Structural implications of primary sequences from a family of Balbiani ring-encoded proteins inChironomus

Summary DNA sequencing has revealed an internal, tandemly repetitive structure in the family of giant polypeptides encoded by three types of Balbiani ring (BR) genes, in three different species ofChironomus. Each major BR repeat can be subdivided into two halves: a region consisting of short subrepeats and a more constant region that lacks obvious subrepeats. Comparative predictions of secondary structure indicate that an -helical segment is consistently present in the amino-terminal half of the constant region in all known BR proteins. Comparative predictions, coupled with consideration of the known phosphorylation of serine and threonine residues in BR proteins, suggest that the -helical structure may also extend into the carboxy-terminal half of the constant region, possibly interrupted by -turn(s). However, it is also possible that the structure is variable, and that a -strand is present in that half in some cases. All of the constant regions conserve one methionine and one phenylalanine residue, as well as all four cysteines; these residues presumably play roles in the packing or cross-linking of aligned constant regions. The structure of the subrepeat region is not clear, but the prevalence of a tripeptide pattern (basic-proline-acidic) suggests some type of structural regularity, possibly an extended helix. The possible significance of these conserved molecular features is discussed in the context of how they may serve the elasticity, insolubility, and hydrophilicity of the fibrils and threads formed by the BR polypeptides.     

Colchicine inhibits plasmodium formation and disrupts pathways of sporopollenin secretion in the anther tapetum ofTradescantia virginiana L.

Summary During an earlier investigation, microtubules were observed at the periphery of invasion processes in the developing syncytial tapetum ofTradescantia virginiana L. They were also associated with membranous sacs that accumulate adjacent to tetrads, with putative fusion sites where the tapetal plasmodium is initiated, and, in postmeiotic stages, with the perispore membrane that encloses the developing spore cells. Colchicine was administered to developing flower buds to investigate the roles of these microtubules. The results indicate that microtubules neither initiate nor guide the tapetal invasion of the loculus. The treatments, however, resulted in absence of cell coat from invasion processes and prevention of cell fusion. They also inhibited polarized migration of membrane sacs and removed the associated microtubules. The development of an organized secretory apparatus at the perispore membrane was disrupted, with subsequent disordered deposition of sporopollenin in the extracellular spaces of the partially-fused plasmodium. The results suggest that microtubules participate in the formation and internal spatial organization of the tapetal plasmodium, and establishment of a secretory surface that normally produces sporopollenin at the tapetum-microspore interface.     

Choline stimulates synthesis of extracellular proteins in Trichoderma reesei QM 9414

A correlation between intracellular phospholipid levels and the rate of exoprotein synthesis was investigated in the filamentous fungus Trichoderma reesei QM 9414 during growth on cellulose. When the incubation temperature was varied between 20 and 37°C, the exoprotein synthesis rate correlated with the total cellular amount of phospholipids, but not with an individual phospholipid component. In contrast, when phospholipid bases were added exogenuously, a significant stimulation of exoprotein synthesis was observed with choline. The addition of the surfactant Tween 80—which also stimulates exoprotein secretion in T. reesei QM 9414—prevented choline stimulation. Optimal stimulation occurred around 20 mM choline. Choline stimulated exoprotein synthesis in general as shown by increased activities of several extracellular enzymes. Mycelia required preincubation for at least 20 h before stimulation of choline could be seen. Mycelia pregrown in the absence or presence of choline were equally effective in formation of -glucosidase upon induction with methyl--d-glucoside, and the addition of choline to the induction medium had no effect. Choline did not alter the osmotic stability of protoplasts of T. reesei. Electron microscopic examinations and analysis of chemical constituents as well as marker enzymes from choline grown and non-choline grown mycelia revealed higher contents of mitochondria and endoplasmic reticula in choline grown mycelia. The possibility is discussed that choline may stimulate exoprotein synthesis by increasing the cellular content of endoplasmic reticula.     

Comparison of the properties of active Ca2+ transport by the islet-cell endoplasmic reticulum and plasma membrane

The properties of active or ATP-dependent calcium transport by islet-cell endoplasmic reticulum and plasma membrane-enriched subcellular fractions were directly compared. These studies indicate that the active calcium transport systems of the two membranes are fundamentally distinct. In contrast to calcium uptake by the endoplasmic reticulum-enriched fraction, calcium uptake by islet-cell plasma membrane-enriched vesicles exhibited a different pH optimum, was not sustained by oxalate, and showed an approximate 30-fold greater affinity for ionized calcium. A similar difference in affinity for calcium was exhibited by the Ca²⁺-stimulated ATPase activities which are associated with these islet-cell subcellular fractions. Consistent with the effects of calmodulin on calcium transport, calmodulin stimulated Ca²⁺-ATPase in the plasma membranes, but did not increase calcium-stimulated ATPase activity in the endoplasmic reticulum membranes. The physiological significance of the differences observed in calcium transport by the endoplasmic reticulum and plasma membrane fractions relative to the regulation of insulin secretion by the islets of Langerhans is discussed.     

Characteristics and determinants of osmotic lysis in chromaffin granules

(1) Using isolated bovine chromaffin granules, we demonstrate that osmotic lysis is not a random process and establish the osmotic pressure dependence of osmotic lysis in chromaffin granules, the so-called osmotic fragility curve. (2) We show by measuring the release of constituents of the granule core and correlating these with changes in spectroscopic parameters (turbidity and endogenous catecholamine fluorescence), that the latter can be safely used to measure lysis. (3) Within a particular granule population, noradrenaline granules lyse at higher osmolarities than adrenaline granules, suggesting a higher core osmolarity of the noradrenaline granules. (4) The size distribution of chromaffin granules as a function of lysis was determined by the use of whole mount electron microscopy. It is shown that the mean size of chromaffin granules decreases as a function of lysis. (5) On the basis of theoretical considerations three alternative models of the sequence of osmotic lysis in chromaffin granules are proposed. The experimental results best support a model which postulates that during partial osmotic lysis, granule membranes reseal into smaller vesicles after graded release of contents. The osmotic fragility would represent several cycles of lysis and resealing and would not be a reflection of the distribution of osmotic pressures in the granule population.     

3',5'-Cyclic Adenosine Monophosphate- and Ca2+-Calmodulin-Dependent Endogenous Protein Phosphorylation Activity in Membranes of the Bovine Chromaffin Secretory Vesicles: Identification of Two Phosphorylated Components as Tyrosine Hydroxylase and Protein Kinase Regulatory Subunit Type II

Abstract: Membranes of the secretory vesicles from bovine adrenal medulla were investigated for the presence of the endogenous protein phosphorylation activity. Seven phosphoprotein bands in the molecular weight range of 250,000 to 30,000 were observed by means of the sodium dodecyl sulphate electrophoresis and autoradiography. On the basis of the criteria of molecular weight, selective stimulation of the phosphorylation by cyclic AMP (as compared with cyclic GMP) and immunoprecipitation by specific antibodies, band 5 (molecular weight 60,300) was found to represent the phosphorylated form of the secretory vesicle-bound tyrosine hydroxylase. The electrophoretic mobility, the stimulatory and inhibitory effects of cyclic AMP in presence of Mg²⁺ and Zn,²⁺ respectively, and immunoreactivity toward antibodies showed band 6 to contain two forms of the regulatory subunits of the type II cyclic AMP-dependent protein kinase, distinguishable by their molecular weights (56,000 and 52,000, respectively). Phosphorylation of band 7 (molecular weight 29,800) was stimulated about 2 to 3 times by Ca²⁺ and calmodulin in the concentration range of both agents believed to occur in the secretory tissues under physiological conditions.     

Neurotensin interacts with carbachol, secretin, and caerulein in the stimulation of the exocrine pancreas of the rat in vitro

In the present investigation the effect of neurotensin on pancreatic secretion of isolated pancreatic lobules from the rat was examined. We found a dose- and time-dependent stimulation of amylase release beginning with a concentration of 10(-9) M neurotensin. This response was potentiated by the cholinergic agonist carbachol, the gastrointestinal peptide secretin, and the CCK analogue caerulein. As we found neurotensin-immunoreactive nerves within the pancreas and as neurotensin-like immunoreactivity is present in the circulation (found previously), neurotensin may well be a further peptide taking part in the regulation of exocrine pancreatic secretion either as a hormone or a neurotransmitter. Neurotensin would then cooperate with cholinergic mechanisms, secretin, and CCK.     

The role of the Asp-32 residue of cholecystokinin in gastric acid secretion and gastrin receptor recognition

The aspartic acid residue at the penultimate position is known to be essential for the hormonal activity of CCK and gastrin on gastric acid secretion. This residue was successively replaced by beta-aspartic acid, beta-alanine, and glutamic acid in the C-terminal heptapeptide of CCK 27-33. The analogues obtained were tested on rat gastric acid secretion and for recognition by gastrin receptors. The replacement by beta-aspartic or beta-alanine decreased gastric secretion and gastrin receptor recognition. In contrast, replacement by glutamic acid affected these two parameters less. The nature of the N-blocking group (Boc or Z) also influenced these activities, Boc derivatives being more potent than Z derivatives. The results were compared to those previously obtained on pancreatic secretion and on stimulation of gall bladder contraction where the modifications were found capable of differentiating between cholecystokinin, pancreozymin and gastrin activities.     

Apokrine Sekretion der peritrophischen Membran von Chironomus thummi piger Str. (Diptera)

Zusammenfassung Am Beispiel der Larven von Chironomus thummi piger werden die Zellen, die die peritrophische Membran abscheiden, licht- und elektronenmikroskopisch untersucht. Es liegen zwei Zelltypen vor, die einerseits durch ihren Reichtum an granulären E.R.-Schläuchen (ER-Zellen), andererseits durch ihren hohen Gehalt an Mitochondrien (M-Zellen) charakterisiert sind. Die ER-Zellen zeigen Veränderungen, die in vielerlei Hinsicht der klassischen Auffassung der apokrinen Sekretion entsprechen und sich nicht in ein modernes Schema der Sekretionsmorphologie einordnen lassen. Die Zellen gehen im Verlauf der Sekretabgabe weder völlig zugrunde, noch bleiben sie vollständig erhalten. Die M-Zellen, die im Gegensatz zu den ER-Zellen keine Sekretionsgranula besitzen, weisen ähnliche Umwandlungen auf. Das fertige Sekretionsprodukt — die peritrophische Membran — entstammt einmal vorgeformten Sekretionsgranula, zum anderen der umgewandelten, abgeschnürten oberen Zellhälfte. Die peritrophische Membran besteht aus zwei Schichten, wovon die lumenseitige, auf Längsschnitten quergestreifte Lage (Wabentextur) vermutlich aus den Sekretionsgranula hervorgeht und die andere längsgefaserte Lage auf die Umwandlung von Zytoplasma zurückzuführen sein dürfte. Die Mikrovilli der Sekretionszellen sind in keiner Weise für die Strukturierung der Wabentextur verantwortlich.

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On the apocrine secretion in the formation of the peritrophic membrane of chironomus thummi piger Str

Summary Taking the larvae of Chironomus thummi piger as an example, the cells secreting the peritrophic membrane have been investigated with the light- and electron-microscope. Two cell-types can be distinguished which in one case are characterized by abundant rough E.R.-tubules (ER-cells) and in the other case by large quantities of mitochondria (M-cells). The ER-cells undergo changes which in many respects correspond to the individual stages of the classic apocrine secretion, and thus do not fit into a modern scheme of the morphology of secretion. In the course of the discharge of the secretory product the cell neither becomes completely necrotic nor remains totally intact. The M-cells, which do not contain secretion granules like the ER-cells, show similar changes. The final product — the peritrophic membrane — is formed on the one hand by membrane bound secretion granules and on the other hand by the transformed and pinched off upper half of the cell. The peritrophic membrane consists of two layers, the one of which, facing the lumen of the midgut and exhibiting a honey-comb-texture, presumably is formed by the secretion granules, whereas the other layer arises from transformed cytoplasm. The microvilli of the secretory cells are not responsible for the formation of the honey-comb-pattern.

Die Untersuchungen wurden mit Unterstützung durch die Max-Planck-Gesellschaft und die Deutsche Forschungsgemeinschaft durchgeführt.     

Cyproheptadine metabolites inhibit proinsulin and insulin biosynthesis and insulin release in isolated rat pancreatic islets

The contribution of drug metabolites to cyproheptadine (CPH)-induced alterations in endocrine pancreatic -cells was investigated by examining the inhibitory activity of CPH and its biotransformation products, desmethylcyproheptadine (DMCPH), CPH-epoxide and DMCPH-epoxide, on hormone biosynthesis and secretion in pancreatic islets isolated from 50-day-old rats. Measurement of (pro)insulin (proinsulin and insulin) synthesis using incorporation of ³H-leucine showed that DMCPH-epoxide, DMCPH and CPH-epoxide were 22, 10 and 4 times, respectively, more potent than CPH in inhibiting hormone synthesis. The biosynthesis of (pro)insulin was also inhibited by CPH and DMCPH-epoxide in islets isolated from 21-day-old rat fetuses. The inhibitory action of CPH and its metabolites was apparently specific for (pro)insulin, and the synthesis of other islet proteins was not affected. Other experiments showed the metabolites of CPH were active in inhibiting glucose-stimulated insulin secretion but were less potent than the parent drug in producing this effect. CPH and its structurally related metabolites, therefore, have differential inhibitory activities on insulin synthesis and release. The observation that CPH metabolites have higher potency than CPH to inhibit (pro)insulin synthesis, when considered with published reports on the disposition of the drug in rats, indicate that CPH metabolites, particularly DMCPH-epoxide, are primarily responsible for the insulin depletion observed when the parent compound is given to fetal and adult animals.Abbreviations CPH cyproheptadine - CPH-epoxide cyproheptadine-10-11-epoxide - DMCPH desmethylcyproheptadine - DMCPH-epoxide desmethylcyproheptadine-10,11-epoxide - HPLC high-performance liquid chromatography - KBB Krebs biocarbonate buffer Recipient of a Society of Toxicology Predoctoral Research Fellowship.Present address: Department of Biochemistry, The University of Hong Kong, Hong Kong.