Early changes in mitochondrial monoamine oxidase activity of rat liver during 2-acetylaminofluorene feeding

The activities of mitochondrial type A and B monoamine oxidase were determined in the liver of rats fed a diet containing 2-acetylaminofluorene (AAF). Three days after the initiation of AAF-feeding, there was a significant decrease of type B monoamine oxidase activity without affect on type A enzyme. The decreased activity of type B monoamine oxidase, which reached a minimum after three weeks, was sustained for as long as AAF-feeding was continued. Sex-related difference in response to AAF was seen in the rat with respect to the onset and the intensity of the decreased type B monoamine oxidase activity, male rats being more sensitive to the carcinogen than female rats. In contrast to the in vivo effect, AAF showed a potent inhibitory effect on type A monoamine oxidase, rather than on type B enzyme, when added in vitro. The pI₅₀ values were estimated to be 7.5 against type A monoamine oxidase and 4.1 against type B enzyme, respectively. The in vitro inhibition of both types of monoamine oxidase by AAF was competitive. The K_i values for AAF were calculated to be 9.51 · 10^?9 M for type A monoamine oxidase and 1.30 · 10^?5 M for type B enzyme, respectively. In accordance with the potent inhibitory effect of AAF on type A monoamine oxidase in vitro, a single administration of the carcinogen, at a dose of 50 mg/kg, resulted in a marked and temporal decrease of the enzyme activity in the mitochondria of male rat liver. Recovery of the decreased type B monoamine oxidase activity was slow, and the enzyme activity did not return to control levels, even if rats were fed the basal diet for 2 or 4 weeks after the cessation of AAF-feeding.     

Apterichtus keramanus, a new snake-eel from Okinawa, southern Japan (Ophichthinae, Ophichthidae)

A new snake-eel,Apterichtus keramanus, is described on the basis of a single 276-mm TL specimen trawled from the coast of Kerama Islands, Okinawa Prefecture, Japan. The species is unique in the genus in having the posterior nostril opening entirely inside the mouth and a dark band running from the anteroventral margin of the eye to the upper lip.     

Plant-growth regulators from common starfish (<Emphasis Type="Italic">Asterias amurensis</Emphasis> Lütken) waste

Starfish waste has been shown to be an effective compost material not only in the promotion of plant growth but also in terms of having insecticidal activity. In the present study, plant growth regulation by chemicals from starfish was examined. The aqueous fraction from a hot water extract of the starfish Asterias amurensis Lütken showed plant-growth activity, while the aqueous fraction from a methanol extract inhibited growth of Brassica campestris. The lipophilic fraction from the methanol extract also exhibited a plant growth-promoting effect. The active components from each extract were identified. Asterubine from the hot water extract promoted plant growth. A ceramide from the lipophilic fraction showed root growth promoting effect, and three glucocerebrosides had promotive effects on the entire plant. Asterosaponins were identified as the main growth inhibitors in the aqueous fraction of the methanol extract. These active compounds from starfish waste could be analyzed as potential plant growth regulators in agricultural applications in the future.     

MiR-376c Down-Regulation Accelerates EGF-Dependent Migration by Targeting GRB2 in the HuCCT1 Human Intrahepatic Cholangiocarcinoma Cell Line

MicroRNA miR-376c was expressed in normal intrahepatic biliary epithelial cells (HIBEpiC), but was significantly suppressed in the HuCCT1 intrahepatic cholangiocarcinoma (ICC) cell line. The biological significance of the down-regulation of miR-376c in HuCCT1 cells is unknown. We hypothesized that miR-376c could function as a tumor suppressor in these cells. To test this hypothesis, we sought the targets of miR-376c, and characterized the effect of its down-regulation on HuCCT1 cells. We performed proteomic analysis of miR-376c-overexpressing HuCCT1 cells to identify candidate targets of miR-376c, and validated these targets by 3′-UTR reporter assay. Transwell migration assays were performed to study the migratory response of HuCCT1 cells to miR-376c overexpression. Furthermore, microarrays were used to identify the signaling that were potentially involved in the miR-376c-modulated migration of HuCCT1. Finally, we assessed epigenetic changes within the potential promoter region of the miR-376c gene in these cells. Proteomic analysis and subsequent validation assays showed that growth factor receptor-bound protein 2 (GRB2) was a direct target of miR-376c. The transwell migration assay revealed that miR-376c significantly reduced epidermal growth factor (EGF)-dependent cell migration in HuCCT1 cells. DNA microarray and subsequent pathway analysis showed that interleukin 1 beta and matrix metallopeptidase 9 were possible participants in EGF-dependent migration of HuCCT1 cells. Bisulfite sequencing showed higher methylation levels of CpG sites upstream of the miR-376c gene in HuCCT1 relative to HIBEpiC cells. Combined treatment with the DNA-demethylating agent 5-aza-2′-deoxycytidine and the histone deacetylase inhibitor trichostatin A significantly upregulated the expression of miR-376c in HuCCT1 cells. We revealed that epigenetic repression of miR-376c accelerated EGF-dependent cell migration through its target GRB2 in HuCCT1 cells. These findings suggest that miR-376c functions as a tumor suppressor. Since metastasis is the major cause of death in ICC, microRNA manipulation could lead to the development of novel anti-cancer therapy strategies for ICC.     

Ultrastructural changes of the midgut epithelial cells in feeding and moulting nymphs of the tick Haemaphysalis longicornis

The midgut epithelial cells in nymphs fed on laboratory rabbits were examined during feeding and after detachment. The midgut epithelium at the unfed stage consisted of digestive cells of lower activity, containing such nutritive substances as protein, lipid and glycogen. As feeding proceeded, the cells became active in intracellular digestion. At the middle of the feeding stage, the spent digestive cells derived from the active digestive cells began to be replaced by the new digestive cells of lower activity. After detachment, the pinocytotic activity of the above cells increased greatly, and the digestive activity increased to some extent. As a result, many large endosomes were formed by fusion of numerous pinosomes. Thereafter, endosomes decreased in size as digestion proceeded and there was an increase of haematin granules. On day 7 after detachment, the new digestive cells of lower activity, belonging to the 'nutritional reserve' type, appeared adjacent to the spent digestive cells which had almost exhausted all endosomes, and these new cells had completely replaced the spent cells by day 3 after moulting.     

Involvement of protein kinase C in Ca(2+)-independent contraction of rat uterine smooth muscle

The contribution of protein kinase C to the contraction by oxytocin of rat uterine longitudinal smooth muscle in Ca(2+)-free solution was investigated. Immunological analysis revealed that type II (beta) and III (alpha) protein kinase C subspecies were present in rat uterine smooth muscle. The pretreatment of a diacylglycerol kinase inhibitor R59022 to accumulate diacylglycerol potentiated the Ca(2+)-independent contraction. The contractile activity was diminished with the depletion of protein kinase C, when the contraction was evoked repeatedly by oxytocin during the prolonged exposure to a tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate. These results suggested the involvement of protein kinase C in oxytocin-induced contraction in Ca(2+)-free solution.     

Effects of temperature on development and growth in the tick,Haemaphysalis longicornis

As a part of ecological studies onHaemaphysalis longicornis, the effects of controlled temperatures (12, 15, 20, 25 and 30°C; 100% RH) on development and growth of the tick were investigated and the critical low temperature for each stage in the life cycle was estimated. As the temperature became low, the periods of preoviposition, oviposition, egg hatching (incubation) and moulting were prolonged. At 12°C, however, oviposition, egg hatching and moulting of the larva and nymph did not occur. The critical low temperatures for oviposition, egg hatching (developmental zero) and larval and nymphal moulting which were calculated theoretically from the regression equations, were 11.1, 12.2, 10.2 and 11.8°C, respectively. The temperature also affected the egg productivity and hatch-ratio. The number of deposited eggs per mg of body weight decreased markedly at 15°C, and the hatch-ratio was lowered with dropped temperatures.     

Elongation and shortening of time required for entry into S phase after release from G 1 and G 0 arrests in temperature-sensitive mutants of rat 3Y1 Cells

Temperature-sensitive (ts) mutants of rat 3Y1 fibroblasts representing four separate complementation groups (3Y1tsD123, 3Y1tsF121, 3Y1tsG125, and 3Y1tsH203) are arrested mainly in the G1 phase when cells of randomly proliferating population at 33.8 degrees C are shifted to 39.8 degrees C (temperature arrest). We examined the time lag of the cellular entry into the S phase after release at 33.8 degrees C, both from the temperature arrest and from the arrest at 33.8 degrees C at a confluent cell density (density arrest). In the temperature-arrested cells, as the duration of temperature arrest increased, the time lag of entry into S phase after shift down to 33.8 degrees C was prolonged, in all four mutants. These observations suggest that the four different functional lesions, each causing arrest in the G1 phase, are also responsible for prolongation of the time lag of entry into the S phase in cells arrested in the G1 phase. The prolongation of the time lag in the temperature-arrested cultures was accelerated at a higher cell density, in medium supplemented with a lower concentration of serum, and at a higher restrictive temperature. In the density-arrested cells, as the duration of pre-exposure to 39.8 degrees C was increased, the time lag of entry into S phase at 33.8 degrees C after release from the arrest was drastically prolonged, in all four mutants. In 3Y1tsF121, 3Y1tsG125, and 3Y1tsH203, when the density-arrested cells were prestimulated by serum at 39.8 degrees C for various periods of time, the time lag of entry into S phase after release from the density arrest at 33.8 degrees C was initially shortened, and then, prolonged progressively as the period of prestimulation increased. These findings, taken together with other data, show that all four ts defects affect cells in states ranging from the deeper resting to mid- or late-G1 phase. It is suggested that events represented by these four mutants are required for entry into the S phase and normally operate in parallel but not in sequence in cells in states ranging from the deeper resting to the mid- or late-G1 phases, though they may affect each other.     

Highly frequent single amino acid substitution in mammalian elongation factor 2 (EF-2) results in expression of resistance to EF-2-ADP-ribosylating toxins

Toxin-resistant polypeptide chain elongation factor 2 cDNA has been cloned from a mutant hamster cell line with only non-ADP-ribosylatable elongation factor 2. The mutation conferring resistance to diphtheria toxin and Pseudomonas aeruginosa exotoxin A is a G-to-A transition in the first nucleotide of codon 717. Codon 715 encodes a histidine residue that is modified post-translationally to diphthamide, which is the target amino acid for ADP-ribosylation by both toxins. Transfection of mouse L cells with a recombinant elongation factor 2 cDNA differing from the wild-type only by this G-to-A transition confers resistance to P. aeruginosa exotoxin A. The degrees of toxin-resistant protein synthesis of stable transfectants are dependent on the ratio of non-ADP-ribosylated elongation factor 2 to wild-type elongation factor 2, not the amount of non-ADP-ribosylated elongation factor 2. The mutation creates a new Mbo II restriction site in the elongation factor 2 gene. Several independently isolated diphtheria toxin-resistant Chinese hamster ovary cell lines show the same alteration in the Mbo II restriction pattern.     

The mode of entry of herpes simplex virus type 1 into Vero cells

The mode of entry of herpes simplex virus type 1 (HSV-1) into Vero cells was investigated quantitatively with biological techniques. The entry of virus occurred rapidly when the virus-adsorbed cells were incubated at 37 C. The kinetics of virus entry was found to be similar to that of the process of uncoating, indicating that the uncoating of HSV-1 occurs simultaneously with the entry of virus into the cell. Experiments with ammonium chloride revealed that acidity in endosomes is not necessary for the entry or uncoating of HSV-1, in contrast with the cases of enveloped RNA viruses. In addition, endocytosis of the virus seems to be one of the processes of entry for HSV-1. However, the kinetics of endocytosis showed that the cell-bound virus is endocytosed gradually and suggested that the endocytosis of HSV-1 does not lead the virus to an uncoating process. These results are most consistent with a mechanism of entry for HSV-1 involving fusion of the viral envelope with the plasma membrane of the host cell.