Bead rings at the endoplasmic reticulum-golgi complex boundary: morphological changes accompanying inihibition of intracellular transport of secretory proteins in arthropod fat body tissue

Golgi complex beads are 10-nm particles arranged in rings on the smooth surface of rough endoplasmic reticulum (ER) makind the forming face of the Golgi complex (GC). In arthropod cells they stain specifically with bismuth. Their morphology has been studied after treatment with reagents known to interfere with GC function. Inhibitors of oxidative phosphorylation (antimycin A, cyanide, and anoxia), but not an inhibitor of glycolysis (iodoacetate), both cause the bead rings to collapse and the GC saccules to round up, and inhibit transition vesicle (TV) formation. Cycloheximide blocks protein synthesis on ribosomes but does not stop TV formation or disrupt bead rings, even after prolonged treatment (6 h) to allow emptying of the rough ER cisternae. Thus the collapse of bead rings is not attributable to inhibition of protein synthesis, and the ring structure of beads does not require continued protein synthesis and secretion for its maintenance. Valinomycin has effects on the GC similar to those of antimycin A, but {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, monensin, and lasalocid do not affect bead ring structure or TV formation. These results are consistent with valinomycin’s secondarily uncoupling mitochondria, which collapses bead rings and prevents TV formation. Thus inhibitors of oxidative phosphorylation do not influence the beads through cation movement. Because mononsin and lasalocid block secretion at the level of the condensing vacuoles, bead rings are not influenced by blocks in secretion distal to them or by the backup of secretory material. These experiments are consistent with inhibitors of oxidative phosphorylation collapsing bead rings by decreasing intracellular ATP. The concomitant block to TV formation and the collapse of bead rings suggests that integrity of the bead rings is essential for the transport of secretory material from the rough ER to the GC.     

Sensitive gas chromatographic assay for the quantitation of bretylium in plasma,urine and myocardial tissue

A sensitive analytical method has been developed for the quantitation of bretylium in plasma, urine and myocardial tissue. Bretylium and the internal standard, UM-360 (o-iodobenzyltrimethylammonium), are extracted and isolated as the iodide salts. Sodium benzenethiolate is added and the mixture heated to 100° for one hour. This results in the formation of 2-bromobenzyl phenyl thioether and 2-iodobenzyl phenyl thioether, which can be separated and quantitated by gas chromatography. Good reliability and reproducibility can be obtained using electron-capture detection with quantities of bretylium as small as 1 ng.     

Involvement of smooth and coated vesicles in the function of the contractile vacuole complex of some cryptophycean flagellates

The contractile vacuole complex of cryptophycean flagellates comprises the contractile vacuole, a pore and a vesicular spongiome. A minority of spongiome vesicles bear a 15-nm coat on the cytoplasmic surface of the membrane. The coat superficially resembles a clathrin coat. The majority of vesicles are smooth surfaced. Both types of vesicles are found at the same time. Smooth vesicles can be seen in profile suggesting vesicle-vesicle and vesicle-vacuole fusion. It is suggested that smooth vesicles are involved in the segregation of fluid from the cytoplasm and in filling the vacuole. Coated elements exist only as independent vesicles and as coated pits in the contractile vacuole membrane. There is no evidence of fusion of coated vesicles. It is suggested that coated vesicles function to retrieve specific membrane components from the contractile vacuole.     

Hydrogen peroxide causes the fatal injury to human fibroblasts exposed to oxygen radicals

Oxygen radicals are suspected as being a cause of the cellular damage that occurs at sites of inflammation. The phagocytic cells that accumulate in areas of inflammation produce superoxide, hydrogen peroxide, hydroxyl radical, and probably singlet oxygen in the extracellular fluid. The mechanism by which these oxygen molecules kill cells is unknown. To determine which of the oxygen species is responsible for the cellular killing, we exposed human fibroblasts in culture to oxygen radicals generated by the enzymatic action of xanthine oxidase upon acetaldehyde. Using the amount of chromium-51 released from labeled fibroblasts as an index of cellular death, we found that cells were protected only by interventions that reduce hydrogen peroxide concentration. Agents that inactivate superoxide, hydroxyl radical, and singlet oxygen were ineffective in limiting oxygen radical-induced cellular death.     

Heparin releases monosomes and polysomes from rough endoplasmic reticulum

Incubation of membrane-bound polyribosomes isolated from murine myeloma cells with heparin caused release of material which sedimented in the polysome, monosome and ribosomal subunit regions of linear sucrose gradients. The released material corresponded to approximately one half that which could be released by treatment with heparin plus Triton X-100. The action of heparin appeared to be related to its polyanionic nature. The use of heparin as a ribonuclease inhibitor in the separation and isolation of free and membrane-bound polysomes could cause artificial accumulation of detached polysomes in the free polysome fraction.     

Biochemical genetics of Chinese hamster cell mutants with deviant purine metabolism: isolation and characterization of a mutant deficient in the activity of phosphoribosylaminoimidazole synthetase.

A new purine-requiring mutant of Chinese hamster ovary cells (CHO-Kl) is described. This mutant, Ade-G, grows on aminoimidazole carboxamide, hypoxanthine, or adenine. It complements all eight of our other previously described Ade- mutants. Biochemical analysis of de novo purine synthesis in whole cells suggests that Ade-G is capable of the first four reactions of de novo purine biosynthesis and that it synthesizes and accumulates phosphoribosylformylglycinamidine (FGAM). Direct enzyme assay in cell-free extracts confirms that Ade-G is defective in phosphoribosylaminoimidazole synthetase activity and does not convert FGAM to phosphoribosylaminoimidazole (AIR), the next intermediate in the de novo biosynthetic pathway.     

Production of group- and type-specific antigens during non-permissive infection of dog kidney cells with herpes simplex virus type 2.

Infection of a continuous cell line of dog kidney origin (MDCK) with herpes simplex virus type 2 (HSV-2) resulted in production of little to no new infectious virus. Serial subculture of MDCK cells inoculated with HSV-2 did not permit establishment of carrier cell cultures, as assessed by negative results of plaque assays for infectious virus and radioimmunoassay (RIA) for viral antigens. Group- and type-specific antigens were detected in lysates of non-permissive MDCK cells inoculated with HSV-2 and tested by RIA at 24 hours post-inoculation. Polypeptides produced in permissive (Vero) and non-permissive (MDCK) cell systems were labeled with [¹⁴C]-amino acids and analyzed by polyacrylamide slab gel electrophoresis and autoradiography. During non-permissive infection, two polypeptides of large molecular weight, not found in uninfected MDCK cells, one of which commigrated with a major HSV-2 structural polypeptide, were synthesized and reproducibly detected.     

The metabolism of cycloartenol,lanosterol, 24-methylenecholesterol and fucosterol in Chlorella ellipsoidea

Lanosterol and cycloartenol labelled with tritium at C-2, and 24-methylenecholesterol and fucosterol labelled with tritium at C-2 and C-4 were fed to actively growing cultures of Chlorella ellipsoidea. Lanosterol and cycloartenol were converted to each of the five desmethyl sterols of C. ellipsoidea. Lanosterol was more efficiently incorporated than cycloartenol.Although there was some evidence for the reduction of the 24-methylene group, it was apparent that 24-methylene-cholesterol was converted primarily to the C₂₉ sterols, clionasterol and poriferasterol. Labelled fucosterol was reduced at the 24(28) double bond, producing clionasterol.