Rearrangement of the surface antigen gene of hepatitis B virus integrated in the human hepatoma cell lines.

The rearrangement of integrated HBV DNA sequences in three different hepatoma cell lines, huH-1, huH-2, KG-55-T from Japanese patients, were studied by blot hybridization using whole HBV genome or a HBsAg or HBcAg DNA as a probe. The characteristic existence of multiple integration sites of HBV DNA sequences in each HindIII-restricted hepatoma cell DNA was revealed by the HBV genome probe. Detection of the isolated HBsAg gene in the HindIII fragment indicates that the integration of HBV DNA was not always related to the maintenance of the whole viral genome, and that movement of the HBsAg gene to another location occurred by rearrangement. On the other hand, the presence of the HBV DNA sequence without the intact HBcAg gene was shown in some of the HindIII fragments, when the HBcAg gene, probe was used, but a HindIII fragment, containing only the HBcAg gene, was not detected so far. The absence of the intact HBcAg gene suggests that the viral genome may lose a part of the HBcAg gene in the process of integration. This is consistent with recent findings of Ogston et al. (1982) that in Woodchuck hepatocellular carcinoma viral sequences are extensively rearranged.     

Denitrification, Nitrate Reduction, and Oxygen Consumption in Coastal and Estuarine Sediments

Denitrification and consumption of oxygen and nitrate in sediments from Tama Estuary, Odawa Bay, and Tokyo Bay were measured in an experimental sediment-water system. Filtered seawater containing [¹⁵N]nitrate flowed continuously over undisturbed sediments, and the concentrations of O₂, nitrate, and nitrite in the influent and effluent and of ¹⁵N₂ in the effluent were monitored. Under steady-state conditions, the rate of nitrate consumption was the same order of magnitude as the rate of oxygen consumption in Tama Estuary sediments, whereas the former rate was one order of magnitude lower than the latter rate in Odawa Bay and Tokyo Bay sediments. Denitrification accounted for 27 to 57% of the nitrate consumption.     

Regulation of indoleamine 2,3-dioxygenase activity in the small intestine and the epididymis of mice

Indoleamine 2,3-dioxygenase activity was found to be ubiquitously distributed in various tissues of mice, such as brain, lung, stomach, intestine, and epididymis. The highest enzyme activity was detected in the alimentary canal and the epididymis. Developmental and daily rhythmic changes of indoleamine 2,3-dioxygenase activity and the effects of various regulatory factors were studied with the supernatant fractions derived from the small intestine and the epididymis. The enzyme activity in these two tissues was absent during the first 2 weeks (the weaning period). From the third week, there was a rapid increase in activities and a maximum was reached when the mice were 8 to 10 weeks of age (adolescence). The enzyme activity in the small intestine then gradually diminished to zero level at 30 weeks of age (prime) or later, while that in the epididymis remained at the high level throughout 69 weeks of age (senescence). The enzyme activity of the small intestine from mice fed during the hours 9:00–13:00 showed daily rhythmic changes; high in the daytime and low at night. Under night feeding (21:00–1:00), the enzyme activity was high at night and low in the daytime. The epididymal enzyme activity showed no daily fluctuations by either feeding schedule. With regard to the developmental and daily rhythmic changes, indoleamine 2,3-dioxygenase activity in the small intestine was similar to that of hepatic tryptophan 2,3-dioxygenase. However, in contrast to the hepatic tryptophan 2,3-dioxygenase activity, indoleamine 2,3-dioxygenase activity in the small intestine and the epididymis was not affected by adrenalectomy or intraperitoneal administration of adrenal steroid or tryptophan.     

Subcellular action of Neocarzinostatin. Intracellular incorporation, DNA breakdown and cytotoxicity

The subcellular site of action of a proteinaceous antitumor antibiotic neocarzinostatin (NCS) was studied using normal human lymphocytes, Epstein-Barr virus transformed lymphoblastoid cells, osmotically burst lymphoblastoid cells, and colicin E1 plasmid DNA. The rate of DNA strand break in these different types of DNA was found to be in the following order: Colicin DNA > burst cell DNA > lymphoblastoid cell DNA > normal lymphocyte DNA. Furthermore, fluorescence microscopy revealed that lymphoblastoid cells incorporated more fluorescein isothiocyanate labeled NCS than normal cells. High uptake of NCS in lymphoblastoid cells coincided with a high killing rate; low uptake of NCS in lymphocytes resulted in very little cell killing. Uptake velocity using fluorescein diacetate (FDA) also showed that the lymphoblastoid cells exhibited a higher uptake of FDA coinciding with a higher killing rate. The cell killing activity of NCS appears to be closely associated with the rate of intracellular uptake of NCS and subsequent direct degradation of DNA by the drug. This notion is reinforced by the reported finding that the dose required for DNA strand scission is only about 1/100 of that for the inhibition of cap formation. Thus DNA strand scission, rather than the cell membrane, appears to be the primary target of NCS. Enhanced incorporation of many substances is commonly observed upon transformation of cells by viruses, and our present results may provide an important clue toward the explanation of the selective toxicity toward tumor cells of NCS.     

Phosphorylation of mouse thymocyte CD4 and CD8: regulation of surface expression.

Murine T cell surface antigens, CD4 and CD8 are phosphorylated in response to phorbol 12-myristate 13-acetate, a protein kinase C activator, but not phosphorylated after concanavalin A, Ca2+ ionophore or dibutyryl-cAMP treatment. We examined the cell surface expression of both antigens and show that surface CD4 on CD4+CD8+ and CD4+CD8- thymocytes is rapidly decreased after PMA treatment, while CD8 expression is unaffected. Prolonged PMA treatment, which down-regulates protein kinase C, allows CD4 reexpression only in the CD4+CD8- population, suggesting that different mechanisms of cell surface antigen expression are operating in the two thymocyte subpopulations.     

Immunohistochemical evidence that rat FSH cells contain beta II-subspecies of protein kinase C.

Immunocytochemical double-staining analysis revealed that in the rat anterior pituitary 86% of cells containing the beta II-subspecies of protein kinase C also contained follicle stimulating hormone (FSH), and that 22% of these FSH cells expressed the beta II-subspecies. These findings suggest a close relationship between the beta II-subspecies of protein kinase C and FSH regulation.     

Vasoactive intestinal peptide enhances dopamine accumulation in primary cell culture of rat hypothalamus.

The effect of vasoactive intestinal peptide (VIP) and PHI-27 on dopamine accumulation in cultured rat hypothalamic cells was investigated. VIP enhanced [3H]dopamine accumulation dose dependently. This effect was significant at 10(-8)-10(-5) M VIP with a concomitant increase in intracellular cyclic AMP (cAMP), and reached its plateau level at 10(-6) M VIP. VIP increased [3H]dopamine accumulation significantly within 15 min. PHI-27 and dibutyryl cAMP ((Bu)2-cAMP) also enhanced [3H]dopamine accumulation. These results suggest that VIP enhances dopamine accumulation in hypothalamic cells by increasing intracellular cAMP.     

Thermoluminescence and flash-induced oxygen yield in herbicide resistant mutants of the D1 protein in Synechococcus PCC7942.

Several strains of Synechococcus PCC7942 carrying point mutations in the gene psbA were studied by thermoluminescence and polarographic measurement of flash-induced oxygen yield. The following results were obtained: (a) Replacement of Ser-264 in D1 by Ala (mutant Di1) or Gly (mutant G264) resulting in DCMU and atrazine resistance leads to a downshift of the thermoluminescence (TL) B-band peak temperature from 40 degrees C in wild-type thylakoids to about 30 degrees C. In dark adapted samples of both mutants the TL and oxygen yield pattern induced by a train of single turnover flashes were strongly damped indicative of a high miss factor. (b) In contrast to Ser-264 mutants, replacement of Phe-255 in D1 by Tyr (mutant Tyr5) induced strong resistance to atrazine but not to DCMU and did not affect the peak termperature of the B-band and the flash-induced TL and oxygen yield patterns. In this respect mutant Tyr5 resembles the wild type. (c) No significant differences have been found between strains with single site mutations in psbAI and normal psbAII/psbAIII genes, and strains with same mutations in psbAI but additional deletion of psbAII and psbAIII. Obviously in strains were psbAI is present, PS II complexes containing gene products of psbAII and psbAIII are not assembled in detectable amounts. (d) Strains with double mutations at positions 264 and 255 display a downshift of the B-band peak temperature. Their oscillatory patterns of B-band intensity and oxygen yield are highly damped. This behaviour is similar to strains D1 and G264 which are modified at position 264 only. We extend reports on additivity of mutation effects on herbicide binding to binding of QB. (e) Mutations at the QB site not only influence the binding of QB and herbicides but also change the thermoluminescence quantum yield and the lifetimes of the redox states S2 and S3 of the water oxidase. This finding might indicate long ranging effects on Photosystem II exerted by structural modifications of the QB site. From these data we conclude that Ser-264 is essential for binding of atrazine, DCMU and QB, whereas Phe-255 is involved in atrazine binding and its substitution by Tyr does not markedly affect QB or DCMU binding in Synechococcus PCC7942.