2,4-Diamino-6-hydroxypyrimidine (DAHP) suppresses cytokine-induced VCAM-1 expression on the cell surface of human umbilical vein endothelial cells in a BH(4)-independent manner

2,4-Diamino-6-hydroxypyrimidine (DAHP) is considered a specific inhibitor of BH(4) biosynthesis and is widely used in order to elucidate the possible biological function of BH(4) in various cells. In the present study, we found that both the synthesis of tetrahydrobiopterin (BH(4)) and expression of vascular cell adhesion molecule 1 (VCAM-1) were increased in human umbilical vein endothelial cells (HUVEC) treated with proinflammatory cytokines. Thus we examined the effects of DAHP to clarify whether BH(4) might be involved in the expression of VCAM-1 in HUVEC. DAHP reduced the levels of both BH(4) and VCAM-1 induced by TNF-alpha and IFN-gamma. However, the dose-response curves of DAHP for the suppression of the VCAM-1 level and that of BH(4) level were markedly different. Supplementation with sepiapterin failed to restore the depressed VCAM-1 level, although it completely restored the BH(4) level. Furthermore, DAHP significantly reduced the VCAM-1 level under the experimental conditions using TNF-alpha alone, which failed to induce BH(4) production. Taken together, these results indicate that DAHP inhibited the expression of VCAM-1 in a BH(4)-independent manner in HUVEC. In the present study, we also found that DAHP significantly suppressed the accumulation of cytokine-induced NF-kappaB (p65) in the nucleus as well as the mRNA levels of VCAM-1 and GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme of BH(4) synthesis. The data obtained in this study suggest that DAHP reduced VCAM-1 and GTPCH protein synthesis at least partially via suppressing the NF-kappaB level in the nucleus of HUVEC.     

Serum cholesterol-decreasing effect of heat-moisture-treated high-amylose cornstarch in cholesterol-loaded rats

Rats were fed on a diet containing cholesterol (Chol) at a level corresponding to the standard Chol intake in humans, and the influence of heat-moisture-treated high-amylose cornstarch (HHA) on their serum Chol level was investigated. HHA decreased the serum level of Chol in rats fed on the diet containing 0.1% Chol, which corresponds to a Chol intake in humans of 800 mg/d, although the liver levels of Chol increased in these rats. HHA did not influence the fecal excretion of Chol/bile acids. It is possible that the decrease in serum Chol level in the rats fed on the high-Chol diet can be attributed to the promotion of Chol uptake in the liver.     

Neocarpenteles: a new ascomycete genus to accommodate Hemicarpenteles acanthosporus

A new genus, Neocarpenteles, the Trichocomaceae (Eurotiales), and a new combination, N. acanthosporum, are proposed to accommodate Hemicarpenteles acanthosporus Udagawa et Takada. The fungus is characterized by sclerotioid, nonostiolate, unilocular stromata in which asci gradually produce, outward from the center, lenticular ascospores with two equatorial crests and convex surfaces ornamented by triangular ridges and microtubercles, and an Aspergillus anamorph with uniseriate aspergilla. It has the Q-10 system as the major ubiquinone. Received: May 1, 2001 / Accepted: July 4, 2001     

Cordyceps owariensis f. viridescens and its new Nomuraea anamorph

Cordyceps owariensis f. viridescens forma nov. (Nom. Jap., Amami-yarinohosemitake) is described from an underground nymph of Platypleura kuroiwae collected in Amami-oshima Island, southwestern Japan. Cultural isolations were made from ascospores of the fresh material of this fungus, and consequently a new hyphomycete was developed as its anamorph. Nomuraea owariensis is described to accommodate this anamorphic state. This is the first report on the association of a Nomuraea anamorph with cicadicolous Cordyceps species. Received: June 6, 2001 / Accepted: December 18, 2001     

Secretion of L-glutamate from osteoclasts through transcytosis

Osteoclasts are involved in the catabolism of the bone matrix and eliminate the resulting degradation products through transcytosis, but the molecular mechanism and regulation of transcytosis remain poorly understood. Upon differentiation, osteoclasts express vesicular glutamate transporter 1 (VGLUT1), which is essential for vesicular storage and subsequent exocytosis of glutamate in neurons. VGLUT1 is localized in transcytotic vesicles and accumulates L-glutamate. Osteoclasts secrete L-glutamate and the bone degradation products upon stimulation with KCl or ATP in a Ca2+-dependent manner. KCl- and ATP-dependent secretion of L-glutamate was absent in osteoclasts prepared from VGLUT1-/- knockout mice. Osteoclasts express mGluR8, a class III metabotropic glutamate receptor. Its stimulation by a specific agonist inhibits secretion of L-glutamate and bone degradation products, whereas its suppression by a specific antagonist stimulates bone resorption. Finally, it was found that VGLUT1-/- mice develop osteoporosis. Thus, in bone-resorbing osteoclasts, L-glutamate and bone degradation products are secreted through transcytosis and the released L-glutamate is involved in autoregulation of transcytosis. Glutamate signaling may play an important role in the bone homeostasis.     

Discovery of novel 7-membered cyclic amide derivatives that inhibit 11beta-hydroxysteroid dehydrogenase type 1

A series of novel 5-trans-hydroxyadamantan-2-yl-5,6,7,8-tetrahydropyrazolo[4,3-c]azepin-4(1H)-ones that inhibit 11beta-hydroxysteroid dehydrogenase type 1 are described. We discovered these 7-membered cyclic amide derivatives by introducing a distinctive linker through pharmacophore analysis of known ligands included in X-ray co-crystal structures. Further optimization using docking studies led to highly potent inhibitors 15b and 27, which furthermore showed the potent efficacy in in vivo studies.     

Cold adaptation of the terrestrial isopod,Porcellio scaber,to subnivean environments

The terrestrial isopod, Porcellio scaber, was susceptible to subzero temperature: both freezing and chilling were injurious. The level of cold hardiness against chilling and freezing showed different patterns in their seasonal variation. The lower lethal temperature causing 50% mortality, an indicator of the tolerance to chilling, ranged from-1.37°C in August to-4.58°C in December. The whole body supercooling point, the absolute limit of freeze avoidance, was kept at about-7°C throughout the year. The winter decrease in lower lethal temperature was concomitant with an accumulation of low molecular weight carbohydrates which are possible protective reagents against chilling injury, whereas the less seasonally variable supercooling point seemed to be associated with the year-round presence of gut content. Food derivatives may act as efficient ice nucleators. The different trend in seasonal changes between lower lethal temperature and supercooling point may be related to the microclimate of the hibernacula in subnivean environments, where the winter temperature became lower than the lower lethal temperature in the summer active phase, but remained higher than the summer supercooling point.Abbreviations LLT₅₀ lower lethal temperature inducing 50% mortality - SCP supercooling point - T _a ambient air temperature - T _s soil surface temperature     

Possible involvement of focal adhesion kinase,p125FAK,in osteoclastic bone resorption

Involvement of tyrosine phosphorylation in osteoclastic bone resorption was examined using osteoclast-like multinucleated cells prepared from co-cultures of mouse osteoblastic cells and bone marrow cells in the presence of 1α,25-dihydroxyvitamin D₃. When osteoclast-like cells were plated on culture dishes in the presence of 10% fetal bovine serum, they were sharply stained in their peripheral region by anti-phosphotyrosine antibody. Western blot analysis revealed that 115-to 130-kD proteins were tyrosine-phosphorylated in osteoclast-like cells. Using immunoprecipitation and immunoblotting, one of the proteins with 115–130 kD was identified as focal adhesion kinase (p125^FAK), a tyrosine kinase, which is localized in focal adhesions. Immunostaining with anti-p 125^FAK antibody revealed that p125^FAK was mainly localized at the periphery of osteoclast-like cells. Herbimycin A, a tyrosine kinase inhibitor, not only suppressed tyrosine phosphorylation of p125^FAK but also changed the intracellular localization of p125^FAK and disrupted a ringed structure of F-actin-containing podosomes in osteoclast-like cells. Antisense oligodeoxynucleotides to p125^FAK inhibited dentine resorption by osteoclast-like cells, whereas sense oligodeoxynucleotides did not. These results suggest that p125^FAK is involved in osteoclastic bone resorption and that tyrosine phosphorylation of p125^FAK is critical for regulating osteoclast function.     

The relationship between physical property and function of highly activated mutants of thermolysin

Thermolysin mutants having a variety of amino acid at the 119th position are designed by considering electrostatic field effect upon the active area. The most activated mutant has five times higher hydrolytic activity than the wild type. Negative correlation between the activity and the thermal stability is observed. A combined effect of the flexibility of the substrate binding site and the negative electrostatic field around the site is suggested as a key to enhance the activity.