Modifying effects of 1'-acetoxychavicol acetate (ACA) and the novel synthetic retinoids Re-80, Am-580 and Am-55P in a two-stage carcinogenesis model in female rats

Effects of dietary administration of 1'-acetoxychavicol acetate (ACA) and the novel synthetic retinoids 4-[1-hydroxy-3-oxo-3-(5,6,7,8-tetrahydro-3-hydroxy-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid (Re-80); 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoic acid (Am-580); and 6-[(3,5-di-tert-butylphenyl) carbamoyl]nicotinic acid (Am-55P) were examined using a two-stage rat carcinogenesis model. A total of 190 female SD rats was treated sequentially with 1,2-dimethylhydrazine (DMH, s.c.); 7,12-dimethylbenz(a)anthracene (DMBA, i.g.); and 2,2'-dihydroxy-di-n-propylnitrosamine (DHPN, in the drinking water) during the first three weeks (DDD-initiation), and an additional 60 rats received the vehicle alone (non-initiation). One week after the completion of the initiation period, they were divided into nine groups and administrated Re-80 (at dose levels of 1.0 or 0.4 ppm), Am-580 (20 or 4 ppm), Am-55P (20 ppm), ACA (100 ppm), all-trans-retinoic acid (10 or 2 ppm) or no supplement in the diet for 33 weeks, until survivors were euthanatized at week 37 weeks. After DDD-initiation, all-trans-retinoic acid at the high dose delayed the development of mammary tumors. The multiplicity of colon tumors in the group fed Am-55P and the incidences of nephroblastomas with ACA or Am-580 were decreased as compared with the control values, but the other chemicals had no modifying effects on tumor development in any organs. Thus, among ACA and the novel synthetic retinoids tested, only Am-55P showed a weak inhibitory effect on a neoplasm of general interest under the present experimental conditions.     

Activation of protein kinase C induces internalization of the GABA(C) receptors expressed in Xenopus oocytes

In a previous study, we showed that the protein kinase C (PKC) activator 4beta-phorbol 12-myristate 13-acetate (PMA) inhibited the gamma-aminobutyric acid (GABA)-gated currents in Xenopus oocytes expressing human rho1 GABA(C) receptors. To investigate whether the inhibition of currents was due to a decrease in efficacy or in the potency of rho1 GABA(C) receptor, concentration-response curves for GABA were compared before and after PMA treatment. The EC50 concentrations of GABA obtained during the maximally inhibited period were not statistically different from the concentrations obtained before PMA treatment (1.74 +/- 0.33 and 1.45 +/- 0.28 microM, respectively). These results indicate that the inhibition depends on a change in number or conductance of active receptor channels, but not on a change in affinity for GABA. To allow histochemical detection of rho1 GABA(C) receptors, we constructed a receptor tagged at the C-terminal position with human c-myc epitope. Electrophysiologically, the tagged receptors showed almost the same sensitivities for GABA and PMA as those of wild-type rho1 GABA(C) receptors. Immunohistochemistry with anti-myc antibody detected a dense concentration of tagged receptors at the surface area of Xenopus oocytes. Transient exposure to PMA reduced the density of immunofluorescence at the surface area and increased it in the subsurface area. These results suggest that the stimulation of protein kinase C leads to internalization of rho1 GABA(C) receptors expressed in Xenopus oocytes.     

Detection of the putative E2 protein of hepatitis C virus in human liver

The question was asked whether a predicted envelope protein, considered to be processed from the polyprotein precursor encoded by the putative E2/NS1 region of the hepatitis C virus (HCV) genome, may be observed in HCV-infected humans. Two polyclonal antibodies against recombinant E2/NS1 proteins were prepared and their reactivity tested against liver extracts from HCV-infected patients by immunoblotting analysis. A band corresponding to a size of 44 kDa was detected in liver extracts from patients who were positive for the HCV-specific antibody anti-C100-3 but not in liver extracts from patients who did not have anti-C100-3 antibody. Additionally, no band was detected using preimmune sera or antisera which had been preabsorbed with recombinant E2/NS1 proteins. Deglycosylation studies demonstrated that the 44 kDa protein was a glycosylated form of a 38 kDa protein which corresponds to the predicted molecular weight of the putative E2/NS1 protein. These results suggest that the 44 kDa protein is a product of the E2/NS1 region. Frequent observation of the 44 kDa band in cases of chronic active hepatitis C suggests a correlation between the expression of this protein and the progression of hepatitis. © 1994 Wiley-Liss, Inc.     

Construction and characterization of centric circular and acentric linear chromosomes in fission yeast

Summary Gene disruption and gap repair of chromosomal DNA have been frequently employed techniques in yeast genetics. To extend the possibility of using these gene manipulations for larger genomic regions, we have examined the maximal sizes of chromosomal DNA disrupted or repaired in vivo. Here we report a simple, potentially general, method for selectively deleting a 150 kb region, or gap-filling a 100 kb region, in the fission yeast genome. This enables the generation of acentric linear chromosomes by deletion, or the cloning of large functional centromeric DNAs into circular minichromosomes by gap-filling. The fidelity of the resulting gap-filling is high, judging from partial-digestion mapping of gap-repaired DNAs. By analysing a series of such circular minichromosomes, we conclude that only a part of the repetitive centromeric region, including the central domain, is essential for mitotic and meiotic chromosome segregation. Acentric linear chromosomes, although unstable, could be maintained, indicating that it may be possible to construct an acentric vector for large DNA fragments in this organism.     

Administration of interleukin -5 or -10 activates peritoneal B-1 cells and induces autoimmune hemolytic anemia in anti-erythrocyte autoantibody-transgenic mice

Activation mechanisms of B-1 (Ly-1 B) cells have been suggested to be different from those of conventional B cells. To assess the role of various interleukins (IL) in the activation of B-1 cells, we injected IL-4, IL-5 or IL-10 into nonanemic anti-red blood cells (RBC) autoantibody-transgenic mice, in which conventional B cells are clonally deleted but peritoneal B-1 cells persist without secreting Ig. Intraperitoneal or intramuscular injection of IL-5 or IL-10, but not IL-4, increased the number of antibody-producing peritoneal B-1 cells by four- to five-fold, resulting in increased anti-RBC serum autoantibody and induction of hemolytic anemia. These results suggest that IL-5 or IL-10 may play an important role in the terminal differentiation of B-1 cells into antibody-producing cells in vivo.     

Intensely positively charged perineuronal nets in the adult rat brain as detected by staining with anionic iron colloid

Ferric chloride, when boiled with ammonium thiocyanate, ammonia and cacodylic acid, is converted into a fine anionic iron colloid which consists of 1.0-1.5 nm electron dense granules and gives a distinct Prussian blue reaction (OHTSUKA and MURAKAMI, 1986). Light microscopy of tissue sections stained with this fine anionic iron colloid at pH values of 6.0, 7.0 and 8.0 showed that the healthy adult rat brain contains a considerable number of neurons which possess an intensely positively charged perineuronal net. This net was most clearly demonstrable by staining with the anionic iron colloid at a pH value of 8.0, at which ionizations of almost all cationic sites of the tissue elements were obliterated. Transmission electron microscopy of ultrathin sections stained at a pH value of 8.0 showed that the anionic iron colloid was preferentially deposited in the perineuronal tissue spaces. These findings indicate that the intensely positively charged perineuronal net contains some strongly basic substances such as guanidino compounds, and occupies the perineuronal (perisynaptic) tissue space.     

Comparison of thermosensitivity in the preoptic/anterior hypothalamic area and the midbrain of the rabbit

Summary To characterize the role of the midbrain temperature-sensitive structure in thermoregulation, the relative importance of thermosensitivity in the hypothalamus and the midbrain was studied in terms of heat production and heat loss in the rabbit. It was found that altering the local temperature in the midbrain had no influence at all on heat loss from the ear surface and also on heat production, while cooling and warming of the preoptic/anterior hypothalamic area induced appropriate thermoregulatory responses.