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The denaturation of bacteriorhodopsin by various organic solvents was studied using absorption, circular dichroism (CD) and fluorescence measurements. Organic solvents with a hydrogen-bonding group caused the release of retinal. The CD measurements showed that the helical structure was maintained even in the denatured state, whereas its tertiary structure was destroyed. The change in fluorescence intensity of tryptophan and fluorescent retinal also confirmed that the tertiary structure was destroyed. Comparison of the denaturation efficiency of various organic solvents showed that the concentration at denaturation was inversely proportional to the partition coefficient of the denaturant. This inverse proportionality clearly indicated that denaturation was determined by the concentration of denaturants which partitioned into the hydrophobic region of the membrane. It was discussed from the experimental results that the tertiary structure of bacteriorhodopsin was stabilized by the hydrogen-bonding networks between side chains of the helices. The results obtained from analysis of the amino acid sequence were also consistent with the hydrogen-bonding mechanism for the formation of the tertiary structure.

Three forms of 20-kDa myosin light chain (MLC), unphosphorylated, monophosphorylated, and diphosphorylated MLC (designated 20K, 20K-P, and 20K-PP) were demonstrated in thrombin-stimulated human platelets by two different gel electrophoretic methods: in the presence of glycerol urea or in two dimensions (isoelectric and sodium dodecyl sulfate). The diphosphorylation of platelet 20-kDa MLC increased, dose dependently, up to 0.4 U/ml thrombin and reached 25% of platelet 20-kDa MLC. After mono- or diphosphorylated 20-kDa MLC from thrombin-stimulated platelets was digested with trypsin, the analysis using two-dimensional peptide mapping demonstrated that two different sites were phosphorylated by MLC kinase and protein kinase C, as noted in the case of 12-O-tetradecanoylphorbol-13-acetate-stimulated platelets (M. Naka, et al. (1983) Nature (London) 306, 490-492). The more rapid monophosphorylation was catalyzed preferentially by MLC kinase while the slower and additional phosphorylation was catalyzed mainly by protein kinase C. These results suggest the importance of distinguishing multiple site phosphorylation of 20-kDa MLC in thrombin-activated human platelets.

Cilostazol (6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2(1H)- qui nolinone, OPC-13013), a new antithrombotic agent, has a potent inhibitory action on platelet aggregation. The cellular and subcellular distribution of this compound was investigated. Autoradiographic studies using tritiated cilostazol showed that this compound penetrates the cell membrane of Chinese hamster ovary cells, clone K1 (CHO-K1 cells) and is distributed mainly in the cytoplasma. These results suggest that this compound exhibits its pharmacological action through specific inhibition of cytoplasmic cyclic AMP phosphodiesterase.

To elucidate the effects of coronary thrombolytic therapy in acute myocardial infarction, we observed serially the degree of left ventricular (LV) wall motion immediately after on day 1, and on days 7, 14, 21 and 28 after thrombolytic therapy, in 22 patients with acute anteroseptal myocardial infarction. Base-line coronary arteriography revealed significant lesions in the proximal portions of the left anterior descending artery of all the patients. The patients were categorized according to results of thrombolytic therapy as Group I-a: seven patients with spontaneous or successful recanalization within three hours of onset of chest pain; Group I-b: nine patients with successful recanalization between three and seven hours, with a mean of 4.8 hours from onset; and Group II: six patients in whom thrombolytic therapy was unsuccessful and infarct-related vessels remained totally occluded. The LV wall motion index (WMI) was defined as the sum of point scores for the degrees of regional wall motion at nine segments on serial two-dimensional echocardiograms, and used for quantitative assessments of LV function. Results were as follows: On day 1, immediately after thrombolytic therapy, the WMI of Group I-a was smaller than that of Group II. However, there was no significant difference between Groups I-a and I-b and between Groups I-b and II. These findings suggest that LV function cannot be recovered immediately after recanalization of occluded arteries unless recanalization occurs exceptionally early. Percent improvement of the WMI from days 1 to 28 in Group I-a, 65 +/- 14%, was significantly greater than that in Group I-b, 31 +/- 18%. However, Group II did not show significant improvement in the WMI. The WMI in Group I-a decreased significantly from days 1 to 7 (9.0 +/- 1.6 vs 7.1 +/- 1.8, p less than 0.05); whereas, the WMI in Group I-b showed no significant decrease until day 21. On day 1, the regional wall motion of the antero-apical wall was akinetic or dyskinetic in all patients studied. On day 28, it improved in six of seven patients in Group I-a, while it remained akinetic or dyskinetic in all patients in Groups I-b and II.(ABSTRACT TRUNCATED AT 400 WORDS)

Imino acid and related alicyclic amine concentrations in blood and urine of mammals including humans were concurrently determined by a selected ion monitoring technique. Nanomole levels of proline and pipecolic acid, and pyrrolidine and piperidine as well, were found in human urine. Proline levels but not pipecolic acid levels were higher in blood of humans than in urine. Pyrrolidine and piperidine levels in blood of humans were picomole levels and much lower than those in urine. Similar tendencies were also recognized when these 4 compounds were analyzed using animal blood and urine, although the levels were generally higher in animals than in humans. Significantly high concentrations of the imino acids and the amines were found also in animal semen.

To investigate whether early reperfusion (less than or equal to 3 hours) preserves aerobic myocardial metabolism in acute myocardial infarction, we examined serial changes in trans-cardiac lactate extraction after thrombolysis in 43 patients with acute antero-septal myocardial infarction. In the chronic phase, we also determined abnormally contracting segments as an index of infarct size and regional ejection fraction as an index of chronic regional cardiac function. In the early reperfusion group (less than or equal to 3 hours), positive lactate extraction was restored and there were small abnormally contracting segments and a high regional ejection fraction. The intermediate reperfusion group (3-5 hours), however, had sustained anaerobic lactate extraction, large abnormally contracting segments and a low regional ejection fraction. The late reperfusion (greater than 5 hours) group showed apparently aerobic lactate extraction, but had large abnormally contracting segments and a low regional ejection fraction. Thus, early reperfusion preserves aerobic lactate metabolism and good chronic ventricular function.

This study investigated the interactive activation process of homograph (H :e.g. "jam") and context word (c :e.g. "strawberry"). Subjects studied a list of H-C or C-H pairs, where presentation intervals between H and C were varied (0, 0.6, or 1.2 s). Then they were tested for the recognition memory of H in a changed context (e.g. "jam-traffic") in Exp. 1, or in the same context (e.g. "jam-raspberry") in Exp. 2. The recognition memory of H as a function of presentation interval was obtained. An interesting phenomenon was that the recognition memory of H in H-C pairs at the 1.2 s interval was poorer than that of the 0.6 s interval, when tested in a changed context, but better in the same context. This suggests that both meanings of H were activated at the 0.6 s interval, but one of them related to C was selected at the 1.2 s interval. Results were discussed in terms of the spreading activation model of semantic memory.

Large and small starch granules were isolated and characterized from mature barley kernels with waxy endosperms. The large granules of any given waxy cultivar contained more amylose than the small granules of the same cultivar. It was also found that large granules contained a greater amount of long amylopectin B chains and had a lower fraction III:fraction II ratio, one of the structural characteristics of amylopectin, than the small granules in the same cultivar. Small granules showed a wider range of gelatinization and smaller heat of gelatinization by differential scanning calorimetry.

Calcium ion-dependent interaction with purified calmodulin (CaM), of a potent inhibitor of histamine release from mast cells, trans-4-guanidinomethylcyclohexanecarboxylic acid p-tertbutylphenylester (GMCHA), was investigated using 5-(dimethylamino)-1-naphthalenesulfonyl-calmodulin (dansyl-CaM). GMCHA undergoes a fluorescence increase with the Ca2+-dansyl-CaM complex but there is no significant change in the dansyl-CaM fluorescence with GMCHA, up to a 10 microM in the absence of calcium ion. This suggests that binding of GMCHA to CaM is Ca2+-dependent on the apparent Kd is approximately 1 microM. GMCHA suppressed the fluorescence of the hydrophobic probe 8-anilino-1-naphthalenesulfonate (ANS), in the presence of the Ca2+-CaM complex, with an IC50 value of 3 microM. [3H]N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) bound to the purified CaM was displaced, in a concentration dependent manner by GMCHA, the Ki value of GMCHA against the binding of W-7 to CaM was 2.3 microM, and there was a selective inhibition of the Ca2+-CaM induced activation of enzymes such as myosin light chain kinase. Increasing the CaM concentration in the presence of Ca2+ overcame the GMCHA-induced inhibition of myosin light chain kinase activation, with a Ki value of 2.7 microM. GMCHA at these concentrations is effective in inhibiting the release of histamine from mast cells. Moreover, this compound suppresses platelet secretion and relaxes vascular strips, at concentrations similar to those seen with the CaM interacting action and characteristic of CaM antagonists such as W-7. GMCHA also inhibits the Ca2+, CaM-dependent myosin light chain phosphorylation of human platelets. These results suggest that GMCHA, a potent inhibitor of histamine release from mast cells, suppresses platelet secretion, relaxes vascular smooth muscle and inhibits Ca2+, CaM-dependent protein phosphorylation, all at similar concentrations.

It is very important to evaluate the size of myocardial infarction and reversibility of ischemic myocardium in order to determine the severity and prognosis of patients with acute myocardial infarction. We performed thallium-201 redistribution myocardial scintigraphy and first pass radionuclide angiography in 12 patients with acute myocardial infarction. In eight cases, left ventricular wall motion was improved after nitroglycerin infusion (improved group), but in four cases it was unchanged (non-improved group). Six of the improved group showed a positive redistribution image, while in the non-improved group, no patient showed such an image. Ejection fraction increased after nitroglycerin infusion in the improved group, but unchanged in the non-improved group. There was no significant difference in hemodynamic parameters between the two groups at the time of admission, and after treatment hemodynamic parameters changed in the same way in both groups. In coronary angiography, three collateral channels were observed in the improved group, but none in the non-improved group. The severity of coronary artery stenosis in the two groups was almost identical. In eight patients, their regional ventricular wall motion improved after nitroglycerin administration, but there were four patients whose regional ventricular wall motion was unchanged. In both groups, hemodynamic and coronary angiographic findings were not different. But there were many positive redistribution images and collateral channels in the improved group. This result shows that reversible ischemia is indispensable for the improvement of regional wall motion. There was a case which showed a positive redistribution image on acute phase Tl-201 scintigraphy and a transient defect on the chronic phase exercise Tl-201 scintigraphy. But after the improvement of ischemia by aorto-coronary bypass operation, the defect on Tl-201 scintigraphy decreased and the wall motion improved. Neither redistribution on acute phase scintigraphy nor a transient defect on chronic phase exercise scintigraphy was observed in the non-improved group. From these data we conclude that the improvement of regional wall motion depends on reversible ischemia.

The beneficial effects of an antagonist of the thromboxane A2/prostaglandin endoperoxide receptor, 7-[2 alpha,4 alpha-(dimethylmethano)-6 beta-(2-cyclopentyl-2 beta- hydroxyacetamido)-1 alpha-cyclohexyl]-5(Z)-heptenoic acid (ONO-3708) on thrombosis were examined. ONO-3708 at 0.1-3 microM inhibited the human platelet aggregation induced by thromboxane A2, prostaglandin H2, collagen, ADP (secondary phase) and epinephrine (secondary phase) without affecting prostanoid synthesis and the content of cyclic AMP in platelets. The in vivo effects, on coronary thrombosis in this case, were examined in two canine models. ONO-3708, 3 to 300 micrograms/kg i.v., prevented dose dependently the coronary thrombosis induced by partial obstruction of the coronary artery. ONO-3708, 3 micrograms/kg per min i.v., significantly prevented electrically stimulated coronary thrombosis without affecting systemic blood pressure and heart rate. These results indicate that the thromboxane A2/prostaglandin endoperoxide receptor could play an important role in the pathogenesis of thrombosis and that ONO-3708 may have therapeutic advantages in preventing thrombosis.

Myosin 20 K-Da light chain phosphorylation in human platelets was found to be catalyzed by MLCK in the early phase during collagen activation. The effect of newly synthesized selective inhibitor of MLCK, ML-9, on collagen induced platelet activation was investigated. ML-9 delayed the time course of the myosin 20 K-Da light chain phosphorylation, sequentially led to a delay in aggregation, secretion and phosphorylation of the 40K-Da peptide, in a dose-dependent fashion. It is proposed that the MLCK catalyzed phosphorylation of myosin 20 K-Da light chain may be an initial response and if so may influence the sequent reactions in the activation of platelets with collagen.

Age-related alterations in pituitary-thyroid function were studied in 173 female patients with simple goitre and in 70 normal female subjects. They were divided into 4 groups according to age: A group, less than 19 years; B group, 20 to 29 years; C group, 30 to 39 years; D group, 40 to 59 years. Serum triiodothyronine (T3) concentrations decreased progressively but insignificantly with age in female patients with simple goitre and in normal female subjects, whereas serum thyroxine (T4) concentrations remained constant throughout the studied age range. Only in female patients with simple goitre, did basal serum TSH concentrations show a tendency to increase with age. However, thyrotrophin-releasing hormone (TRH)-stimulated increase of serum TSH was progressively augmented with age both in female patients with simple goitre and in normal female subjects; the magnitude of change was greater in the former group. As reflected by acute increases of serum T3 and T4 concentrations, thyroidal responsiveness to endogenous TSH was progressively depressed with age in female patients with simple goitre and in normal female subjects. This age-related thyroidal refractoriness to TSH was more apparent when the changes were expressed as delta T3 (stimulated T3 - basal T3)/delta TSH (maximum TSH after TRH - basal TSH), and delta T4 (stimulated T4 - basal T4)/delta TSH. Delta T4/delta TSH was lower in female patients with simple goitre than in normal female subjects in all age groups. However, the difference was significant only for delta T4/delta TSH in group A. Thyroidal responsiveness to exogenous TSH also gradually declined with age in female patients with simple goitre.(ABSTRACT TRUNCATED AT 250 WORDS)