Possible influence of lysophospholipase on the production of 1-acyl-2-acetylglycerophosphocholine in macrophages.

The rate of production of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) and 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (acylPAF) was measured in macrophages following the incorporation of [3H]acetate. Upon activation by A23187, guinea pig alveolar macrophages incorporated [3H]acetate into PAF, but a little radioactivity was found in acylPAF. However, labeling of acylPAF and PAF with [3H]acetate was greatly enhanced in A23187-stimulated alveolar macrophages that had been pretreated with phenylmethanesulphonyl fluoride (PMSF). [3H]PAF was predominantly converted to 1-[3H]alkyl-2-acyl glycerophosphocholine, but [14C]acylPAF rapidly hydrolyzed to 14C-labeled free fatty acid by the incubation with lysates prepared from macrophages. The deacetylation of [14C]acylPAF and [3H]PAF by acetylhydrolase and also the hydrolysis of [14C]lysoPC by lysophospholipase were strongly inhibited in macrophages that had been pretreated with PMSF, while PMSF failed to inhibit the activities of acetyltransferase and acyltransferase. The relative proportions of PAF and acylPAF were quite different in different types of cells. In contrast to alveolar macrophages, peritoneal macrophages, neutrophils and spleen cells from guinea pigs incorporated 2-4 times more [3H]acetate into acylPAF than into PAF. The presence of high levels of acylPAF in peritoneal macrophages was confirmed by GLC-MS analysis. The activities of lysophospholipase, acetylhydrolase and acetyltransferase were measured in alveolar and peritoneal macrophages to determine whether the preferential formation of acylPAF as compared to PAF in peritoneal macrophages was due to differences in these activities between alveolar and peritoneal macrophages. The activity of acetylhydrolase of peritoneal macrophages was almost the same as that in alveolar macrophages. The activity of acetyltransferase in peritoneal macrophages was about half of that in alveolar macrophages. However, the activity of lysophospholipase in peritoneal macrophages was one-sixth of that in alveolar macrophages. These results suggest that lysophospholipase is one of the primary factors involved in the control of the production of acylPAF in activated cells, and that it acts by modulating the availability of lysoPC for the synthesis of acylPAF. Furthermore, high levels of activity of lysophospholipase allow the preferential formation of PAF, via the rapid hydrolysis of lysoPC which would act as a competitive inhibitor of the incorporation of acetate into lysoPAF.     

Conformations of dibucaine and tetracaine in small unilamellar phosphatidylcholine vesicles as studied by nuclear Overhauser effects in 1H nuclear magnetic resonance spectroscopy.

Conformations of dibucaine and tetracaine in small unilamellar phosphatidylcholine vesicles have been investigated by nuclear Overhauser effects (NOEs) in 1H nuclear magnetic resonance spectroscopy. Two-dimensional NOE and chemical exchange correlated spectroscopy (NOESY) and rotating frame NOE spectroscopy (ROESY) methods have been applied for obtaining the NOEs. In the NOESY spectra, NOEs between protons within the drug were overwhelmed by spin diffusion even at a short mixing time. This observation reduced the usefulness of the NOESY method on the one hand, however, on the other hand it facilitated remarkably in revealing signals due to the drug, hidden in the broad resonances of the membranes. In the ROESY spectra, the spin diffusion phenomena were less effective; accordingly the conformations of the drugs interacting with membranes were determined by the ROESY method. The observed NOE data showed that dibucaine takes more than two conformations and that both dibucaine and tetracaine are present as a dimer in the membranes. Molecular dynamics calculations supported these findings.     

Ultrastructural localization of laminin on in vivo embryonic, neonatal, and adult rat cardiac myocytes and in early rat embryos raised in whole-embryo culture.

The temporal and spatial distribution of the basement membrane component laminin was examined in vivo in developing rat hearts at 11.5 and 15 days of embryonic development (ED), and in neonates and adults, by pre-embedding ultrastructural immunocytochemistry. In addition, the patterns observed at 11.5 days ED were compared to the distribution of laminin in embryos maintained in whole-embryo culture. At 11.5 days ED laminin was localized in punctate patches on the surface of the plasma membrane, with large gaps between areas of staining. The development of myocytes and localization of laminin in the whole embryo-cultured embryos was similar to that found in the in vivo embryos. At 15 days ED, laminin localization was limited to distinct patches of developing extracellular matrix material associated with the sarcolemma. Gaps between areas of localization were shorter than in the 11.5-day hearts. In neonates, distribution of laminin localization was more extensive with fewer gaps and was associated with the developing basement membrane. In adult hearts, laminin was localized along the entire length of the basement membrane and was heaviest in areas of morphological specialization, such as Z-bands, where collagen bundles contacted the sarcolemma.     

Convenient synthesis of 18-hydroxylated cortisol and prednisolone.

18,20-Epoxy-11 beta,17 alpha,20 beta,21-tetrahydroxypregn-4-en-3-one was synthesized by the application of hypoiodite reaction to the cortisol acetonide. The intermediary 18-iodo derivative was converted to the 11-oxo steroid by chromic acid prior to silver ion-assisted solvolysis. Removal of the protective group with hydrochloric acid was finally carried out to give the desired 11 beta,17 alpha,18,21-tetrahydroxypregn-4-ene-3,20-dione as the hemiacetal form. 18,20-Epoxy-11 beta-17 alpha,20 beta,21- tetrahydroxypregna-1,4-dien-3-one was also prepared from prednisolone through a similar reaction sequence.     

A subunit of yeast site-specific endonuclease SceI is a mitochondrial version of the 70-kDa heat shock protein

Endo.SceI of Saccharomyces cerevisiae is a heterodimeric site-specific endonuclease, which is distinguishable from prokaryotic restriction endonucleases in the mode of recognition of its cleavage site. We have used monoclonal antibodies specific to the larger subunit (75 kDa) of Endo.SceI to isolate the gene for the subunit (ENS1) from S. cerevisiae. Unexpectedly, ENS1 was found to encode a 70-kDa heat shock protein-related polypeptide and to be identical to recently cloned SSC1. Subcellular fractionation experiments on yeast cells revealed that the primary target site of the larger subunit is mitochondria, where almost all the Endo.SceI activity is localized. Molecular genetic analysis of ENS1 demonstrated its indispensability for growth and the requirement of a high level of its expression at the sporulation and germination stages. The data suggest that ENS1 plays an important role, especially at these differentiation stages.     

Detection of the associated state of membrane proteins by polyacrylamide gradient gel electrophoresis with non-denaturing detergents Application to band 3 protein from erythrocyte membranes

Polyacrylamide gradient gel electrophoresis was carried out in micellar solutions of various detergents which differ in degree of potency to denature proteins. From the application of this method to band 3 protein from erythrocyte membranes, it was suggested that the procedure was useful in studying the molecular state of membrane proteins.The electrophoretic behaviors of human and bovine band 3 protein did not show any species specificity in either a denature state and a state resembling the native state. As well as in nonionic detergent solutions, the dimeric and tetrameric structures of bovine band 3 protein were preserved in sodium deoxycholate solution, in which protein complexes maintained in nonionic detergent solutions are frequently dissociated. Even in dodecyltrimethylammonium bromide solution, which is a denaturant for water-soluble proteins, part of the band 3 protein was still present as the oligomer. The results suggest that the oligomeric form of band 3 protein is the stable structure and that the dimer and tetramer possibly coexist in membranes.     

Collagenolytic activity of carrageenin granuloma in rats.

Collagenolytic activity at various phases of the development of carrageenin granuloma was investigated by measuring the amount of dialysable hydroxyproline formed during incubation in vitro of minced granulation tissue. Collagenolytic activity reached a maximum at day 8 after carrageenin injection and then decreased gradually, while collagen synthetic activity was rapidly decreased from day 4 to day 11. The significance of dialysable hydroxyproline in native collagen breakdown of carrageenin granuloma is discussed.     

Studies on the circadian rhythm of phosphoenolpyruvate carboxykinase. III. Circadian rhythm in the kidney.

Phosphoenolypyruvate carboxykinase [EC 4.1.1.21] activity in rat kidney shows a circadian rhythm with the highest activity between 0200 h and 0800 h and the lowest activity between 1400 h and 2000 h. The rhythm was observed in both sexes and throughout the year. Actinomycin D and cycloheximide effectively blocked the circadian increase in enzyme activity. These findings suggest that the circadian increase in phosphoenolypyruvate carboxykinase activity is due to net synthesis of enzyme protein through newly synthesized mRNA. In experiments with kidney cortex slices, gluconeogenesis from the radioactive precursor, [14C]malic acid, was considerably higher at 0200 h than at 1400 h, varying in parallel with the change in the enzyme activity.