Multiple discontinuous ligand-mimetic antibody binding sites define a ligand binding pocket in integrin alpha(IIb)beta(3)

Integrin alpha(IIb)beta(3), a platelet fibrinogen receptor, is critically involved in thrombosis and hemostasis. However, how ligands interact with alpha(IIb)beta(3) has been controversial. Ligand-mimetic anti-alpha(IIb)beta(3) antibodies (PAC-1, LJ-CP3, and OP-G2) contain the RGD-like RYD sequence in their CDR3 in the heavy chain and have structural and functional similarities to native ligands. We have located binding sites for ligand-mimetic antibodies in alpha(IIb) and beta(3) using human-to-mouse chimeras, which we expect to maintain functional integrity of alpha(IIb)beta(3). Here we report that these antibodies recognize several discontinuous binding sites in both the alpha(IIb) and beta(3) subunits; these binding sites are located in residues 156-162 and 229-230 of alpha(IIb) and residues 179-183 of beta(3). In contrast, several nonligand-mimetic antibodies (e.g. 7E3) recognize single epitopes in either subunit. Thus, binding to several discontinuous sites in both subunits is unique to ligand-mimetic antibodies. Interestingly, these binding sites overlap with several (but not all) of the sequences that have been reported to be critical for fibrinogen binding (e.g. N-terminal repeats 2-3 but not repeats 4-7, of alpha(IIb)). These results suggest that ligand-mimetic antibodies and probably native ligands may make direct contact with these discontinuous binding sites in both subunits, which may constitute a ligand-binding pocket.     

Particle size and shape of thermally denatured bovine serum albumin by light scattering

Solutions of bovine serum albumin in various stages of thermal denaturation, ranging from very early to fairly advanced, were prepared in a light scattering cell. The molecular weights and the molecular sizes of the subsequent aggregate particles were determined by the light scattering method. In the early stages of thermal denaturation, in the presence of sodium chloride, the aggregate particles were fairly stable, growing essentially in rods, relatively uniform in size. In the absence of sodium chloride, the aggregate particles extended fairly widely and were not uniform in size even in the early stages.     

Cloning and nucleotide sequence of a novel 28-kDa protein from the mantle muscle of the squid Todarodes pacificus with homology to tropomyosin

In recent studies, we found autodegradation of collagen from the mantle muscle of the squid Todarodes pacificus and also that the 28- and 25-kDa proteins are closely related to this phenomenon [Connect. Tissue Res. 45 (2004) 109-121]. We obtained partial sequences of three internal portions of this protein, which suggested that 25-kDa protein is a partially degraded form of the 28-kDa protein. We determined the full cDNA sequence of this protein by the degenerate polymerase chain reaction (PCR) using the information of amino acid sequences. The deduced amino acid sequence corresponding to the 212-bp cDNA contained all of the amino acid identified from the 28-kDa protein. Rapid amplification of cDNA ends (RACE) and squid mantle muscle RNA allowed cloning of the full 522-bp sequence, corresponding to a protein of 174 amino acids. A database search indicated that this is a new protein that shares 27-34% identity with tropomyosins from various animals. Structural prediction suggested that it possesses heptad repeats that form coiled-coil structures. We expressed a recombinant protein encoded by the 212-bp cDNA in Escherichia coli and used it to generate a polyclonal antibody. Western blotting with this antibody showed that the 28-kDa protein is expressed in fin, tentacle, and mantle muscle, but not in liver.     

Statins selectively inhibit leukocyte function antigen-1 by binding to a novel regulatory integrin site

The beta2 integrin leukocyte function antigen-1 (LFA-1) has an important role in the pathophysiology of inflammatory and autoimmune diseases. Here we report that statin compounds commonly used for the treatment of hypercholesterolemia selectively blocked LFA-1-mediated adhesion and costimulation of lymphocytes. This effect was unrelated to the statins' inhibition of 3-hydroxy-3-methylglutaryl coenzyme-A reductase; instead it occurred via binding to a novel allosteric site within LFA-1. Subsequent optimization of the statins for LFA-1 binding resulted in potent, selective and orally active LFA-1 inhibitors that suppress the inflammatory response in a murine model of peritonitis. Targeting of the statin-binding site of LFA-1 could be used to treat diseases such as psoriasis, rheumatoid arthritis, ischemia/reperfusion injury and transplant rejection.     

Population dynamics of the beech caterpillar, <Emphasis Type="Italic">Syntypistis punctatella</Emphasis>, and biotic and abiotic factors

The beech caterpillar, Syntypistis punctatella (Motschulsky) (Lepidoptera: Notodontidae), often causes extensive defoliation of beech forests in Japan. Outbreaks have often occurred synchronously among different areas at intervals of 8–11 years. Synchrony of outbreaks was considered to be caused by synchrony of weather. Populations of this insect exhibit periodical dynamics in both outbreak and nonoutbreak areas. Factors that might influence the population dynamics of the beech caterpillar were classified from the point of view of the natural bioregulation com-plex, which includes a coleopteran predator, Calosoma maximowiczi, avian predators, parasitoids, entomopathogenic fungi, and delayed induced defensive response (DIR) of beech trees. Because such periodic population dynamics are believed to be caused by one or more delayed density-dependent factors, delayed density-dependent mortality has been identified as a likely source of population cycles. The DIR and pathogenic diseases showed a high order of density dependence. An infectious pathogen, Cordyceps militaris, was considered to be the most plausible agent responsible for periodic dynamics of the beech caterpillar population because insect diseases were effective in cases in which the S. punctatella population started to decrease without reaching outbreak densities, but DIR was not. Conspicuous defoliation caused by this insect tends to occur at certain elevations, where forests are composed of pure stands of beech trees. I propose three different hypotheses to explain this phenomenon: the diversity–stability hypothesis, the resource concentration hypothesis, and the altitudinal soil nutrient hypothesis. Received: November 20, 1999 / Accepted: August 3, 2000     

Effect of triton WR-1339 on peroxisomal enzymes of rat liver

The effect of Triton WR-1339 on peroxisomal enzymes of rat liver was studied. The dose vs. response relationships of peroxisomal enzyme activities to Triton WR-1339 were first examined 3.5 days after injection. Catalase activity was reduced to 50% of that of the control at a dose of 200 mg per 100 g body weight; it was found that the decrease depended on the dose of this compound. Urate oxidase activity was not significantly affected. D-Amino acid oxidase activity showed intermediate behavior. The activities of these enzymes were found to be reduced more markedly at 2 days than at 3.5 days after injection, and subsequently the levels of the activities recovered. At 2 days after injection of a dose of 200 mg per 100 g body weight, the activities of catalase, D-amino acid oxidase and urate oxidase had decreased to 40, 60 and 60%,respectively, of the control values.It was found that the decreases in the activities of these enzymes caused by Triton WR-1339 had occurred in the large granule fraction, but not in the cytoplasm.Measurement of the specific activity, Ouchterlony gel diffusion and quantitative immunoprecipitation suggested that there was a similarity between the Triton WR-1339-treated and untreated rats in the nature of purified catalases.These results suggest that Triton WR-1339 depresses the activities of liver peroxisomal enzymes, especially the catalase activity.     

Automated Software Analysis of Fetal Movement Recorded during a Pregnant Woman’s Sleep at Home

Fetal movement is an important biological index of fetal well-being. Since 2008, we have been developing an original capacitive acceleration sensor and device that a pregnant woman can easily use to record fetal movement by herself at home during sleep. In this study, we report a newly developed automated software system for analyzing recorded fetal movement. This study will introduce the system and compare its results to those of a manual analysis of the same fetal movement signals (Experiment I). We will also demonstrate an appropriate way to use the system (Experiment II). In Experiment I, fetal movement data reported previously for six pregnant women at 28-38 gestational weeks were used. We evaluated the agreement of the manual and automated analyses for the same 10-sec epochs using prevalence-adjusted bias-adjusted kappa (PABAK) including quantitative indicators for prevalence and bias. The mean PABAK value was 0.83, which can be considered almost perfect. In Experiment II, twelve pregnant women at 24-36 gestational weeks recorded fetal movement at night once every four weeks. Overall, mean fetal movement counts per hour during maternal sleep significantly decreased along with gestational weeks, though individual differences in fetal development were noted. This newly developed automated analysis system can provide important data throughout late pregnancy.     

Impaired insulin signaling in endothelial cells reduces insulin-induced glucose uptake by skeletal muscle

In obese patients with type 2 diabetes, insulin delivery to and insulin-dependent glucose uptake by skeletal muscle are delayed and impaired. The mechanisms underlying the delay and impairment are unclear. We demonstrate that impaired insulin signaling in endothelial cells, due to reduced Irs2 expression and insulin-induced eNOS phosphorylation, causes attenuation of insulin-induced capillary recruitment and insulin delivery, which in turn reduces glucose uptake by skeletal muscle. Moreover, restoration of insulin-induced eNOS phosphorylation in endothelial cells completely reverses the reduction in capillary recruitment and insulin delivery in tissue-specific knockout mice lacking Irs2 in endothelial cells and fed a high-fat diet. As a result, glucose uptake by skeletal muscle is restored in these mice. Taken together, our results show that insulin signaling in endothelial cells plays a pivotal role in the regulation of glucose uptake by skeletal muscle. Furthermore, improving endothelial insulin signaling may serve as a therapeutic strategy for ameliorating skeletal muscle insulin resistance.