Signal transduction pathways mediated by glycoprotein Ia/IIa in human platelets: comparison with those of glycoprotein VI

Human platelets were activated either by glycoprotein (GP) Ia/IIa agonist (rhodocytin) or by a GPVI agonist (collagen-related peptide, CRP), and the intracellular signal transduction pathways were compared in the presence of various inhibitors. Rhodocytin isolated from Calloselasma rhodostoma venom was verified as a GPIa/IIa agonist, based on the inhibitory effects of three mAbs directed against GPIa. Platelet activation mediated by GPIa/IIa led to overt platelet aggregation, elevation of intracellular Ca2+, and tyrosine phosphorylation of several proteins, similar to that of GPVI. p72(syk) and phospholipase Cgamma2 (PLCgamma2) tyrosine phosphorylation were also observed with GPIa/IIa-mediated platelet aggregation, although they peaked slightly later than that of GPVI. In contrast to GPVI-mediated platelet activation, most of these phenomena induced by GPIa/IIa activation were markedly suppressed by acetylsalicylic acid (ASA) or cytochalasin D. These findings suggest that the requirements for thromboxane A2 (TXA2) production and actin polymerization, which are the characteristics of collagen-induced platelet activation, are derived from the GPIa/IIa-mediated signal transduction, but not from that of GPVI.     

Local delivery of soluble TNF-alpha receptor 1 gene reduces infarct size following ischemia/reperfusion injury in rats

Apoptosis in the myocardium is linked to ischemia/reperfusion injury, and TNF-alpha induces apoptosis in cardiomyocytes. A significant amount of TNF-alpha is detected after ischemia and reperfusion. Soluble TNF-alpha receptor 1 (sTNFR1) is an extracellular domain of TNF-alpha receptor 1 and is an antagonist to TNF-alpha. In the present study, we examined the effects of sTNFR1 on infarct size in acute myocardial infarction (AMI) following ischemia/reperfusion. Male Wistar rats were subjected to left coronary artery (LCA) ligation. After 30 min of LCA occlusion, the temporary ligature on the LCA was released and blood flow was restored. Immediately after reperfusion, a total of 200 microg of sTNFR1 or LacZ plasmid was injected into three different sites of the left ventricular wall. At 6 h, 1 and 2 days after reperfusion, the TNF-alpha bioactivity in the myocardium was significantly higher in rats receiving LacZ plasmid than in sham-operated rats, whereas sTNFR1 plasmid significantly suppressed the increase in the TNF-alpha bioactivity. The sTNFR1 plasmid significantly reduced DNA fragmentation and caspase activity compared to the LacZ plasmid. Finally, the sTNFR1 expression-plasmid treatment significantly reduced the area of myocardial infarction at 2 days after ischemia/reperfusion compared to LacZ plasmid. In conclusion, the TNF-alpha bioactivity in the heart increased from the early stage of ischemia/reperfusion, and this increase was thought to contribute in part to the increased area of myocardial infarction. Suppression of TNF-alpha bioactivity with the sTNFR1 plasmid reduced the infarct size in AMI following ischemia and reperfusion.     

Internal architecture, origin-insertion site, and mass of jaw muscles in Old World hamsters

The jaw muscle (i.e., masticatory, suprahyoid, and extrinsic tongue) anatomy and mass were examined in four genera of Old World hamsters (cricetine murids), Mesocricetus, Cricetulus, Tscherskia, and Phodopus. The masseter was the largest and most complicated of the muscles examined. In the superficial layer, a few ventral fibers form a small medially turned portion with an insertion site more similar to those of sciurids than of other murids. In Mesocricetus, the superficial layer has a discrete anteroventral portion that has not been reported for other murid rodents. Examination of the fiber attachment sites indicated that the deep layer contains four parts and the medial layer contains three parts. The deep layer originates from two aponeuroses that are firmly connected to each other at their anterior ends and lie along the zygomatic arch. The aponeurosis of insertion for the deep layer is situated along the masseteric ridge and the dorsal border of the angular process, but is absent in its middle part, consistent with reports in two relatives, sigmodontine and arvicoline murids. In cricetine murids, unlike in other rodents, fibers insert on the dorsal narrow strip of the posterior mandibular aponeurosis, not on its broad medial aspect. The relative mass of some masticatory and suprahyoid muscles is related to body mass. Small species (Cricetulus and Phodopus) have relatively larger masseter and mylohyoid muscles and smaller temporalis and geniohyoid muscles than large species (Mesocricetus and Tscherskia).     

Effects of chloramphenicol on photosynthesis, protein profiles and transketolase activity under extremely high CO2 concentration in an extremely-high-CO2-tolerant green microalga, Chlorococcum littorale

An extremely-high-CO2-tolerant alga, Chlorococcum littorale, showed high quantum efficiency of PSII (PhiII) in the light at 40% CO2, as well as at 5% CO2. However, PhiII decreased greatly when chloramphenicol (CAP) was added at 40% CO2, while no such decrease was observed at 5% CO2. Cycloheximide showed no effect on PhiII at either 5% or 40% CO2. The amount of a 76 kDa polypeptide (p76) on SDS-PAGE decreased markedly in the presence of CAP at 40% CO2 but not at 5% CO2. A partial amino acid sequence of p76 was 71-100% identical (10-14 identical residues out of 14 amino acids determined) to those of transketolases (TKLs) reported in higher plants and a cyanobacterium. In agreement with these observations, the TKL activity in C. littorale was decreased by CAP at 40% CO2, but not at 5% CO2. The transient decrease in TKL activity caused by CAP under 40% CO2 was well correlated with that in PhiII. These results indicate that the addition of CAP directly or indirectly influences the stability of TKL in C. littorale at 40% CO2, but not at 5% CO2, and that photosynthetic activity was reduced by a decrease in TKL activity.     

Attenuation of a delayed increase in the extracellular glutamate level in the peri-infarct area following focal cerebral ischemia by a novel agent ONO-2506

A novel agent, ONO-2506 [(R)-(-)-2-propyloctanoic acid, ONO Pharmaceutical Co. Ltd.] was previously shown to mitigate delayed infarct expansion through inhibition of the enhanced production of S-100beta, while inducing a prompt symptomatic improvement that attained a significant level as early as 24h after drug administration. To elucidate the mechanism underlying the prompt symptomatic improvement, the present study aimed to examine whether ONO-2506 modulates the level of extracellular glutamate ([Glu]e) in the rat subjected to transient middle cerebral artery occlusion (tMCAO). In this model, it had been shown that ONO-2506 reduces the infarct volume, improves the neurological deficits, and enhances the mRNA expression of glial glutamate transporters (GLT-1 and GLAST). The [Glu]e levels in the ischemic cortices were continuously measured using intracerebral microdialysis. The alterations in the [Glu]e levels in the sham-operated and tMCAO-operated groups with or without drug administration were compared. In the tMCAO groups, the [Glu]e level increased during tMCAO to a similar extent, returned to normal on reperfusion, and increased again around 5h. In the saline-treated group, however, the [Glu]e level further increased from 15 h on to reach about 280% of the normal level at 24h. This secondary increase in the [Glu]e level in the late phase of reperfusion was prevented by ONO-2506. The intracerebral infusion of glutamate transporter inhibitor, l-trans-pyrrolidine-2,4-dicarboxylic acid, at 24h after tMCAO induced an increase in the [Glu]e level, which was marked in both the sham-operated and ONO-2506-treated groups, but much less pronounced in the saline-treated group. The above results suggest that functional modulation of activated astrocytes by pharmacological agents like ONO-2506 may inhibit the secondary rise of [Glu]e level in the late phase of reperfusion, leading to amelioration of delayed infarct expansion and neurological deficits.     

Expression of lysosomal protective protein/cathepsin A in a stably transformed human neuroblastoma cell line during bi-directional differentiation into neuronal and Schwannian cells

Human neuroblastoma GOTO cell lines were established that stably express recombinant human lysosomal protective protein/cathepsin A (PPCA) cDNA by transfection. Intracellular cathepsin A (acid serine carboxypeptidase) activity increased four-fold compared with in those of the parent and mock-transfected cell lines. The immunoreactive 54 kDa precursor/zymogen and mature 32/20 kDa two-chain forms were produced in the cells. The amount of the latter form expressed in the GOTO cells was significantly larger than those in the PPCA-overexpressing CHO cell lines previously established. The intracellular proteins showed a typical lysosomal granular distribution and the glycosylated 54 kDa precursor was secreted into the culture medium without the addition of an alkalizing agent. The PPCA-overexpressing cell lines also retained the ability to differentiate bi-directionally as well as the parent cells; into neuronal cells on induction by dibutyryl cAMP in serum-free medium and into Schwannian cells on induction by bromodeoxyuridine. During the course of differentiation into neuronal and Schwannian cells, the intracellular cathepsin A activity further increased two and five times, respectively, which was associated with an increase in the expression of the 32/20 kDa two-chain form. The glycosylated precursor proteins were taken up via the mannose 6-phosphate receptors, and the cathepsin A, alpha-neuraminidase and beta-galactosidase (beta-Gal) activities deficient in the fibroblasts derived from a patient with PPCA deficiency (galactosialidosis) were restored. These results suggest that the bi-directional differentiation of GOTO cell lines stably expressing the recombinant human PPCA gene could be a model system for analyzing the functions of PPCA in peripheral neuronal cells and Schwannian cells as well as the recombinant PPCA could be a useful source for enzyme replacement therapy (ERT) for galactosialidosis patients.