Direct demonstration of involvement of the adaptor protein ShcA in the regulation of Ca2+-induced platelet aggregation

Platelet aggregation is mediated by conformational change of integrin alpha(IIb)beta(3). Tyrosine-phosphorylation of cytoplasmic domain of beta(3) upon platelet activation has been demonstrated to play a critical role in this process. Recently, the adaptor protein ShcA has been shown to bind to the tyrosine-phosphorylated beta(3), while it remains open whether ShcA plays any role in platelet aggregation. Here, we show that ShcA bound to tyrosine-phosphorylated beta(3)-tail peptide through its phosphotyrosine-binding domain in vitro. Then, we examined the involvement of ShcA in platelet aggregation by a previously established in vitro assay using platelets permeabilized with streptolysin-O, where exogenous addition of platelet cytosol is required for reconstitution of the Ca(2+)-induced aggregation. When ShcA was specifically depleted with anti-ShcA antibody from the cytosol, this ShcA-depleted cytosol lost the aggregation-supporting activity, which was rescued by addition of purified recombinant ShcA. Thus, ShcA is essential for the Ca(2+)-induced platelet aggregation.     

Loss and recovery of the blood-nerve barrier in the rat sciatic nerve after crush injury are associated with expression of intercellular junctional proteins

The blood-nerve barrier in peripheral nerves is important for maintaining the environment for axons. Breakdown of the barrier by nerve injury causes various pathologies. We hypothesized that the breakdown and recovery of the blood-nerve barrier after injury are associated with the changes in the expression of intercellular junctional proteins. To test this hypothesis, we induced crush injuries in the rat sciatic nerve by ligation and analyzed spatiotemporal changes of claudin-1, claudin-5, occludin, VE-cadherin, and connexin43 by immunoconfocal microscopy and morphometry and compared them with changes in the permeability of the blood-nerve barrier by intravenous and local administration of Evans blue-albumin (EBA). On day 1 after removal of the ligature EBA leaked into the connective tissue in the endoneurium and then the leakage gradually decreased and disappeared on day 7. On day 1 claudin-1, claudin-5, occludin, VE-cadherin, and connexin43 had totally disappeared from the perineurium and endoneurium. Thereafter, claudin-1, claudin-5, occludin, and VE-cadherin recovered from day 2, whereas connexin43 was redetected on day 5. These results indicate that the breakdown and following recovery of the blood-nerve barrier are closely associated with changes in the expression of claudins, occludin, VE-cadherin, and connexin43 and that the recovery time course is similar but nonidentical.     

PCR method for detecting trace amounts of buckwheat (Fagopyrum spp.) in food

Buckwheat often causes severe allergic reactions, even when its ingestion level is extremely low. Therefore, buckwheat is listed in several countries as a common food allergen. In addition to common buckwheat and Tartarian buckwheat that are cultivated and consumed widely, wild buckwheat may be potentially allergenic. Food containing undeclared buckwheat poses a risk to patients with the buckwheat allergy. We describe in this report a PCR method to detect buckwheat DNA by using primers corresponding to the internal transcribed spacer region and the 5.8S rRNA gene. The method is buckwheat-specific and compatible with both cultivated and wild buckwheat of the Fagopyrum spp. Its sensitivity was sufficient to detect 1 ppm (w/w) of buckwheat DNA spiked in wheat DNA. This method should benefit food manufacturers, clinical doctors, and allergic patients by providing information on the presence of buckwheat contamination in food.     

E2F1 and c-Myc potentiate apoptosis through inhibition of NF-kappaB activity that facilitates MnSOD-mediated ROS elimination

Overexpression of c-Myc or E2F1 sensitizes host cells to various types of apoptosis. Here, we found that overexpressed c-Myc or E2F1 induces accumulation of reactive oxygen species (ROS) and thereby enhances serum-deprived apoptosis in NIH3T3 and Saos-2. During serum deprivation, MnSOD mRNA was induced by NF-kappaB in mock-transfected NIH3T3, while this induction was inhibited in NIH3T3 overexpressing c-Myc or E2F1. In these clones, E2F1 inhibited NF-kappaB activity by binding to its subunit p65 in competition with a heterodimeric partner p50. In addition to overexpressed E2F1, endogenous E2F1 released from Rb was also found to inhibit NF-kappaB activity in a cell cycle-dependent manner by using E2F1(+/+) and E2F1(-/-) murine embryonic fibroblasts. These results indicate that E2F1 promotes apoptosis by inhibiting NF-kappaB activity.     

The effects of bestatin, a microbial aminopeptidase inhibitor, on epidermal growth factor-induced DNA synthesis and cell division in primary cultured hepatocytes of rats

We investigated the effects of microbial protease inhibitors, in particular the aminopeptidase inhibitor bestatin, on DNA synthesis and cell division induced by epidermal growth factor (EGF) in hepatocytes. Although bestatin did not significantly affect binding of EGF to hepatocytes, it inhibited EGF-induced DNA synthesis and cell division. DNA synthesis in rat hepatocytes was maximal 24-26 h after EGF addition to the medium. The time required for maximal DNA synthesis was not affected if bestatin was removed less than 12 h after addition, but synthesis was partially inhibited if bestatin was added to the medium several hours after EGF addition, depending on the time of bestatin addition. Our results suggest that bestatin arrests the new cell cycle induced by EGF at about 12 h after the initiation. Considering also our results obtained by employing other protease inhibitors, we concluded that specific proteases play important roles in hepatocyte DNA synthesis and cell division induced by EGF.     

Coevolutionary dynamics in one-to-many mutualistic systems

“One-to-many” mutualisms are often observed in nature. In this type of mutualism, each host individual can interact with many symbionts, whereas each individual symbiont can interact with only one host individual. Partner choice by the host is a potentially critical mechanism for maintaining such systems; however, its long-term effects on the coevolution between the hosts and symbionts have not been completely explored. In this study, I developed a simple mathematical model to describe the coevolutionary dynamics between hosts and symbionts in a one-to-many mutualism. I assumed that each host chooses a constant number of symbionts from a potential symbiont population, a fraction of which are chosen through preferential choice on the basis of the cooperativeness of the symbionts and the rest are chosen randomly. Using numerical calculations, I found that mutualism is maintained when the preferential choice is not very costly and the mutation rate of symbionts is large. I also found that symbionts that receive benefits from hosts without a return (cheater symbionts) and hosts that do not engage in preferential partner choice (indiscriminator hosts) can coexist with mutualist symbionts and discriminator hosts, respectively. The parameter domain of pure mutualism, i.e., free from cheater symbionts and indiscriminator hosts, can be narrower than the whole domain where the mutualism persists.     

LPT1 encodes a membrane-bound O-acyltransferase involved in the acylation of lysophospholipids in the yeast Saccharomyces cerevisiae

Phospholipids are major components of cellular membranes that participate in a range of cellular processes. Phosphatidic acid (PA) is a key molecule in the phospholipid biosynthetic pathway. In Saccharomyces cerevisiae, SLC1 has been identified as the gene encoding lysophosphatidic acid acyltransferase, which catalyzes PA synthesis. However, despite the importance of PA, disruption of SLC1 does not affect cell viability (Nagiec, M. M., Wells, G. B., Lester, R. L., and Dickson, R. C. (1993) J. Biol. Chem. 268, 22156-22163). We originally aimed to identify the acetyl-CoA:lyso platelet-activating factor acetyltransferase (lysoPAF AT) gene in yeast. Screening of a complete set of yeast deletion clones (4741 homozygous diploid clones) revealed a single mutant strain, YOR175c, with a defect in lysoPAF AT activity. YOR175c has been predicted to be a member of the membrane-bound O-acyltransferase superfamily, and we designated the gene LPT1. An Lpt1-green fluorescent protein fusion protein localized at the endoplasmic reticulum. Other than lysoPAF AT activity, Lpt1 catalyzed acyltransferase activity with a wide variety of lysophospholipids as acceptors, including lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidylinositol, and lysophosphatidylserine. A liquid chromatography-mass spectrometry analysis indicated that lysophosphatidylcholine and lysophosphatidylethanolamine accumulated in the Deltalpt1 mutant strain. Although the Deltalpt1 mutant strain did not show other detectable defects, the Deltalpt1 Deltaslc1 double mutant strain had a synthetic lethal phenotype. These results indicate that, in concert with Slc1, Lpt1 plays a central role in PA biosynthesis, which is essential for cell viability.     

Crystallization and preliminary X-ray diffraction studies of C-phycocyanin from a red alga, Porphyra tenera

C-Phycocyanin from a red alga, Porphyra tenera, has been crystallized by the vapor-diffusion procedure. Both orthorhombic and hexagonal forms were obtained from ammonium sulfate solutions, whereas only the orthohombic form was selectively grown from sodium citrate solutions. The orthorhombic crystals are more suitable for further crystallographic work; their space group is P2(1)2(1)2(1), with unit-cell dimensions of a = 105, b = 121, and c = 184 A. The asymmetric unit comprises two (alpha beta)3 trimer molecules of C-phycocyanin. These crystals diffract X-rays up to about 3 A resolution.