4,Gly(ol)5]enkephalin (DAMGO), H-Tyr-D-Arg-Phe-beta-Ala-OH (TAPA) or H-Tyr-D-Arg-Phe-beta-Ala-NH2 (TAPA-NH2) against substance P (SP)- or capsaicin-elicited nociceptive behaviors was investigated in mice. DAMGO, TAPA or TAPA-NH2 given intrathecally inhibited the nociceptive behaviors elicited by intrathecally administered SP or capsaicin, and these antinociceptive effects were completely eliminated by intrathecal co-administration with D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), a selective mu-opioid receptor antagonist. Pretreatment subcutaneously with naloxonazine, a selective mu1-opioid receptor antagonist, partially attenuated the antinociceptive effect of TAPA-NH2, but not DAMGO and TAPA, against SP. However, the antinociception induced by TAPA, but not DAMGO and TAPA-NH2, against capsaicin was significantly inhibited by naloxonazine. On the other hand, co-administration intrathecally with Tyr-D-Pro-Trp-Gly-NH2 (D-Pro2-Tyr-W-MIF-1), a selective mu2-opioid receptor antagonist, significantly attenuated the antinociceptive effects of DAMGO, but not TAPA and TAPA-NH2, against capsaicin, while the antinociceptions induced by three opioid peptides against SP were significantly inhibited by D-Pro2-Tyr-W-MIF-1. These results suggest that differential inhibitory mechanisms on pre- and postsynaptic sites in the spinal cord contribute to the antinociceptive effects of the three mu-opioid peptides.