Intraretinal isolation of P3 subcomponents in the isolated rabbit retina after treatment with sodium aspartate.
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Amoebae belonging to the genus Acanthamoeba are potentially pathogenic to humans, causing mainly amoebic keratitis. Pathogenic ability of the 15 known Acanthamoeba genotypes is under investigation. We report that four out of five cases of amoebic keratitis studied in Greece, present T4 sequence type, while the remaining one presents T5 sequence type (Acanthamoeba lenticulata), which is the second most frequent genotype found among environmental samples. Thus, it is confirmed, for the first time to our knowledge, that A. lenticulata can cause keratitis. However the reason that it is under represented in clinical samples compared to environmental ones is unknown.
Although members of the protein tyrosine phosphatase (PTP) family are known to play critical roles in various cellular processes through the regulation of protein tyrosine phosphorylation in cooperation with protein tyrosine kinases (PTKs), the physiological functions of individual PTPs are poorly understood. This is due to a lack of information concerning the physiological substrates of the respective PTPs. Several years ago, substrate-trap mutants were developed to identify the substrates of PTPs, but only a limited number of PTP substrates have been identified using typical biochemical techniques in vitro. The application of this strategy to all the PTPs seems difficult, because the substrates identified to date were restricted to relatively abundant and highly tyrosine phosphorylated cellular proteins. Therefore, the development of a standard method applicable to all PTPs has long been awaited. We report here a genetic method to screen for PTP substrates which we have named the "yeast substrate-trapping system." This method is based on the yeast two-hybrid system with two essential modifications: the conditional expression of a PTK to tyrosine-phosphorylate the prey protein, and screening using a substrate-trap PTP mutant as bait. This method is probably applicable to all the PTPs, because it is based on PTP-substrate interaction in vivo, namely the substrate recognition of individual PTPs. Moreover, this method has the advantage that continuously interacting molecules for a PTP are also identified, at the same time, under PTK-noninductive conditions. The identification of physiological substrates will shed light on the physiological functions of individual PTPs.
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The determination of the relationship between ligand affinity and bioactivity is important for the understanding of receptor function in biological systems and for drug development. Several physiological and pathophysiological functions of bradykinin (BK) are mediated via the B2 receptor. In this study, we have examined the relationship between B2 receptor (soluble and membrane-bound) binding of BK peptidic antagonists, inhibition of calcium signalling at a cellular level, and in vitro inhibition of ileum contraction. Only human systems were employed in the experiments. Good correlations between the studied activities of BK antagonists were observed for a variety of different peptidic structures. The correlation coefficients (r) were in the range of 0.905 to 0.955. In addition, we analyzed the effect of the C-terminal Arg9 removal from BK and its analogs on B2 receptor binding. The ratios of binding constants (Ki(+Arg)/Ki(-Arg)) for the Arg9 containing compounds and the corresponding des-Arg9 analogs varied from about 10 to 250,000. These ratios strongly depend on the chemical structures of the compounds. The highest ratios were observed for two natural agonist pairs, BK/des-Arg9-BK and Lys0-BK/des-Arg9-Lys0-BK.
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A new experimental porcine model for creating selective ischemia of a specific part of the myocardium while the rest of the myocardium remains free of ischemia has been used to study the electrocardiographic (ECG) changes deriving from selective ischemia of the right ventricular (RV) free wall. A patch was stitched to the ventricle to produce selective myocardial ischemic injury. In a preliminary study of nine pigs, selective ischemia of the left ventricular free wall in five and of the RV free wall in four animals was induced, and a postmortem dye injection was performed to evaluate blood flow in the area of ischemia. In an ECG study of 20 pigs, the baseline ECG was recorded with use of the standard leads I-III, aVR, aVL, and aVF, left precordial leads (V1-V6), and leads V4R, V3H, and V4H and 1 hour after inducing ischemia, the ECG study was repeated. Our experimental model produced ischemic injury in which the location and surface area were known antemortem. In the 20 pigs, ST-segment changes were recorded in leads V1-V3, V3H, and V4H. In only four pigs (20%) was ST-segment elevation recorded in lead V4R. The results show that the ECG signs of selective ischemia of the RV free wall may imitate the signs of anterior or anterolateral infarction of the left ventricle. In this study, elevation of the ST-segment in lead V4R was not pathognomonic of for RV ischemia. This model is a new tool for studying hemodynamic and ECG changes of selective univentricular or biventricular ischemic injury.
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