Abortive Infection of L Cells by Influenza B Virus: Defect in Bud Formation

Host-dependent restriction of influenza B virus replication in L cells was analysed in comparison with productive infection in MDCK or 1–5C-4 cells. The synthesis and intracellular distribution of virus-specific proteins and the production of cytoplasmic ribonucleoproteins in nonpermissive L cells were similar to those in permissive MDCK cells. However, an electron microscopic study of infected L cells showed neither extracellular virions nor budding virus particles on the cell surface, in contrast to MDCK cells which produced numerous virus particles. PAGE analysis of the plasma membrane isolated from the cells demonstrated no significant difference in the composition of viral polypeptides between permissive 1-5C-4 and nonpermissive L cells. It was noted that the abortiveness of influenza B virus infection in L cells may be due to a defect in host cell function involved in the initiation of virus budding.     

Solution Radioactivated by Hadron Radiation Can Increase Sister Chromatid Exchanges

When energetic particles irradiate matter, it becomes activated by nuclear reactions. Radioactivation induced cellular effects are not clearly understood, but it could be a part of bystander effects. This investigation is aimed at understanding the biological effects from radioactivation in solution induced by hadron radiation. Water or phosphate buffered saline was activated by being exposed to hadron radiation including protons, carbon- and iron-ions. 1 mL of radioactivated solution was transferred to flasks with Chinese hamster ovary (CHO) cells cultured in 5 mL of complete media. The induction of sister chromatid exchanges (SCE) was used to observe any increase in DNA damage responses. The energy spectrum and the half-lives of the radioactivation were analyzed by NaI scintillation detector in order to identify generated radionuclides. In the radioactivated solution, 511 keV gamma-rays were observed, and their half-lives were approximately 2 min, 10 min, and 20 min. They respectively correspond to the beta+ decay of ¹⁵O, ¹³N, and ¹¹C. The SCE frequencies in CHO cells increased depending on the amount of radioactivation in the solution. These were suppressed with a 2-hour delayed solution transfer or pretreatment with dimethyl sulfoxide (DMSO). Our results suggest that the SCE induction by radioactivated solution was mediated by free radicals produced by the annihilated gamma-rays. Since the SCE induction and DMSO modulation are also reported in radiation-induced bystander effects, our results imply that radioactivation of the solution may have some contribution to the bystander effects from hadron radiation. Further investigations are required to assess if radioactivation effects would attribute an additional level of cancer risk of the hadron radiation therapy itself.     

Differences in lectin binding patterns of normal human endometrium between proliferative and secretory phases

Lectin binding patterns in normal human endometrium were examined by light and electron microscopy using seven different lectins (ConA, WGA, RCA, PNA, UEA-1, DBA, and SBA). For light microscopic observations, criteria based on the incidence and intensity of cells positive for the lectin staining were adopted to evaluate the different staining patterns of the proliferative and secretory endometria obtained by the avidin-biotin-peroxidase complex (ABC) technique. At the light microscopic level, ConA, WGA, and RCA stained endometrial glandular cells in both phases. The number of PNA-positive cells with the binding sites entirely limited to the apical surface tended to be reduced slightly in the secretory phase. UEA-1 weakly stained the apical surface of glandular cells in the proliferative phase but not in the secretory phase. Among the lectins used in this study, DBA and SBA displayed remarkable changes between the phases. That is, in the proliferative phase they produced only a faint or slight positive stain at the apical surface, but the incidence and intensity of DBA- and the SBA-positive glandular cells increased in the secretory phase. By electron microscopy, the reaction product of ConA was observed in the plasma membrane, endoplasmic reticulum, nuclear envelope, and the Golgi apparatus, and the binding sites of RCA and DBA were observed in the plasma and Golgi membranes. Between both phases, the reactivity of ConA and RCA showed almost no change. However, the secretory endometrial cells containing the DBA-positive Golgi apparatus were markedly increased in number compared with the proliferative ones bearing the lectin-positive organelles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of acetone glycerol acyl esters by immobilized lipase ofMucor miehei

Summary The applications of immobilized lipase ofMucor miehei for the synthesis of acetone glycerol acyl ester from acetone glycerol and fatty acid, which is the first step for monoglyceride production was investigated. With a high oleic acid to acetone glycerol ratio (O/A, mol/mol), a high catalytic activity was observed under low water content in the reaction mixture. By the combination of high O/A ratio (>3) and removal of water which was produced during the reaction, the conversion degree was increased to almost 100%. With the O/A ratio of 3, the approximate half-life of the immobilized lipase and productivity of ester was estimated to be 20 days and 869 g product/g immobilized enzyme per 2 half-lives, respectively.     

Establishment and characterization of a human chorionic gonadotropin (hCG) producing cell line (RTSG) from an ovarian epithelial cancer

A new cell line designated RTSG established in vitro from the pleural effusion of a patient with metastatic ovarian epithelial cancer has been subcultured 46 times for more than 2 years. The cells grew in a monolayered sheet, showing a tendency to pile up, with the population doubling in 48 hrs. Electron-microscopically, desmosomes were characteristically observed, suggesting the cells were of epithelial origin. Chromosomal analysis revealed aneuploidy with a tetraploid mode. The heterotransplanted tumors in nude mice were histopathologically classified as a poorly differentiated adenocarcinoma, whereas the original tumor consisted mainly of mucinous and serous cystadenocarcinoma and only partly of poorly differentiated adenocarcinoma. The cells secreted hCG (38.8 mIU/day/10(6) cells) and beta-hCG (6.1 ng/day/10(6) cells) in spent medium. Immunocytologic +-and-histochemical staining for tumor markers of the original tumor, the cultured cells and the transplanted tumors also revealed the localization of not only hCG and beta-hCG but also CA19-9 and CA-125 whose values had been elevated in the preoperative serum (hCG: 10 mIU/ml, CA19-9: 6,400 U/ml, CA-125: 225 U/ml). Results of PAS, Alcian-blue and Mucicarmine strains indicated that most of the PAS-positive substances in the cultured cells and the transplanted tumors were consistent with glycogen while the original tumor mainly contained mucin except for the lesion of poorly differentiated adenocarcinoma with glycogen. These results suggested that the cultured cells might originate from poorly differentiated adenocarcinoma cells in the original tumor.     

Inhibition by Cyclic AMP of Phorbol Ester-Potentiated Norepinephrine Release from Guinea Pig Brain Cortical Synaptosomes

The involvement of Ca2+/phospholipid-dependent protein kinase (protein kinase C, PKC) and cyclic AMP-dependent protein kinase in the K+-evoked release of norepinephrine (NE) was studied using guinea pig brain cortical synaptosomes preloaded with [3H]NE. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a potent activator of PKC, enhanced the K+-evoked release of [3H]NE, in a concentration-dependent manner, but with no effect on the spontaneous outflow and uptake of [3H]NE in the synaptosomes. The apparent affinity of the evoked release for added calcium but not the maximally evoked release was increased by TPA (10(-7) M). Inhibitors of PKC, polymyxin B, and a more potent inhibitor, staurosporine, counteracted the TPA-induced potentiation of the evoked release. Both forskolin and dibutyryl cyclic AMP (DBcAMP) enhanced the evoked release, but reduced the TPA-potentiated NE release. A novel inhibitor of cyclic AMP-dependent protein kinase, KT5720, blocked both the forskolin-induced increase in the evoked release and its inhibition of TPA-induced potentiation in the evoked release, thereby suggesting that forskolin or DBcAMP counteracts the Ca2+-dependent release of NE by activating cyclic AMP-dependent protein kinase. These results suggest that the activation of PKC potentiates the evoked release of NE and that the activation of cyclic AMP-dependent protein kinase acts negatively on the PKC-activated exocytotic neurotransmitter release process in brain synaptosomes of the guinea pig.     

Silence of streptomycin 6-phosphotransferase gene derived by incubation at a high temperature inStreptomyces griseus

Summary The bald mutants from streptomycin (SM)-producingStreptomyces griseus 2247 obtained by incubation at high temperature (36° C), designated as HT strains, lost resistance to their own antibiotic and scarcely produced the antibiotic. Although SM susceptibility in the mutant was due to loss of SM 6-phosphotransferase activity produced in the cell, the gene coding for the enzyme cloned from an HT strain was surely expressed inS. lividans 1326 as a host. Northern blot analysis showed that the corresponding RNA is not detected in the mutant, indicating that though the gene encoding SM 6-phosphotransferase, at least, the structural gene is not deleted in the cell, the expression is silent.     

Homeostatic Regulation of Membrane Potential by an Electrogenic Ion Pump against Change in the K+ Concentration of the Extra- and Intra-Organ Perfusion Solutions

The dependence of membrane potentials on changes in the extra-cellularK⁺ concentration [K⁺]_e was investigated in potato tuber sliceswith dripping perfusion, and in growing Vigna hypocotyl segmentswith pressurized intra-organ perfusion methods. Only under anoxiawere the membrane potential of potato tuber slices and the electricpotential difference between the parenchyma symplast and xylem(V_px) of Vigna hypocotyl segments depolarized markedly (46 mVand 42 mV/log[K⁺]_e unit, respectively) with increasing [K⁺]_eabove the critical values. The electric potential differencebetween the parenchyma symplast and organ surface (V_ps of thehypocotyl segments remained nearly unchanged up to 30 mEq [K⁺]_e.Under highly aerobic conditions the membrane potentials wererelatively independent of [K⁺]_e except at very high K⁺ concentrations.V_ps showed even hyperpolarization with the increasing KCl concentrationin the perfusion solution that is not in direct contact withthe surface membrane of the parenchyma symplast. The respiration-dependentelectrogenic components of the membrane potentials regularlyincreased with the increasing [K⁺]_e. A voltage-dependent homeostaticcontrol of membrane potential is discussed. (Received August 13, 1984; Accepted December 21, 1984)     

Aberrant porphyrin metabolism in hepatocellular carcinoma

In 15 patients with hepatocellular carcinoma (HCC) and 14 patients with liver cirrhosis (LC), urinary excretions of delta-aminolevulinic acid (ALA), porphobilinogen (PBG), uroporphyrin (UP), coproporphyrin (CP), and erythrocyte contents of CP and protoporphyrin (PP) were examined. In patients with HCC, urinary excretions of ALA and PBG and erythrocyte contents of CP and PP were not increased, but urinary excretions of UP and CP were significantly increased more than those of LC patients. Urinary excretions of UP and CP had no correlations with liver function tests and excretion of UP correlated slightly with blood hemoglobin level. After administration of ALA intravenously, urinary excretions of UP and CP were clearly increased in patients with HCC compared to normal controls. A Red fluorescent area was present at the cancerous area but not in the noncancerous cirrhotic area in a patient with HCC. These results suggest that aberrant porphyrin metabolism occurred in patients with HCC compared to other liver diseases.