Role of Ca2+-activated K+ channel in regulation of NaCl reabsorption in thick ascending limb of Henle's loop

1. Reabsorption of NaCl in the thick ascending limb of Henle's loop involves the integrated function of the Na+,K+,Cl- -cotransport system and a Ca2+-activated K+ channel in the luminal membrane with the Na+,K+-pump and a net Cl- conductance in the basolateral membrane. 2. Assay of K+ channel activity after reconstitution into phospholipid vesicles shows that the K+ channel is stimulated by Ca2+ in physiological concentrations and that its activity is regulated by calmodulin and phosphorylation from cAMP dependent protein kinase. 3. For purification luminal plasma membrane vesicles are isolated and solubilized in CHAPS. K+ channel protein is isolated by affinity chromatography on calmodulin columns. The purified protein has high Ca2+-activated K+ channel activity after reconstitution into vesicles. 4. The purified K+ channel consists of two proteins of 51 and 36 kDa. Phosphorylation from cAMP dependent protein kinase stimulates K+ channel activity and labels the 51 kDa band. The 36 kDa band is rapidly cleaved by trypsin and may be involved in Ca2+ stimulation. 5. Opening of the K+ channel by Ca2+ in physiological concentrations and regulation by calmodulin and phosphorylation by protein kinase may mediate kinetic and hormonal regulation of NaCl transport across the tubule cells in TAL.     

Clinical significance of platelet alloantibodies detected in immunofluorescence test (neonatal alloimmune thrombocytopenia and post-transfusion purpura)

Three cases of neonatal alloimmune thrombocytopenia and one patient with post-transfusion purpura could be diagnosed only by introducing the platelet immunofluorescence test. Thrombocytopenia was caused by anti-PlA1 platelet alloantibodies detected neither in the agglutination nor by the complement fixation test.     

Fenestrated capillaries in the ventral sebaceous gland of the Djungarian hamster, Phodopus sungorus

The ventral sebaceous gland of the Djungarian hamster is a macroscopically visible organ situated in the midventral area of the abdominal wall. It consists of densely packed acini arranged in lobules with common excretory ducts. The rich vascular network of the gland is characterized by fenestrated capillaries. Fenestrated endothelium has not yet been reported as a characteristic and regular finding within sebaceous glands. Results are discussed with regard to proliferation rate of sebocytes and the demand of fluid and nutrient supply.     

Resolution of the conformational distribution and dynamics of a flexible molecule using frequency-domain fluorometry

We report the first resolution of both the conformational distribution and end-to-end diffusion coefficient of a flexible molecule. This molecular information was recovered using only the donor intensity decay in a single solvent at a single viscosity, as observed by the technique of frequency-domain fluorometry. This technique can be extended to measurements of structural fluctuations of biological macromolecules.     

Chemo-enzymatic synthesis of the Galili epitope Gal(alpha)(1-->3)Galbeta(1-->4)GlcNAc on a homogeneously soluble PEG polymer by a multi-enzyme system.

The alpha-Gal trisaccharide Gal(alpha)(1-->3)Galbeta(1-->4)GlcNAc 11 was synthesized on a homogeneously soluble polymeric support (polyethylene glycol, PEG) by use of a multi-enzyme system consisting of beta-1,4-galactosyltransferase (EC 2.4.1.38), alpha-1,3-galactosyltransferase (EC 2.4.1.151), sucrose synthase (EC 2.4.1.13) and UDP-glucose-4-epimerase (EC 5.1.3.2). In addition workup was simplified by use of dia-ultrafiltration. Thus the advantages of classic chemistry/enzymology and solid-phase synthesis could be united in one. Subsequent hydrogenolytic cleavage afforded the free alpha-Gal trisaccharide.     

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.     

Rapid increases in the membrane expression of neutral endopeptidase (CD10), aminopeptidase N (CD13), tyrosine phosphatase (CD45), and Fc gamma-RIII (CD16) upon stimulation of human peripheral leukocytes with human C5a.

Rapid increases in the membrane expression of C3 receptors on granulocytes and monocytes in response to the anaphylatoxin C5a have previously been described. In this study we demonstrate increases in the membrane expression of neutral endopeptidase (NEP, CD10, CALLA), aminopeptidase N (APN, CD13), tyrosine phosphatase (CD45/CD45Ro) and the Fc R Fc gamma-RIII (CD16) on granulocytes within minutes of treatment with human C5a. Monocytes responded to C5a with increases in CD13 and CD45/CD45Ro. These membrane modulations could be prevented by preincubating the C5a preparations with anti-C5a mAb C17/5 but not by pretreating the cells with cycloheximide. Increases of CD10, CD13, and CD11b but not CD11a (LFA-1) were also observed in leukocytes from patients undergoing hemodialysis with cuprophan membranes. The increase of CD16 on granulocytes was dependent on the presence of plasma during in vitro activation with C5a indicating that plasma contains inhibitors which prevent the previously described loss of Fc gamma-RIII upon stimulation of the cells.