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.     

The effect of temperature on the kinetics of ethanol production by a thermotolerant strain of Kluveromyces marxianus

Summary The batch fermentation kinetics of a novel thermotolerant strain of the yeast Kluveromyces marxianus were evaluated between 30°C and 48°C. The most significant effects of elevated temperature were reductions in overall biomass and ethanol yields. Decreases in the concentration of ethanol attained, and the presence of unutilized substrate suggested increased ethanol inhibition at the higher temperatures studied.     

Internal motional averaging and three-dimensional structure determination by nuclear magnetic resonance.

Dynamic averaging effects from internal motions on interproton distances estimated from nuclear Overhauser effects (NOE) are determined by using a molecular dynamics simulation of lysozyme. Generalized order parameters measuring angular averaging and radial averaging parameters are calculated. The product of these two parameters describes the full averaging effects on cross-relaxation. Analysis of 2778 non-methyl NOE interactions from the protein interior and surface indicates that distances estimated by assuming a rigid molecule have less than 10% error for 89% of the NOE interactions. However, analysis of 1854 methyl interactions found that only 68% of the distances estimated from cross-relaxation rates would have less than 10% error. Qualitative evaluation of distances according to strong, medium and weak NOE intensities, when used to define only the upper bound for interproton separation, would misassign less than 1% of the distance constraints because of motional averaging. Internal motions do not obscure the identification of secondary structure, although some instances of significant averaging effects were found for interactions in alpha-helical regions. Interresidue NOEs for amino acids more than three residues apart in the primary sequence are more extensively averaged than intraresidue or short-range interresidue NOEs. Intraresidue interactions exhibit a greater degree of angular averaging than those involving interresidue proton pairs. An internal motion does not equally affect all NOE interactions for a particular proton. Thus, incorporation of averaging parameters in nuclear magnetic resonance structure determination procedures must be made on a proton-pair-wise basis. On the basis of the motional averaging results, particular fixed-distance proton pairs in proteins are suggested for use as distance references. A small percentage of NOE pairs localized to three regions of the protein exhibit extreme averaging effects from internal motions. The regions and types of motions involved are described.     

Familial DiGeorge syndrome and associated partial monosomy of chromosome 22

Summary Partial monosomy of 22q due to an unbalanced 4;22 translocation was seen in a 2-month-old male with Type I truncus arterious, dysmorphic features, and T-cell abnormalities. The family history revealed a previous sib with Type I truncus arteriosus, thymic aplasia, and parathyroid hypoplasia noted on postmortem examination, consistent with DiGeorge syndrome. Evaluation of the asymptomatic mother of these two patients revealed partial T-cell deficiency and the same unbalanced translocation with deletion of proximal 22qll. These findings provide further evidence that some cases of complete or partial DiGeorge syndrome are associated with monosomy of the proximal long arm of chromosome 22, and they may explain many, if not all, familial cases of the syndrome.Supported in part by National Foundation-March of Dimes Grant No. 2-161/C-331. Funds from the Texas Department of Health through PL94-278 National Genetic Diseases Act, from the Robert J. Kleberg, Jr. Center for Human Genetics, and USPHS Grant No. RR-05425.     

Calcium ion stimulated endogenous protein kinase catalyzed phosphorylation of basic proteins in myelin subfractions and myelin-like membrane fraction from rat brain

Polypeptide composition and endogenous phosphorylation were investigated in the subfractions of rat brain myelin isolated by either discontinuous or continuous sucrose density gradient centrifugation of myelin. Similarly, a myelin-like membrane fraction (SN4) was also studied. Observations were made that strongly indicated the presence of a calcium-stimulated protein kinase in a highly purified myelin preparation and which exclusively phosphorylated myelin basic proteins of the membrane preparation. Adenosine cyclic 3',5'-phosphate (cAMP) stimulated kinase on the other hand was found to be considerably enriched in the myelin-like membrane fraction. Although this latter enzyme is also capable of phosphorylating the basic proteins, its effect was at least 5 times weaker compared to the calcium-stimulated myelin protein kinase. Within the gradient subfractions there appeared a close relation between the amount of basic proteins and their calcium-stimulated phosphorylation; a similar relationship, however, was not obtained in the case of cAMP-dependent phosphorylation of myelin basic proteins. The former (i.e., Ca2+-stimulated phosphorylation) was found to require a protein factor that functionally resembled calmodulin. The results thus raises an interesting possibility of the presence of calmodulin-like proteins and a calcium-stimulated protein kinase in adult myelin membrane from mammalian brain, both of which have been hitherto unrecognized constituents of myelin membranes.     

Purification and properties of human placental acid lipase

Two peaks of lysosomal acid lipase activity were purified from normal human placenta. Acid lipase I, with an estimated molecular weight of 102 500, was purified 1016-fold while acid lipase II, with an estimated molecular weight of 30 600, was purified 3031-fold. The final yields of enzyme activity for acid lipase I and II were 0.9% and 2.2% respectively. The purity of the final preparations was documented by demonstration of a single protein band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Both preparations of the purified enzyme demonstrated activity towards triolein, cholesteryl oleate and the artificial substrate 4-methylumbelliferyl oleate. Examination of Km values, thermal stability, pH optima, and electrophoretic mobility revealed similar properties for the two enzyme peaks. The response of the two enzyme preparations to inhibitors was similar with both being significantly inhibited by 0.2 M NaCl, 0.2 M KCl, 5 mM HgCl2 and 5 mM p-chloromercuribenzoate. The activity of the two preparations as assayed with either triolein or cholesterol oleate was not significantly affected by the addition of bovine serum albumin. In contrast, the 4-methylumbelliferyl oleate activity of both preparations was significantly inhibitred by albumin. These findings support the hypothesis that the same enzyme or enzymes are responsible for the intralysosomal hydrolysis of triacylglycerols and cholesterol esters in human tissues.     

Structure of a hydroxyl radical induced cross-link of thymine and tyrosine

DNA-protein cross-links are formed when living cells or isolated chromatin is exposed to ionizing radiation. Little is known about the actual cross-linked products of DNA and proteins. In this work, a novel hydroxyl radical induced cross-link of thymine and tyrosine has been isolated along with a tyrosine dimer by high-performance liquid chromatography of aqueous mixtures of tyrosine and thymine that had been exposed to hydroxyl radicals generated by ionizing radiation. The isolated compounds have been examined by gas chromatography-mass spectrometry, high-resolution mass spectrometry, and 1H and 13C nuclear magnetic resonance spectroscopy. The structure of the thymine-tyrosine cross-link has been identified as the product from the formation of a covalent bond between the methyl group of the thymine and carbon 3 of the tyrosine ring. In addition, the 3,3' tyrosine dimer was isolated and characterized. The mechanism of the formation of these compounds is discussed. This work presents the first complete chemical characterization of a hydroxyl radical induced DNA base-amino acid cross-link.     

Plant Reactions to Inoculation of Roots with Fungi and Bacteria

The potential of 120 isolates of fungi and bacteria from plant rhizospheres to interfere with plant development and growth was studied in greenhouse experiments. The pure cultured isolates were obtained from plant roots in the field and applied as suspensions to the roots of eight test plant species. 10–20% of the isolates caused distinct symptoms on shoots, growth retardations without other symptoms or growth promotions. Responses of treated plants ranged from death of plants soon after treatment to up to about 40% higher shoot fresh weight than in control plants. Two bacterial isolates induced strong reactions in most of the plant species tested while other isolates showed a more or less pronounced specificity by giving reactions in only some of the plant species tested.