A single gene transfer of gibberellin biosynthesis gene cluster increases gibberellin production in a Fusarium fujikuroi strain with gibberellin low producibility

Fusarium fujikuroi, the causative agent of bakanae disease in rice, produces many kinds of secondary metabolites. Recently, two phylogenetic subgroups (F and G groups) of Japanese F. fujikuroi have been identified and found to have differences in their gibberellin (GA) and fumonisin production. G-group F. fujikuroi produces large amounts of GA, but is a fumonisin nonproducer. F-group produces large amounts of fumonisin, but is a GA low or nonproducer. We investigated the cause of low GA production in the F-group. Genetic mapping suggests that low GA production in the F-group strain Gfc0825009 is due to a GA gene cluster for GA biosynthesis. Analysis of the nucleotide and amino acid sequences of the genes in the GA gene cluster showed >98.4% homology between the F-group strain Gfc0825009 and the G-group strain Gfc0801001. Following a 7-day culture under low nitrogen conditions, we found that expression of P450-1, P450-4, and P450-2 in the cluster increased in the G-group strain and not in the F-group strain. We hypothesized that complementation by GA genes in the G-group strain would be required to increase GA production in the F-group strain. However, we found that this occurred with a single gene complementation of DES, P450-1, P450-4, or P450-2. Simultaneous increase in the expression of P450-1, P450-4, and P450-2 were detected in the complementary transformants. Moreover, the same phenomenon was observed by reintegration of its own P450-1. Our results suggest the presence of unknown regulatory mechanisms of the GA gene cluster in F. fujikuroi.     

Development of a time-resolved fluoroimmunoassay for octopus gonadotropin-releasing hormone

We have developed a time-resolved fluoroimmunoassay (TR-FIA) for octopus gonadotropin-releasing hormone (oct-GnRH) to determine the profiles of oct-GnRH peptide levels in cephalopods. The sensitivity and the intra-assay and inter-assay coefficients of variation were 4.9 pg/well and 6.8 (n = 10) and 2.7% (n = 5), respectively. Anti-oct-GnRH antibody was tested on all known forms of GnRH and found to cross-react with lamprey GnRH-II (27.1%), annelid GnRH (3.36%), tunicate GnRH-I (0.92%), dogfish GnRH (0.51%), and scallop Patinopecten yessoensis GnRH (0.05%). The displacement curve obtained for serially diluted brain extracts of three cephalopods, the spear squid Loligo bleekeri, swordtip squid Loligo edulis, and North Pacific giant octopus Octopus dofleini, paralleled the oct-GnRH standard curve. The presence of oct-GnRH in the central nervous system (CNS) of these cephalopods was further examined by a combination of reverse-phase high performance liquid chromatography and oct-GnRH TR-FIA. CNS extracts from these cephalopods showed peaks with retention times similar to that of synthetic oct-GnRH. These results indicate that this novel oct-GnRH TR-FIA is widely applicable for oct-GnRH measurement in cephalopods.     

Phylogeography of the Japanese eight-barbel loach <Emphasis Type="Italic">Lefua echigonia</Emphasis> from the Yamagata area of the Tohoku district,Japan

The Japanese eight-barbel loach Lefua echigonia, which is a freshwater fish native to Japan, is distributed from the Tohoku to Kinki districts and is divided into six regional populations according to mtDNA analysis. In this study, we investigated L. echigonia collected from several locations in Yamagata Prefecture and neighboring prefectures using mtDNA control region sequences and confirmed the spatial distribution pattern among the new regional population (Yamagata population). The new population was limited to the Mogami river system in the inland area of Yamagata Prefecture and is distinguished from other regional populations by high sequence divergences.     

Studies on Endophytic Actinomycetes ( I ) <Emphasis Type="Italic">Streptomyces </Emphasis>sp. Isolated from Rhododendron and Its Antifungal Activity

To survey endophytic actinomycetes as potential biocontrol agents against fungal diseases of rhododendron, young plants of rhododendron were surface-sterilized for use as an isolation source. Nine, six and two isolates, with distinguishing characteristics based on the macroscopic appearance of colonies, were obtained from roots, stems and leaves, respectively, suggesting that various species of actinomycetes grow in the respective organs of this plant as symbionts or parasites. On an agar medium, only isolate R-5 commonly formed a clear growth-inhibition zone against two major fungal pathogens of rhododendron, Phytophthora cinnamomi and Pestalotiopsis sydowiana, indicating that this isolate can produce antifungal material(s). Acetone extracts of a liquid culture of R-5 had a broad antimicrobial spectrum against Gram-positive bacteria, yeast and filamentous fungi. Isolate R-5 was identified as a Streptomyces sp. based on morphological, physiological and chemotaxonomical characteristics. The present results indicate that isolate R-5 is a suitable candidate for the biocontrol of diseases of rhododendron. Received 25 March 2000/ Accepted in revised form 18 May 2000     

Interaction of orexin/hypocretin-like immunoreactive neurons with melanin-concentrating hormone and α-melanocyte-stimulating hormone neurons in brain of a pleuronectiform fish, barfin flounder

Immunohistochemical localization of orexin/hypocretin in the brain of a pleuronectiform fish, the barfin flounder Verasper moseri was examined as the first step in unraveling the possible function of the hormone in the brain. Orexin-A-like immunoreactive (ir) cell bodies were found to be located in the nucleus posterioris periventricularis (NPPv) of the hypothalamus, and orexin-A-like-ir fibers were detected not only in the hypothalamus but also extensively throughout the brain. The orexin-A-like-ir cell bodies did not project their fibers to the pituitary gland. Since melaninconcentrating hormone (MCH) and α-melanocyte-stimulating hormone (α-MSH) are suggested to regulate food intake in addition to orexin/hypocretin in the teleost fish, it was examined whether neural connections exist between orexin neurons and the MCH and α-MSH neurons in the barfin flounder brain by using double-staining immunohistochemistry. Some orexin-A-like-ir fibers were in close contact with the MCH-ir and α-MSH-ir cell bodies in the hypothalamus. Moreover, a few MCH-ir and α-MSH-ir fibers were in close contact with the orexin-A-like-ir cell bodies in the hypothalamus. These results suggest that reciprocal connections exist between the orexin and MCH neurons and between the orexin and α-MSH neurons in the brain of the barfin flounder.     

Identification and kinetic study of tyrosinase inhibitors found in sake lees

The present study found that the n-hexane extract of freeze-dried sake lees inhibits tyrosinase activity and showed that the constituents isolated from the n-hexane extract are the mixture of triacylglycerols. The inhibitory effects of triolein and trilinolein found as the triacylglycerols were examined using tyrosinases from mushroom and Streptomyces castaneoglobisporus. The IC50 values of the triacylglycerol mixture for the oxidase activity on mushroom and Streptomyces tyrosinases were 20 and 0.14 microg/mL, respectively. The IC50 values of trilinolein for the oxidase activity on mushroom and Streptomyces tyrosinases were 8.4 and 0.1 microM, respectively. However, the inhibitory effect of triolein (IC50=30 microM) was lower than that of trilinolein, even when the Streptomyces tyrosinase was used for the assay. Kinetic analyses indicate that both trilinolein and triolein inhibit the tyrosinase activity noncompetitively. When transformed with a plasmid carrying the Streptomyces tyrosinase gene, the melanin-synthesizing ability of the transformed Escherichia coli host was dose-dependently interfered with by trilinolein.     

Impact of paralarvae and juveniles feeding environment on the neon flying squid (Ommastrephes bartramii) winter–spring cohort stock

In this study, we found that there were significant positive correlations between the catch per unit effort (CPUE, a squid abundance index) for the neon flying squid (Ommastrephes bartramii) winter–spring cohort and the satellite‐derived chlorophyll a concentrations in their spawning grounds located at 140–160°E where 21°C < sea surface temperature < 25°C from February to May. The spawning grounds of the winter–spring cohort are located in a quiet stream region, and a particle tracking experiment, based on the velocity field obtained from an ocean data assimilation system, showed that paralarvae and juveniles aged <90 days remained in their spawning grounds and the chlorophyll a concentration in their habitat had a significant positive correlation with the CPUE. A backward particle tracking experiment also showed that the chlorophyll a concentration in the spawning grounds had a significant positive correlation with the autumn–winter mixed layer depth. Based on these results, we hypothesize that the CPUE interannual variability is caused by variations in the feeding environment of the paralarvae and juveniles, which may be linked to autumn–winter mixed layer depth variations.     

Follicle-stimulating hormone (FSH) stimulates the expression of Pin1, a peptidyl-prolyl isomerase, in the bovine granulosa cells

A peptidyl-prolyl isomerase, Pin 1, has been shown to play a role in the regulation of cell cycle progression, both in vitro and in vivo. However, the involvement of Pin 1 during follicular development is not well understood. The aim of this study was first to investigate the expression of Pin 1 mRNA in the granulosa and theca cells of the follicle at different developmental stages of follicles in the bovine ovary, and second, to examine the effects of follicle-stimulating hormone (FSH) and estradiol (E2) on the expression of Pin 1 in the cultured bovine granulosa cells. Follicles were classified into four groups based on the diameter (dominant follicles >8.5mm in diameter, subordinate follicles <8.5mm in diameter) and the relative levels of E2 and progesterone (P4) (E2:P4>1, estrogen active; E2:P4<1, estrogen inactive): i.e. preovulatory dominant follicles (POFs); E2 active dominant follicles (EADs); E2 inactive dominant follicles (EIDs); small follicles (SFs). The expression of the Pin 1 gene was significantly increased in the granulosa cells of EADs as compared with those of other follicles, whereas its expression in theca cells did not differ among follicles at different developmental stages. The concentration of 5 ng/ml FSH alone and the combination of 1 ng/ml E2 and 5 ng/ml FSH stimulated the expression of the Pin 1 gene in bovine granulosa cells. Our data provide the first evidence that Pin 1 expression in the granulosa cells but not the theca cells changes during follicular development, and that FSH stimulate the expression of the Pin 1 gene. These results suggest that Pin 1 regulates the timing of cell proliferation and may act as an intracellular signal responder in the granulosa cells during bovine follicle development.     

Tetrodotoxin accumulation conflicts with low salinity tolerance in juvenile tiger puffer Takifugu rubripes

We evaluated whether bearing tetrodotoxin (TTX) affects salinity stress in the juvenile tiger puffer Takifugu rubripes. Juveniles of hatchery-reared non-toxic T. rubripes [body weight (BW): 1.7?±?0.2 g, n?=?120] were divided into six tanks and acclimatized to salinity (8.5 ppt) that is equivalent to blood osmolality. Fish in three tanks were fed non-toxic diet, and those in the other three tanks were fed a TTX-containing diet (356 ng/g diet) three times a day until satiation. In each diet treatment, salinity of one tank was kept at 8.5 ppt, and the other two tanks were adjusted to either 1.7 or 34.0 ppt, and fish were reared for another 33 days. Then, we compared survival, growth, TTX accumulation, plasma osmolality, plasma cortisol, and glucose levels among treatments. We detected TTX only in the fish in the TTX-diet groups. Survival was highest at 8.5 ppt (70%) and lowest at 1.7 ppt in the TTX-diet group (20%). The BW was greater at 8.5 ppt, and plasma osmolality was significantly higher at 34.0 ppt than at any other salinities. Plasma cortisol level was significantly higher but glucose level was lower at 1.7 ppt. Possessing TTX at a low salinity may be lethal to tiger puffer juveniles.

     

A comparison of the immunological effects of propofol and isoflurane for maintenance of anesthesia in healthy dogs

Most anesthetics have an immuno-suppressive effect on cellular and neurohumoral immunity, and research shows that total intravenous anesthesia (TIVA) with propofol has a greater immuno-protective effect than inhalational anesthesia in human medicine. However, in veterinary clinics, these effects remain ambiguous. To clarify the details, we focused on propofol and isoflurane, investigating clinical blood hematology and immunological profiles drawn from healthy dogs under and after two anesthesia techniques. Twelve healthy adult beagles were included in this study, randomly assigned to the propofol anesthesia group (group P: n=6) or the isoflurane anesthesia group (group I: n=6). In both groups, the number of lymphocytes in peripheral blood decreased after 2 hr of anesthesia (2 hr), but group P showed significantly less decrease than group I. For T-lymphocyte subsets examined by flowcytometry, the ratio of CD3+, CD4+ and CD8+ lymphocytes in the peripheral blood mononuclear cell (PBMC) of group P at 2 hr also exhibited a high level compared to group I. Moreover, for mRNA expression of cytokines measured by real-time PCR, the IL2 (pro-inflammatory cytokine) of group P showed no decrease like group I. The IL10 (anti-inflammatory cytokine) of group P also showed no increase like group I, while both cytokines maintained nearly the same level until 2 hr. These results suggest that, compared to propofol, isoflurane had more strongly immuno-suppression caused by anesthesia, and propofol itself might have some immuno-protective effects. Thus, TIVA with propofol might benefit immunological support in the perioperative period of dogs.