Calmodulin-binding proteins and calmodulin-regulated enzymes in dog pancreas.

Calmodulin was isolated and purified to homogeneity from dog pancreas. Highly purified subcellular fractions were prepared from dog pancreas by zonal sucrose-density ultracentrifugation and assayed for their ability to bind 125I-calmodulin in vitro. Proteins contained in these fractions were also examined for binding of 125I-calmodulin after their separation by polyacrylamide-gel electrophoresis in SDS. Calmodulin-binding proteins were detected in all subcellular fractions except the zymogen granule and zymogen-granule membrane fractions. One calmodulin-binding protein (Mr 240,000), observed in a washed smooth-microsomal fraction, has properties similar to those of alpha-fodrin. The postribosomal-supernatant fraction contained three prominent calmodulin-binding proteins, with apparent Mr values of 62,000, 50,000 and 40,000. Calmodulin-binding proteins, prepared from a postmicrosomal-supernatant fraction by Ca2+-dependent affinity chromatography on immobilized calmodulin, exhibited calmodulin-dependent phosphodiesterase, protein phosphatase and protein kinase activities. In the presence of Ca2+ and calmodulin, phosphorylation of smooth-muscle myosin light chain and brain synapsin and autophosphorylation of a Mr-50,000 protein were observed. Analysis of the protein composition of the preparation by SDS/polyacrylamide-gel electrophoresis revealed a major protein of Mr 50,000 which bound 125I-calmodulin. This protein shares characteristics with the calmodulin-dependent multifunctional protein kinase (kinase II) recently observed to have a widespread distribution. The possible role of calmodulin-binding proteins and calmodulin-regulated enzymes in the regulation of exocrine pancreatic protein synthesis and secretion is discussed.     

The molecular organization of nerve membranes

Summary The lipid content and composition from an axolemma-rich preparation isolated from squid retinal axons was analyzed.The lipids, which accounted for 45.5% of the dry weight of this membrane, were composed of 22% cholesterol, 66.7% phospholipids and 5.2% free fatty acids. The negatively charged species phosphatidyl ethanolamine (37%), phosphatidyl serine (10%) and lysophosphatidyl ethanolamine (4%) made up 51% of the phospholipids. The amphoteric phosphatidyl choline and sphingomyelin accounted for 39% and 4%, respectively.The relative distribution of fatty acids in each of the isolated phospholipids was studied. The most remarkable feature of these phospholipids was the large proportion of long-chain polyunsaturated fatty acids. The 226 acyl chain accounted for 37% in phosphatidyl ethanolamine, 21.7% in phosphatidyl choline, 17.5% on phosphatidyl serine and 20.3% in sphingomyelin (all expressed as area %).The molar fraction of unsaturated fatty acids reached 65% in phosphatidyl ethanolamine and 42.0 and 44.8% in phosphatidyl choline and phosphatidyl serine, respectively. The double bond index in these species varied between 1.0 and 2.6.The lipid composition of the axolemma-rich preparation isolated from squid retinal axons appears to be similar to other excitable plasma membranes in two important features: (a) a low cholesterol/phospholipid molar ratio of 0.61; and (b) the polyunsaturated nature of the fatty acid of their phospholipids.This particular chemical composition may contribute a great deal to the molecular unstability of excitable membranes.The preceding papers of this series were published inArchives of Biochemistry and Biophysics.     

The Organically Handicapped Child—How Can the Family Physician Help?

The better his understanding of some of the ways in which an organic deficit might affect normal development of the handicapped child, the more able the family physician will be to offer guidance to the family aimed at preventing the development of secondary problems. He can thus be instrumental in helping a child achieve his maximal potential.First, it is important to take into account how the parents'' emotional and intellectual responses to having a defective child may interfere markedly in normal parent-child relationship. Second, ways in which each deficit will limit a child''s exposure to stimuli must not be over-looked. Third, one must consider how a deficit may indirectly distort the normal learning patterns when parents do not make age appropriate demands. Fourth, it is important to understand how specific interference in the area of language skills may cause further developmental retardation. Fifth, one must be aware of special problems that an organically handicapped child must face in the society outside of the family. Last of all, in an older child, one must consider the need for a full scale evaluation to sort out primary and secondary factors in the picture.     

Ultrastruktur und Bildung von Poren im Endothel von porösen und geschlossenen Kapillaren

Zusammenfassung An einer Reihe von normalen und pathologisch veränderten Organen wird die Porenstruktur, Porenbildung und Porenrückbildung elektronenmikroskopisch untersucht. Die Endothelpore ist eine Diskontinuität in der Endothelwand mit einem sehr konstanten Durchmesser von 500 Å. Das Diaphragma ist nur mit der äußeren Membranlamelle am Porenrand verbunden. Diaphragmalose Poren sind etwas größer (Ø650 Å), zeigen einen glatten Porenrand und kommen besonders in verdichteten Endothelteilen vor. Frustrane Poren münden blind in Vakuolen oder liegen in porösen Endothelfalten im Gefäßlumen. Die eigentliche Bildung der Poren geschieht immer in abgeflachten ( 800 Å) Endothelteilen. Die vorbereitende Abflachung geht jedoch in den verschiedenen Endothelzonen (Periksryon, dicker Endothelwand, Cytoplasmainseln) unterschiedliche Wege. Alle diese Vorgänge stellen Vesikulation in besonderer Lage und mit besonderer Fusionsrichtung der Vesikel dar. Wegen dieser Unterschiede wird die Endothelwand in 4 Zonen eingeteilt: Perikaryon; dicke, porenlose Wandteile mit cytoplasmatischen Vesikeln; dünne, porenhaltige Teile ohne Zellorganellen; dicke Cytoplasmainseln, die die porösen Wandteile voneinander trennen. Der Vorgang der Porenrückbildung bleibt unklar. Vielleicht besteht er in der Faltung des Endothels, die zu porösen Vakuolen führt. Die Porenbildung verändert die Endotheloberfläche nicht, kann aber das Cytoplasmavolumen vermindern. Der Aufbau des Diaphragmas sowie der Mechanismus und die auslösenden Faktoren der Porenbildung werden diskutiert.

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Summary Ultrastructure, formation and disappearance of endothelial pores was studied electron microscopically in several normal or experimentally changed organs. Pores being discontinuies in the endothelial wall have a very constant diameter of 500 Å. The diaphragm at the margin of the pore is in contact with only the outer lamella of the unit membrane. Pores without a diaphragm being somewhat larger (Ø 650 Å) show a smoother margin and are to be found in endothelia with dense cytoplasm. Frustrated pores form blind openings in vacuoles or are situated in porous endothelial folds within the vessel lumen. Pores alway are formed in flattened parts of endothelium ( 800 Å). In thick capillary walls the endothelium previously is flattened by vesiculation, which differs in different zones of the wall (Perikaryon, thick continuous endothelium, and cytoplasmic islands in porous capillaries) in location and direction of vesicle fusion. Because of these differences the endothelial wall is divided in 4 zones: Perikaryon; thick parts without pores containing cytoplasmic vesicles etc.; flattened, porous parts containing no cytoplasmic organelles; thick cytoplasmic islands which separate porous parts. The process removing pores is not clear. Perhaps they are removed by folding of the endothelial surface and formation porous vacuoles. Formation of pores dosn't enlarge or reduce the surface, but may reduce the cytoplasmic volume of endothelium. The nature of diaphragm as well as the mechanism and releasing factors of the formation of pores are discussed.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft     

Veränderungen am Hauptstück und peritubulären Kapillaren der Rattenniere nach Hypophysektomie

Zusammenfassung Das Hauptstück des Nephrons hypophysektomierter Ratten wurde elektronenmikroskopisch untersucht. Nach Entfernung der Hypophyse treten folgende Veränderungen auf: Zwischen normal dichten (hellen) Hauptstückzellen erscheinen sog. Dunkle, Zellen, deren Cytoplasma eine hohe Elektronendichte aufweist. Dazwischen finden sich Übergangsformen, deren basale Einfaltungen wie bei den dunklen Zellen erweitert sind. Diese Zellformen unterscheiden sich auch durch die Struktur ihrer Mitochondrien. In den hellen und mitteldichten Zellen sind die Membranteile der basalen Einfaltungen verändert. Es kommt unter Auflösung der cytoplasmatischen Matrix zur Verschmelzung von Membranabschnitten des ER und der Zellmembran. Schließlich bilden sich lamellierte Einschlußkörper, die in die Nähe des Kernes wandern. Analoge Veränderungen der Membranen des Bürstensaumes wurden nur bei den Übergangszellen gesehen. Die Einschlußkörper liegen hier im Zellapex; sie werden als Cytolysosomen betrachtet. Der Bürstensaum der hellen Zellen weist keinen intervillösen Raum auf, weil die Membranen benachbarter Mikrovilli miteinander verschmolzen sind. Die Weite des intervillösen Spaltes wächst mit der Dichte der Zellen. Daneben wurden auch Veränderungen der Basalmembran und der Kapillarwand beobachtet. Am auffälligsten ist die Abschnürung von membranbegrenzten Gebilden von der Lumenoberfläche des Endothels (1 oder 2 Außenmembranen, gekammerte oder mehrphasische Formen). Auflösung der cytoplasmatischen Matrix, Membranverschmelzungen und Abschnürung durch Vesikulation sind an der Bildung beteiligt. Die Veränderungen nach Hypophysektomie werden auf das Fehlen von Nebennierenrindenhormon zurückgeführt.

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Summary Following hypophysectomy changes in proximal convoluted tubules and their capillaries are observed in the rat kidney. Three types of epithelial cells are present: bright, dark and transitional cells. The basal infoldings and the spaces between the microvilli of the brush border are dilated in the dark cells. In bright and transitional cells the membranes of ER and cell membrane fuse; whorls occur in the cytoplasm. Transitional cells show similar changes in the cell apex. In the bright cells the spaces between the microvilli disappear and the outer membrane lamellae form an external compound membrane (Robertson). The capillary endothelium shows vacuoles which are pinched off from the inner surface. Different forms (one or two enveloping membranes, subdivided and polyphasic forms) are observed. Pinching off is caused by vesiculation. Since adrenalectomy is followed by similar alteration of the epithelial cells and capillaries it is suggested that hypophysectomy affects the kidney indirectly by the lack of adrenal hormones.