Effect of solution non-ideality on erythrocyte volume regulation

A non-ideal, hydrated, non-dilute pseudo-binary salt-protein-water solution model of the erythrocyte intracellular solution is presented to describe the osmotic behavior of human erythrocytes. Existing experimental activity data for salts and proteins in aqueous solutions are used to formulate van Laar type expressions for the solvent and solute activity coefficients. Reasonable estimates can therefore be made of the non-ideality of the erythrocyte intracellular solution over a wide range of osmolalities. Solution non-ideality is shown to affect significantly the degree of solute polarization within the erythrocyte intracellular solution during freezing. However, the non-ideality has very little effect upon the amount of water retained within erythrocytes cooled at sub-zero temperatures.     

Raman spectra and vibrational assignments for deuterated membrane lipids. 1,2-Dipalmitoyl phosphatidylcholine-d9 and -d62

Vibrational Raman spectra of polycrystalline 1,2-dipalmitoyl phosphatidylcholine-d9 (fully deuterated choline methyl groups) and 1,2-dipalmitoyl phosphatidylcholine-d62 (fully deuterated acyl chains) were recorded in the 3050- 2800, 2250-2050 and 1800-700 cm-1 regions. The fundamental vibrational modes were assigned primarily on the basis of isotopic frequency shift ratios, group frequency correlations and comparisons with specific model compounds. Since deuterium-substituted lipids provide well-isolated spectral probes, particularly in the carbon-deuterium stretching region, the dependence of the 2250-2050 cm-1 region on lipid phase was examined for the dipalmitoyl phosphatidylcholine-d62 species. The methylene CD2 deformation and twisting modes at 984 and 919 cm-1, respectively, also exhibit intense, isolated vibrational transitions which should prove useful for monitoring molecular order in mixed dueterated and undeuterated lipid systems. Except for the relatively weak choline methyl C-D and C-H stretching modes, the spectrum of 1,2-dipalmitoyl phosphatidylcholine-d9 is not distinguishable from that of the undeuterated system. For both the d9 and undeuterated species, the vibrational modes associated with the lipid head group region are sensitive to slight hydration.     

Interaction of melittin with dimyristoyl phosphatidylcholine liposomes. Evidence for boundary lipid by raman spectroscopy

The interaction of melittin, a polypeptide consisting of 26 amino acid residues, with dimyristoyl phosphatidylcholine bilayers was investigated by vibrational Raman spectroscopy. Spectral peak height intensity ratios, involving vibrational transitions in both the 3000 cm^?1 acyl chain methylene carbon-hydrogen stretching mode region and the 1100 cm^?1 acyl chain carbon-carbon skeletal stretching mode interval, served as temperature profile indices for monitoring the bilayer order-disorder processes. For a lipid : melittin molar ratio of 14 : 1 two order-disorder transitions were observed. In comparison to a gel to liquid crystalline phase transition of 22.5°C for the pure lipid, the lower transition, exhibiting a 2°C width, is centered at 17°C and is associated with a depression of the main lipid phase transition of dimyristoyl phosphatidylcholine. The second thermal transition, displaying a 7°C interval, occurs at approx. 29°C and is associated with the melting behavior of approximately seven immobilized boundary lipids which surround the inserted hydrophobic segment of the polypeptide. For a lipid : melittin molar ratio of 10 : 1 two thermal transitions are also observed at 11 and 30°C. As before, they represent, respectively, the main gel to liquid crystalline phase transition and the melting behavior of approximately four boundary lipids attached to melittin. From these data alternative schemes are suggested for disposing the immobilized lipids around the hydrophobic portion of the polypeptide within the bilayer.     

Studies of lipopolysaccharides from Yersinia pseudotuberculosis subtypes VA and VB

Lipopolysaccharides (LPS) from various strains of Yersinia pseudotuberculosis type V have been isolated and characterised. Differences in sugar composition and serological activity of LPS from various strains within the same subtype of Y. pseudotuberculosis have been revealed.     

Hepatic microsomal epoxide hydrase. Chemical evidence for a single polypeptide chain.

Highly purified hepatic microsomal epoxide hydrase, which had been purified in the presence of proteolytic enzyme inhibitors, was subjected to carboxypeptidase Y digestion, automated Edman degradation, and carbohydrate analysis. Carboxypeptidase Y digestion resulted in the near stoichiometric release of leucine, the COOH-terminal amino acid. Automated Edman degradation permitted the identification of the first 20 amino acid residues of epoxide hydrase. Methionine was identified as the NH2-terminal residue. The NH2-terminal region of epoxide hydrase is similar in hydrophobicity to the NH2-terminal precursor segments of several secretory proteins and the NH2-terminal regions of several microsomal cytochromes P-450. Carbohydrate analyses of the enzyme revealed the presence of 0.5 to 1.0 mol of mannose/50,000 g of protein. These results provide evidence for the presence of a single polypeptide chain in our purified enzyme preparations and suggest that there may be only one enzymic form of epoxide hydrase in microsomes from phenobarbital-treated rats.     

Effects of temperature and molecular interactions on the vibrational infrared spectra of phospholipid vesicles

Infrared spectra were obtained as a function of temperature for a variety of phospholipid/water bilayer assemblies (80% water by weight) in the 3000-950 cm^?1 region. Spectral band-maximum frequency parameters were defined for the 2900 cm^?1 hydrocarbon chain methylene symmetric and asymmetric stretching vibrations. Temperature shifts for these band-maximum frequencies provided convenient probes for monitoring the phase transition behavior of both multilamellar liposomes and small diameter single-shell vesiclesof dipalmitoyl phosphatidylcholine/water dispersions. As examples of the effects of bilayer lipid/cholesterol/water (3 : 1 mol ratio) and lipid/cholesterol/amphotericin B/water (3 : 1 : 0.1 mol ratios) vesicles were examined using the methylene stretching frequency indices. In comparison to the pure vesicle form, the transition width of the lipid/cholesterol system increased by nearly a factor of two (to 8°C) while the phase transition temperature remained approximately the same (41° C). For the lipid/cholesterol/amphotericin B system, the phase transition temperature increased by about 4.5° C (to 45.5°C) with the transition width increasing by nearly a factor of four ($\text{to}\text{≈ 15°}\text{C}$) above that of the pure vesicles. The lipid/cholesterol/amphotericin B data were interpreted as reflecting the formation below 38°C of a cholesterol/amphotericin B complex whose dissociation at higher temperature (38–60°C range) significantly broades the gel-liquid crystalline phase transition.     

Esterification of arylhydroxylamines: Evidence for a specific gene product in mutagenesis

Characterization of a $\text{Salmonella}\text{typhmurium}$ mutant strain (TA98/1,8-DNP₆) resistant to the mutagenicity of nitrated polycyclic aromatic hydrocarbons (nitroarenes) revealed that it was also non-responsive to the mutagenic action of nitroso- and N-hydroxylaminoarenes. The mutant strain was fully sensitive to the mutagenic action of the corresponding hydroxamic acid ester. These results suggest that TA98/1,8-DNP₆ is deficient in a specific esterifying enzyme and that esterification of the penultimate mutagenic metabolites of nitro- and aminoarenes ($\text{e.g.}$, arylhydroxylamines) to form potent electrophiles is controlled by a specific gene.     

Thrombin- and histamine-induced signal transduction in human endothelial cells. Stimulation and agonist-dependent desensitization of protein phosphorylation

Treatment of human endothelial cells with thrombin, histamine, or dioctanoylglycerol (DiC8), a synthetic diacylglycerol, resulted in the rapid and transient phosphorylation of a Mr = 29,000 protein (P29) in a dose-dependent manner. Various tumor promoters also promoted P29 phosphorylation while the adenylate cyclase activator, forskolin, did not. The level of phosphorylation with all three agonists was similar (2.5-4 fold), and analysis of P29 by two-dimensional gel electrophoresis revealed identical patterns in each case. Receptor specificity was demonstrated for the histamine-stimulated changes; pyrilamine (10(-6) M; H1) but not cimetidine (10(-4); H2) blocked the response. The thrombin effect was active site-dependent. Phosphorylation induced by thrombin and histamine occurred within 1 min, peaked between 5 and 10 min, and returned to control levels by 1 h. DiC8-induced phosphorylation occurred more slowly but was also reduced by 1 h while phorbol ester treatment prolonged phosphorylation for at least 4 h. Treatment of these cells with thrombin or histamine for 1 h desensitized P29 to further phosphorylation by the homologous agonist although secondary phosphorylation could occur with heterologous compounds. However, if the primary agonist was removed following the onset of a desensitized state, secondary phosphorylation of P29 could be stimulated by the same compound. These same results were observed with two other phosphoproteins Mr = 18,000 (P18) and 80,000 (P80) which became more highly phosphorylated in response to thrombin treatment and with histamine/thrombin-stimulated prostaglandin I2 production. In contrast, homologous down-regulation of P29 phosphorylation was not observed with DiC8-treated cells, and the decline in phosphorylated P29 was associated with the loss of functional DiC8. The protein kinase inhibitors staurosporine and H-7 blocked P18 and P80 phosphorylation by thrombin but had no effect on P29 phosphorylation by histamine, thrombin, or DiC8 suggesting distinct pathways leading to the phosphorylation of these different proteins. These data suggest that multiple and independent thrombin/histamine-induced events are susceptible to receptor occupancy-dependent homologous down-regulation.     

Differing Neurochemical and Morphological Sequelae of Global Ischemia: Comparison of Single- and Multiple-Insult Paradigms

The purpose of this investigation was to investigate pathomechanisms responsible for the deleterious effects of repeated episodes of brief forebrain ischemia. Halothane-anesthetized male Wistar rats were subjected to either (a) a single 15-min period or (b) three 5-min periods (separated by 1 h) of global forebrain ischemia by bilateral carotid artery occlusions plus hypotension (50 mm Hg), followed by various periods of recirculation. Brain temperature was normothermic throughout. In one series of rats, extracellular levels of glutamate, glycine, and gamma-aminobutyric acid (GABA) were measured in the dorsolateral striatum (n = 6-8 per group) and lateral thalamus (n = 4-6 per group) by microdialysis and HPLC before and during ischemia and during 3-5 h of recirculation. In a parallel series of rats (n = 6 per group), ischemic cell change was quantified at 2 (dark neurons), 24, or 72 h following either single or multiple ischemic insults. A single 15-min ischemic period led to massive glutamate release (13-fold increase; p = 0.001), which returned to normal by 20-30 min of recirculation and remained normal thereafter. By contrast, in rats with three 5-min periods of ischemia, the glutamate level rise with each repeated insult (four- to 4.5-fold; p < or = 0.02) was smaller than that observed during the single 15-min insult, but a late sustained rise (five- to six-fold; p < 0.05) occurred at 2-3 h of recirculation. Brief ischemia-induced elevations of glycine and GABA levels were detected in both the single- and multiple-insult groups, with normalization during recirculation. In contrast, the excitotoxic index, a composite measure of neurotransmitter release ([glutamate] x [glycine]/[GABA]), differed markedly following single versus multiple insults (p = 0.002 by repeated-measures analysis of variance) and increased by seven- to 12-fold (p < 0.05) at 1-3 h following the third insult. The total amount of glutamate released was 3.3-fold higher in the multiple-insult than in the single-insult group (p < 0.02). At 2 h of recirculation, histopathological analysis of dorsolateral striatum showed a significantly greater frequency of dark neurons in the multiple- than in the single-insult group (p < 0.05 by analysis of variance). In the thalamus, a higher frequency of ischemic neurons was seen in the multiple-than in the single-insult group at all intervals studied. Thus, in rats with multiple ischemic insults, accelerated ischemic damage was found in the striatum, and severe ischemic injury was documented in the thalamus.(ABSTRACT TRUNCATED AT 400 WORDS)