Platelet aggregation: inhibition of secondary aggregation by phenol.

Abnormal platelet function in patients with chronic renal failure has been associated with elevated levels of phenol and phenolic acids in serum. In vitro studies show inhibition of secondary aggregation by phenol, suggesting that phenol acts at the platelet release reaction. When platelet rich plasma was incubated with phenol, inhibition was found to decrease with increasing preincubation time at 37 degrees C, but not at 0 degree C. Also, the inhibitory effect of phenol in vitro was overcome by the addition of arachidonic acid. These findings demonstrate inhibition of secondary aggregation by phenol. Thus the site of the inhibitory action of phenol was at the initiation of the secondary wave of platelet aggregation.

Recurrent decreases of erythrocyte electrophoretic mobility in rats exposed to 1.0 R whole-body x-radiation.

Circulating erythrocytes from rats were examined up to 30 weeks post whole-body exposures of 1.0 R for alterations in the expression of net negative surface charge as measured by whole-cell microelectrophoresis in saline sorbitol. Erythrocyte electrophoretic mobility was determined in an apparatus composed of a horizontal transilluminated cylindrical chamber, equipped with a reversible, blacked platinum electrode, immersed in a water bath maintained at 25.0 +/- 1.0 degree C (Rank Brothers). In two separate experiments, recurrent decreases in the expression of net negative surface charge occurred at 10, 17, and 30 weeks post-irradiation. At these times distributional analyses of recorded erythrocyte electrophoretic mobility (EEPM) values revealed a skewing of the normally distributed EEPM population values to lower EEPM. Total sialic acid content released from hydrolyzed erythrocyte membrane preparations revealed no significant differences between erythrocytes from sham and irradiated animals. In vivo post-irradiation labeling of erythrocytes with diisopropyl-[32P] phosphorofluoridate at 4 and 33 weeks (separate experiments) indicated only a minor abbreviated erythrocyte life span at 33 weeks. Therefore, effects from low dose (1.0 R) whole-body irradiation would appear to include a recurrent defect in the expression of the net negative surface charge.

Diazepam receptor: specific nuclear binding of [3H]flunitrazepam.

Autoradiographic localization of [3H]flunitrazepam in nuclei of the rat cerebral cortex was further confirmed by biochemical analysis of specific nuclear binding. Highly purified rat cerebral cortex nuclei were shown to bind [3H]flunitrazepam specifically. The Kd(app) for nuclear binding was 28 nM for the nuclei compared with a Kd(app) of 1.1 nM for binding of [3H] flunitrazepam to synaptosomal membrane fractions of the same tissue. Inhibition of the nuclear binding with inosine and hypoxanthine was greater than inhibition of the synaptic membrane fractions. These results lead to to conclude that specific binding may occur at both the synaptic membrane and the nuclear levels and that different endogenous ligands may compete at each site for binding. Furthermore, the possibility exists for translocation and alteration of the bound ligand complex from membrane site to nuclear site.

Increased specific binding of [3H]diazepam in rat brain following chronic diazepam administration.

Male rats (175 g) were given 30 mg of diazepam in their food daily for 35 days. The animals became drowsy and ataxic from this high dose of drug. After the 35-day dosing, the rats were killed daily, and specific binding of [3H]diazepam and [3H]flunitrazepam was determined in synaptosomal preparations from these and corresponding control rats. On days 3, 4, 6, and 7 after the treatment period the specific binding and specific binding of [3H]diazepam was double that of the control binding and specific binding of [3H]flunitrazepam was 1.67 times that of control. The data indicate that very high doses of diazepam, given for long periods, cause increased specific binding of radiolabeled ligand to brain subfractions. The possible mechanisms and implications are discussed. When lower doses or shorter dosage regimens are used, increased binding is not observed.

Properties of rat cerebral cortex neuronal nuclear surfaces: electrokinetic parameters.

Rat cerebral cortex neuronal nuclei were isolated by a mild technique utilizing sucrose; citric acid was not used in the isolation of the nuclei. These nuclei in 0.0145 M NaC1, 4.5 per cent sorbitol, and 0.6 mM NaHCO3 with pH 7.2 plus or minus 0.1 at 25 degrees C had an electrophoretic morbidity of minus 2.01 mum-s(-1)-V(-1)-cm(-1). The mobility curves for the brain nuclei indicated that the surface had an acid-dissociable group of pK APPROXIMATELY 2.7. Nuclei treated with 50 mg neurominidase/mg particle protein had a mobility of minus 1.4 mum-s(-1)-V(-1)-cm(-1). DNase or RNase at 50 mug/mg protein had no effect on the mobility of the isolated nuclei. Concanavalin A at 50 mug/mg protein decreased the nuclei electrophoretic mobility to minus 1.82 mum-s(-1)-V(-1)-cm(-1). The results are interpreted to mean that the brain nuclear external surface contains terminal sialic acid residues, but it is completely devoid of nucleic acids, and it binds canavalin A.

Electrokinetic properties of isolated cerebral-cortex synaptic vesicles.

Synaptic vesicles isolated from guinea-pig cerebral cortex had an electrophoretic mobility of -3.55mum.s(-1).V(-1).cm in saline-sorbitol, pH7.2, at 25 degrees C (ionic strength 0.015g-ions/1). The mobility was pH-dependent, varied with ionic strength and indicated that the vesicular surface contained weak acidic functions with a pK(a) in the range 3.0-3.8. Although the vesicular surface was determined to be highly negatively charged, treatment with neuraminidase had no effect on mobility and indicated that the relatively strong carboxyl groups of sialic acid do not contribute significantly to vesicular electrokinetic properties. Treatment of synaptic vesicles with trypsin or trypsinized concanavalin A resulted in increases in mobility, but treatment with ribonuclease, deoxyribonuclease, chrondroitinase ABC or hyaluronidase had no significant effect on mobility. Mn(2+) or Ca(2+) was more effective in decreasing vesicle mobility than was Mg(2+), Sr(2+) or Ba(2+). The electrokinetic properties of the synaptic vesicle surface are discussed and contrasted with the properties of the synaptosomal membrane.

Mitochondrial autonomy. Sialic acid residues on the surface of isolated rat cerebral cortex and liver mitochondria.

N-acetylneuraminic acid at the surfaces of rat cerebral cortex and liver mitochondria and derived mitoplasts (inner membrane plus matrix particles) was studied biochemically and electrokinetically. Rat cerebral cortex mitochondria in 0.0145 M NaCl, 4.5% sorbitol, pH 7.2 +/- 0.1, 0.6 mM NaHCO(3), had an electrophoretic mobility of - 2.88 +/- 0.01 micro/sec per v per cm. In the same solution the electrophoretic mobility of rat liver mitochondria was - 2.01 +/- 0.02, of rat liver mitoplasts was - 1.22 +/- 0.07, and of rat cerebral cortex mitoplasts - 0.91 +/- 0.04 micro/sec per v per cm. Treatment of these particles with 50 microg neuraminidase/mg particle protein resulted in the following electrophoretic mobilities in micro/sec per v per cm: rat cerebral cortex mitochondria, - 2.27; rat liver mitochondria, - 1.40; rat cerebral cortex mitoplasts, - 0.78; and rat liver mitoplasts, - 1.10. Rat liver mitochondria, mitoplasts, and outer mitochondrial membranes contained 2.0, 1.1, and 4.1 nmoles of sialic acid/mg protein, respectively. 10% of the liver mitochondrial protein and 27.5% of the sialic acid was solubilized in the mitoplast and outer membrane isolation procedure. Rat cerebral cortex mitochondria, mitoplasts, and outer mitochondrial membranes contained 3.1, 0.8, and 6.2 nmoles sialic acid/mg protein, respectively; 10% of the brain mitochondrial protein and 49 % of the sialic acid was solubilized in the mitoplast and outer membrane isolation solution procedure. Treatment of both the rat liver and cerebral cortex mitochondria with 50 microg neuraminidase (dry weight) /mg protein resulted in the release of about 50% of the available outer membrane sialic acid residues. Treatment of all of the particles with trypsin caused release of sialic acid but did not greatly affect the particle electrophoretic mobility. In each instance, curves of pH vs. electrophoretic mobility indicated that the particle surface contained an acid dissociable group, most likely a carboxyl group of sialic acid with pK(a) approximately 2.7. Treatment of either the rat liver or the cerebral cortex mitochondria with trypsinized concanavalin A did not affect the particle electrophoretic mobility but did cause a decrease in the electrophoretic mobility of L5178Y mouse leukemic cells.