Salt tolerance and glycerol accumulation of a respiration-deficient mutant isolated from the petite-negative, salt-tolerant yeast Zygosaccharomyces rouxii

A respiration-deficient (RD) mutant was isolated from the petite-negative, salt-tolerant yeast Zygosaccharomyces rouxii. One strain among sixteen glycerol-non-utilizing mutants exhibited vigorous liberation of CO2 but no uptake of O2. Furthermore, this strain lacked cytochrome aa3 and had a reduced level of cytochrome b. The few mitochondria found in cells of this strain contained few or no cristae. Salt tolerance and intracellular accumulation of glycerol by the RD strain were almost equal to that of the wild-type strain in media containing NaCl up to 2.5 M. In media with more than 3 M NaCl, the growth of the RD mutant was retarded and the intracellular accumulation of glycerol was depressed in spite of ample production.     

Effects of prostaglandin E2 and D2 on gastric somatostatin and gastrin secretion

The effects of PGE2 and PGD2 on gastric somatostatin and gastrin releases were investigated using the isolated perfused rat stomach. In the presence of 5.5 mM glucose, the infusion of PGE2 elicited a significant augmentation in somatostatin release, but suppressed gastrin secretion from the perfusate. On the other hand, PGD2 did not affect somatostatin release, although the gastrin secretion decreased significantly, the same as after PGE2 infusion. These results suggest that PGE2 and PGD2 may be important in the regulation of gastric endocrine function, but that PGD2 does not affect gastric somatostatin secretion.     

FINE STRUCTURE OF PACINIAN CORPUSCLES IN THE MESENTERY OF THE CAT

Pacinian corpuscles in the mesentery of adult cats were fixed with either glutaraldehyde, osmium tetroxide or permanganate solutions by close intra-arterial injection through the mesenteric artery, and were processed, after electron staining and Epon embedding, for electron microscopy. Better resolution of the corpuscle's ultrastructure was obtained than available heretofore. The myelinated segment of the corpuscle contains blood vessels separated from the axon by collagen fibers and 3 to 4 layers of lamellae. No blood vessels are found in the central core, though access from the vessels is afforded by diffusion through the "cleft" of the inner core. Two cell types are discernible in the inner core hemilamellae; the "clear cells" in which pinocytotic vesicles and organelles abound and reflect the greater metabolic activity of these cells, in contrast to the "dark cells." The ultraterminal is ellipsoidal in form with projections into the "cleft" which give this portion an irregular appearance in section. The terminal and ultraterminal are packed with mitochondria, and "synaptic" vesicles are seen in the ultraterminal. The innermost laminae of the inner core cells are in close apposition to the terminal and break their regular pattern of hemilamellation to surround the small ultraterminal projections at the apical part of the corpuscle.     

Stabile production of a thrombin resistant pro-urokinase derivative (PRO-UKS1) by Namalwa KJM-1 cells adapted to serum-free medium

Pro-UKS1 was designed as a thrombin-resistant derivative of pro-urokinase (pro-UK) by introducing a glycosylation site using site-directed mutagenesis. An expression plasmid for pro-UKS1, pMo1UKS1SEd1-5, was constructed and introduced into Namalwa KJM-1 cells (Hosoiet al., 1988), and cells resistant to G418 and Methotrexate (MTX) were obtained. Amongst them, the highest pro-UKS1 producer (resistant to 500 nM of MTX), clone 41-8, was selected and further characterized. Clone 41-8 was cultured in serum-free ITPSGF medium (Hosoiet al., 1988). Under the conventional conditions, the concentration of pro-UKS1 reached 26 g ml^–1. Addition of glucose and tri-iodothyronine (T₃) improved productivity, and the maximal productivity of pro-UKS1 was 67 g ml^–1 day^–1. In this conditioned medium, content of pro-UKS1 was above 80% of total proteins.Abbreviations BSA bovine serum albumin - dhfr dihydrofolate reductase - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - kb kilobase pairs - kDa kilodaltons - MTX Methotrexate - PBS phosphate buffered saline - pro-UK pro-urokinase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - T₃ tri-iodothyronine - Tween-PBS phosphate buffered saline containing 0.05% Tween 80     

Localization of intestinal sucrase-isomaltase complex on the microvillous membrane by electron microscopy using nonlabeled antibodies

Microvillous vesicles isolated from rabbit small intestine showed a trilaminar membrane with a rather smooth surface, which was apparently not affected by papain solubilizing sucrase-isomaltase complex or by trypsin unable to solubilize it. When microvilous vesicles or trysinized ones were incubated with immunoglobulin G against the sucrase-isomaltase complex or monovalent fragments therefrom, an apparently continuous electron-opaque layer approximately 180 A in width appeared around the external surface of vesicles. Such a layer was not formed on papainized vesicles. Microvillous vesicles and trypsinized ones negatively stained with phosphotungate showed a great number of particles protruding approximately 150 A from the membrane surface, but papainized vesicles did not. The particles existed close to one another and appeared to form a particulate layer 150 A in width on the surface. The antibodies, whether they were divalent or monovalent, increased the width of the layer to approximately 200 A and obscured the fine particulate structure of intact and trypsinized vesicles. Papainized vesicles retained their smooth surface upon interaction with antibodies. These results, together with those with the Triton-solubilized sucrase- isomaltase complex (Nishi and Takesue, 1978), J. Ultra-struct. Res., 62:1- 12), indicate not only that sucrase-isomaltase complexes are located close to one another on the membrane, but also that they or at least their protein portions protrude approximately 150 A from the surface of the trilaminar membrane.     

Topographical studies on intestinal microvillous leucine β-naphthylamidase on the outer membrane surface

Summary The location of leucine -naphthylamidase on the outer surface of the microvillous membrane of rabbit small intestine was examined by analyzing the interaction of antibodies against leucine -naphthylamidase or another microvillous enzyme, sucrase-isomaltase complex, with microvillous vesicles having different relative amounts of these enzymes, in respect to vesicle agglutination, inhibition of enzyme activity, and electron-microscopic morphology. The results obtained indicate that leucine -naphthylamidase, or at least its antigenic sites, protrude about 10 nm from the outer surface of the microvillous membrane.     

Ca2(+)-calmodulin-dependent protein kinase II phosphorylates various types of non-epithelial intermediate filament proteins

We have investigated the actions of Ca2(+)-calmodulin (CaM)-dependent protein kinase II on various types of non-epithelial intermediate filament proteins, vimentin, desmin, glial fibrillary acidic protein (GFAP) and neurofilament triplet proteins. Most of these filament proteins could serve as substrates. The effects of phosphorylation on the filamentous structure of vimentin were investigated in sedimentation experiments and by using electron microscopy. The amount of unassembled vimentin increased linearly with increased phosphorylation. However, the extent of the effect of phosphorylation on the potential to polymerize was also affected by the MgCl2 concentration, under conditions for reassembly. The actions of Ca2(+)-CaM-dependent protein kinase II on non-epithelial intermediate filaments under physiological conditions are given attention.     

Two cage-type lupin alkaloids from Sophora franchetiana

Two new cage-type lupin alkaloids, (?)-tsukushinamine-B and tsukushinamine-C, have been isolated from the fresh epigeal parts of Sophora franchetiana, along with (?)-cytisine, (?)-N-formylcytisine, (?)-rhombifoline, (?)-anagyrine, (?)-baptifoline and (±)-ammodendrine, as well as (?)-tsukushinamine-A. The structures of these novel tsukushinamine-type lupin alkaloids were determined by spectroscopic data and partly by a chemical reaction. Variations of the alkaloid contents in the seeds, seedlings and various parts of S. franchetiana were also examined.