Polar secretion of endothelin-1 by cultured endothelial cells.

The aim of this study was to determine the permeability of endothelial monolayers for endothelin-1 and a possible directionality of the endothelin-1 secretion process. Human umbilical vein endothelial cells were cultured on acellular amniotic membranes, dividing the tissue culture wells into an apical (luminal) and a basolateral (abluminal) compartment. Whereas in the absence of endothelial monolayers 44.9 +/- 2.3 and 43.5 +/- 2.0% of the unilaterally added endothelin-1 permeated from the apical to the basolateral side and from the basolateral to the apical side, respectively, only 6.5 +/- 0.6 and 6.6 +/- 0.4% diffused in the presence of endothelial cells. Analyzing endothelin-1 secretion, approximately 80% of the total amount of synthesized endothelin-1 was found in the basolateral compartment; thrombin (10 units/ml) stimulated the production of endothelin-1 approximately 2-fold, but did not change the relative distribution of endothelin-1 between the apical and basolateral compartments. In the presence of dexamethasone (10(-7) M), a decrease in the level of endothelin-1 was found in the apical compartment, whereas the total amount of endothelin-1 produced was not affected. Dexamethasone did not influence the permeability of human umbilical vein endothelial cell monolayers for endothelin-1. These results strongly support the hypothesis that endothelin-1 is a local paracrine regulator of vasotone.     

Reaction of adrenal glomerular zone to detergents.

The effects have been investigated of chronic exposure to detergents on volumetric and histochemical features of the glomerular zone of adrenal glands in the white rat. Neutral lipids, triglycerides, phospholipids and nucleic acids were analysed. There was an increase in the quantities of all the three fatty substances in the corticocytes of the glomerular zone and the quantity of ribonucleic acid was enhanced, whereas that of deoxyribonucleic acid was reduced. The volumes of the nuclei differed significantly between the experimental and the control animals.     

Crystallization and preliminary X-ray analysis of gamma-aminobutyric acid transaminase

gamma-Aminobutyric acid transaminase from pig liver, an alpha 2 dimeric enzyme of Mr 110,100, has been crystallized by the vapour diffusion method with polyethylene glycol as precipitant. The crystals are monoclinic, space group P2(1), unit cell dimensions a = 82.1 A, b = 230.0 A, c = 70.3 A, beta = 123.9 degrees and diffract to 2.5 A resolution. There are two dimers per asymmetric unit.     

Lung microsomal p-nitrophenol hydroxylase -- characterization and reconstitution of its activity

1. Formation of catechols from benzene and nitrobenzene have been implicated in the carcinogenic activity of these chemicals. In liver, p-nitrophenol, an intermediate of p-nitrobenzene is enzymatically converted to 4-nitrocatechol. 2. For the first time in this study, the presence of a highly active enzyme catalyzing the formation of 4-nitrocatechol from p-nitrophenol was detected in lung microsomes. The average specific activity of lung p-nitrophenol hydroxylase was found to be 0.494 nmol 4-nitrocatechol formed mg prot-1 min-1. 3. The optimum conditions for sheep lung microsomal p-nitrophenol hydroxylase were established. The maximal activity was noted at pH 6.8. The rate of p-nitrophenol hydroxylation was linear up to 2 mg prot/ml of incubation mixture. The maximal rate of 4-nitrocatechol formation was observed with 0.25 mM p-nitrophenol. 4. The Lineweaver-Burk and Eadie-Hofstee plots were found to be curve-linear. Two different Km values were calculated as 11.6 and 71.4 microM from the Lineweaver-Burk plot and as 10.7 and 74.5 microM from the Eadie-Hofstee plot. This suggested that there were either two forms of enzyme or two different enzymes participating in ortho hydroxylation of p-nitrophenol in lung microsomes. 5. Lung microsomal p-nitrophenol hydroxylase activity of sheep was reconstituted in the presence of purified lung microsomal cytochrome P-450, NADPH dependent cytochrome P-450 reductase and synthetic lipid, phosphatidylcholine dilauroyl.     

The effect of haptoglobin on prostaglandin H synthase from ram vesicular glands

It was shown that the ability of sheep and horse haptoglobins differing in their immunological properties to inhibit PGH synthetase is about the same. It was found that haptoglobin inhibits the PGH synthetase-catalyzed enzymatic reaction, the inhibiting effect being non-competitive with respect to the electron donor, adrenaline. The degree of PGH synthetase inhibition by haptoglobin depends on the glycoprotein concentration, incubation time and enzyme activity.     

A method for determining specific circulating immune complexes in patients with acute Sonne dysentery]

The antigen-specific method for the determination of specific immune complexes in the blood serum of patients with acute Sonne dysentery has been developed on the basis of the enzyme-linked immunosorbent assay with the use of reagents manufactured in the USSR. The possibility of the early prognostication of prolonged carrier state by using the proposed method for the determination of the level of specific circulating immune complexes has been shown.     

Probing the Conformation of FhaC with Small-Angle Neutron Scattering and Molecular Modeling

Probing the solution structure of membrane proteins represents a formidable challenge, particularly when using small-angle scattering. Detergent molecules often present residual scattering contributions even at their match point in small-angle neutron scattering (SANS) measurements. Here, we studied the conformation of FhaC, the outer-membrane, β-barrel transporter of the Bordetella pertussis filamentous hemagglutinin adhesin. SANS measurements were performed on homogeneous solutions of FhaC solubilized in n-octyl-d17-βD-glucoside and on a variant devoid of the α helix H1, which critically obstructs the FhaC pore, in two solvent conditions corresponding to the match points of the protein and the detergent, respectively. Protein-bound detergent amounted to 142 ± 10 mol/mol as determined by analytical ultracentrifugation. By using molecular modeling and starting from three distinct conformations of FhaC and its variant embedded in lipid bilayers, we generated ensembles of protein-detergent arrangement models with 120–160 detergent molecules. The scattered curves were back-calculated for each model and compared with experimental data. Good fits were obtained for relatively compact, connected detergent belts, which occasionally displayed small detergent-free patches on the outer surface of the β barrel. The combination of SANS and modeling clearly enabled us to infer the solution structure of FhaC, with H1 inside the pore as in the crystal structure. We believe that our strategy of combining explicit atomic detergent modeling with SANS measurements has significant potential for structural studies of other detergent-solubilized membrane proteins.     

Normal vision can compensate for the loss of the circadian clock

Circadian clocks are thought to be essential for timing the daily activity of animals, and consequently increase fitness. This view was recently challenged for clock-less fruit flies and mice that exhibited astonishingly normal activity rhythms under outdoor conditions. Compensatory mechanisms appear to enable even clock mutants to live a normal life in nature. Here, we show that gradual daily increases/decreases of light in the laboratory suffice to provoke normally timed sharp morning (M) and evening (E) activity peaks in clock-less flies. We also show that the compound eyes, but not Cryptochrome (CRY), mediate the precise timing of M and E peaks under natural-like conditions, as CRY-less flies do and eyeless flies do not show these sharp peaks independently of a functional clock. Nevertheless, the circadian clock appears critical for anticipating dusk, as well as for inhibiting sharp activity peaks during midnight. Clock-less flies only increase E activity after dusk and not before the beginning of dusk, and respond strongly to twilight exposure in the middle of the night. Furthermore, the circadian clock responds to natural-like light cycles, by slightly broadening Timeless (TIM) abundance in the clock neurons, and this effect is mediated by CRY.