Isolation and identification of EG-VEGF/prokineticins as cognate ligands for two orphan G-protein-coupled receptors

Endocrine gland-derived vascular endothelial growth factor (EG-VEGF, identical to prokineticin 1) is a novel peptide recently identified as a selective mitogen for endocrine gland endothelial cells. The present study demonstrates that EG-VEGF/prokineticin 1 and a peptide closely related to EG-VEGF, prokineticin 2, are cognate ligands of two orphan G-protein-coupled receptors designated ZAQ (=EG-VEGF/PK-R1) and I5E (=EG-VEGF/PK-R2). EG-VEGF/prokineticin 1 and prokineticin 2 induced a transient increase in intracellular calcium ion concentration ([Ca(2+)](i)) with nanomolar potency in Chinese hamster ovary (CHO) cells expressing EG-VEGF/PK-R1 and -R2 and bind to these cells with high affinity and with different receptor selectivity. EG-VEGF/prokineticins provoke rapid phosphorylation of p44/42 MAP kinase and DNA synthesis in the bovine adrenal capillary endothelial cells (BACE). The mRNAs of both EG-VEGF/PK-R1 and -R2 were expressed in BACE. The identification of the receptors for EG-VEGF/prokineticins may provide a novel molecular basis for the regulation of angiogenesis in endocrine glands.     

Changes in osteoclasts after irradiation with carbon ion particles

We examined the effects of radiation on decreases in osteoclast numbers after regional irradiation of rats with carbon ions and gamma rays. Male Wistar rats were subjected to hind-leg irradiation with carbon ions (290 MeV/u) or gamma rays at doses of 15, 22.5, or 30 Gy. The effects of carbon ions and gamma rays on osteoclasts were studied using histologic and morphometric methods. At doses of 15 Gy and 22.5 Gy, osteoclast numbers increased transiently until day 5 after irradiation and then decreased rapidly in both the carbon ion and gamma ray irradiation groups. The carbon ion group showed reduced osteoclast size compared with the gamma ray group. Carbon ion irradiation had a more marked effect on osteoclast activity, and suppressed maturation to a greater extent than gamma irradiation. These observations suggest that carbon ion irradiation induces differential modulation of osteoclast growth factor expression.     

Diversity and rarity hotspots and conservation of butterfly communities in and around the Aokigahara woodland of Mount Fuji,central Japan

In central Japan, Aokigahara woodland is considered to be one of the most natural areas around Mount Fuji and a core area in the conservation of the biodiversity of Mount Fuji. We chose butterflies as an indicator species of biodiversity and examined six communities in and around the woodland in 2000 using transect counts to examine and search for diversity and rarity hotspots and their associated landscapes. The results showed that butterfly species richness and species diversities H 1/ were significantly higher in forest-edge sites than in forest-interior and/or open-land sites, and variation in the total number of species among these three landscape types was well accounted for by ecologically specialist species, such as landscape specifics, oligovoltines, narrow diet feeders and low-density species. Thus, the species regarded as vulnerable to extinction, including Red List species, were observed more often in forest-edge sites than in forest-interior and/or open-land sites. As a result, in the study area, diversity and rarity hotspots were found in forest-edge landscapes. The reasons why butterfly diversity and rarity hotspots were established in forest-edge landscapes were analyzed and interpreted from several points of view, including disturbance level, landscape elements and plant species richness. From these results, and the fact that some species were confined to forest-interior sites, we conclude that it is very important to conserve and manage forest-edge habitats (considered to be semi-natural) as well as forest-interior habitats (considered to be the most natural) to maintain the diversity of butterfly communities and preserve the various types of threatened species in and around the Aokigahara woodland.     

Internal ultrastructure of spores of microsporidans isolated from the Silkworm,Bombyx mori

Nosema bombycis, two Nosema spp., and a Pleistophora sp. were propagated in the silkworm and the fine structures of their spores were studied. The morphology of the polaroplast, the appearance of the nucleus, and the number of coils in the polar filament differed among the spores of the four species. The spores of the three Nosema species, however, had several identical components; e.g., the polaroplast was made up of two parts, they had two nuclei, and the ribosome arrangement was similar. On the other hand, the spore of Pleistophora sp. had a polaroplast composed of three parts, a single nucleus, and ribosomes arranged around the polar filament. Thus the fine structures of the spore differentiate microsporidan species.     

Cutting edge: T cells monitor N-myristoylation of the Nef protein in simian immunodeficiency virus-infected monkeys

The use of the host cellular machinery is essential for pathogenic viruses to replicate in host cells. HIV and SIV borrow the host-derived N-myristoyl-transferase and its substrate, myristoyl-CoA, for coupling a saturated C(14) fatty acid (myristic acid) to the N-terminal glycine residue of the Nef protein. This biochemical reaction, referred to as N-myristoylation, assists its targeting to the plasma membrane, thereby supporting the immunosuppressive activity proposed for the Nef protein. In this study, we show that the host immunity is equipped with CTLs capable of sensing N-myristoylation of the Nef protein. A rhesus macaque CD8(+) T cell line was established that specifically recognized N-myristoylated, but not unmodified, peptides of the Nef protein. Furthermore, the population size of N-myristoylated Nef peptide-specific T cells was found to increase significantly in the circulation of SIV-infected monkeys. Thus, these results identify N-myristoylated viral peptides as a novel class of CTL target Ag.     

The Cerberus/Dan-family protein Charon is a negative regulator of Nodal signaling during left-right patterning in zebrafish

We have isolated a novel gene, charon, that encodes a member of the Cerberus/Dan family of secreted factors. In zebrafish, Fugu and flounder, charon is expressed in regions embracing Kupffer's vesicle, which is considered to be the teleost fish equivalent to the region of the mouse definitive node that is required for left-right (L/R) patterning. Misexpression of Charon elicited phenotypes similar to those of mutant embryos defective in Nodal signaling or embryos overexpressing Antivin(Atv)/Lefty1, an inhibitor for Nodal and Activin. Charon also suppressed the dorsalizing activity of all three of the known zebrafish Nodal-related proteins (Cyclops, Squint and Southpaw), indicating that Charon can antagonize Nodal signaling. Because Southpaw functions in the L/R patterning of lateral plate mesoderm and the diencephalon, we asked whether Charon is involved in regulating L/R asymmetry. Inhibition of Charon's function by antisense morpholino oligonucleotides (MOs) led to a loss of L/R polarity, as evidenced by bilateral expression of the left side-specific genes in the lateral plate mesoderm (southpaw, cyclops, atv/lefty1, lefty2 and pitx2) and diencephalon (cyclops, atv/lefty1 and pitx2), and defects in early (heart jogging) and late (heart looping) asymmetric heart development, but did not disturb the notochord development or the atv/lefty1-mediated midline barrier function. MO-mediated inhibition of both Charon and Southpaw led to a reduction in or loss of the expression of the left side-specific genes, suggesting that Southpaw is epistatic to Charon in left-side formation. These data indicate that antagonistic interactions between Charon and Nodal (Southpaw), which take place in regions adjacent to Kupffer's vesicle, play an important role in L/R patterning in zebrafish.     

Effect of inhaled indomethacin on distilled water-induced airway epithelial cell swelling.

We evaluated the mechanism of the anti-asthmatic effect of inhaled indomethacin (Indo) by using an animal model (guinea pigs) of airway inflammation. After being exposed to either ozone or room air at identical flow rates (5 l/min) for 2 h, guinea pigs were anesthetized, tracheostomized, and lung resistance (RL) was subsequently measured. Guinea pigs inhaled either saline or Indo (1.5 mg/ml) for 1 min before undergoing an ultrasonically nebulized distilled water (UNDW) inhalation test. RL increased significantly after 10 min of UNDW inhalation in the room air and ozone groups but more so in the ozone group. This increase in RL was significantly suppressed by pretreatment with Indo. In the morphometric assessment of airway mucosa, a significant swelling of the epithelial cells after UNDW inhalation was observed in both the room air and ozone groups but especially so in the ozone group. This increase was also suppressed with Indo pretreatment. These results suggest that the increase in RL and the swelling of airway epithelial cells induced by inhaled UNDW in ozone-exposed guinea pigs was suppressed by pretreatment of inhaled Indo and that this suppression may be one of the reasons for the anti-asthmatic effect of inhaled Indo.     

Localization of acetylcholine-related molecules in the retina: implication of the communication from photoreceptor to retinal pigment epithelium

It has been long speculated that specific signals are transmitted from photoreceptors to the retinal pigment epithelium (RPE). However, such signals have not been identified. In this study, we examined the retinal expression and localization of acetylcholine-related molecules as putative candidates for these signals. Previous reports revealed that α7 nicotinic acetylcholine receptors (nAChRs) are present in the microvilli of RPE cells that envelope the tips of photoreceptor outer segments (OS). Secreted mammalian leukocyte antigen 6/urokinase-type plasminogen activator receptor-related protein-1 (SLURP-1) is a positive allosteric modulator of the α7 nAChR. Therefore, we first focused on the expression of SLURP-1. SLURP-1 mRNA was expressed in the outer nuclear layer, which is comprised of photoreceptor cell bodies. SLURP-1 immunoreactivity co-localized with rhodopsin and S-opsin in photoreceptor OS, while choline acetyltransferase (ChAT) and high affinity choline transporter (CHT-1) were also expressed in photoreceptor OS. Immunoelectron microscopy identified that the majority of SLURP-1 was localized to the plasma membranes of photoreceptor OS. These results provide evidence that SLURP-1 is synthesized in photoreceptor cell bodies and transported to photoreceptor OS, where SLURP-1 may also be secreted. Our findings suggest that photoreceptor OS communicate via neurotransmitters such as ACh and SLURP-1, while RPE cells might receive these signals through α7 nAChRs in their microvilli.     

Chemical synthesis of polysaccharides. 7. Enzymatic hydrolysis of (1 → 6)-α-DL-glucopyranan (DL-dextran)

Hydrolysis of (1 → 6)-α-DL -glucopyranan (synthetic DL -dextran) by an endo-dextranase from a Penicillium species was examined in an acetate buffer solution (pH 5.3) at 37°C. Three samples of different tacticities (isotactic dyad content, 55, 63, and 72%) were employed with a clinical dextran for comparison. Colorimetric determination of the reducing end units of the saccharides produced during hydrolysis showed that the maximum degrees of hydrolysis based on the D-glucose units, (D.H.)_D, for the DL -dextrans were 21.4, 27.8, and 33.0% in the order of increasing isotacitic dyad content, whereas the (D.H.)_D value for the clinical dextran was 51.9%. A statistical treatment of the enzymatic hydrolysis is proposed to interpret the experimental results.