Species identification of upstream fatminnow <Emphasis Type="Italic">Rhynchocypris oxycephalus</Emphasis> and downstream fatminnow <Emphasis Type="Italic">Rhynchocypris lagowskii</Emphasis>, based on PCR-RFLP of mitochondrial DNA

The morphological similarity between upstream fatminnow Rhynchocypris oxycephalus and downstream fatminnow Rhynchocypris lagowskii makes it difficult to discriminate accurately between these species in rivers where they coexist. For easy and precise identification of these two species, we developed a genetic discrimination method based on PCR-RFLP analysis for specimens from the Inohzawa River watershed in the Izu Peninsula, central Honshu, Japan. This genetic method was applied to the species identification of the fatminnows from two other watersheds, the Kano and Kawazu Rivers, flowing across the peninsula from north to south. We present the genetic evidence for the restricted distribution of R. oxycephalus and the ubiquitous distribution of R. lagowskii in the peninsula.     

Structural analysis of sphingophospholipids derived from Sphingobacterium spiritivorum, the type species of genus Sphingobacterium

The unique feature of the genus Sphingobacterium is the presence of sphingophospholipids and ceramides, besides diacylglycerophospholipids. As major cellular lipid components, five kinds of sphingophospholipids were purified from Sphingobacterium spiritivorum ATCC 33861(T), the type species of genus Sphingobacterium. They were identified as ceramide phosphorylethanolamines (CerPE-1 and CerPE-2), ceramide phosphoryl-myo-inositols (CerPI-1 and CerPI-2), and ceramide phosphorylmannose (CerPM-1). The ceramide of CerPE-1, CerPI-1, and CerPM-1 was composed of 15-methylhexadecasphinganine (isoheptadeca sphinganine, iso-C17:0) and 13-methyltetradecanoic acid (isopentadecanoic acid, iso-C15:0), whereas that of CerPE-2 and CerPI-2 was composed of isoheptadeca sphinganine and 2-hydroxy-13-methyltetradecanoic acid (2-hydroxy isopentadecanoic acid, 2-OH iso-C15:0). These sphingophospholipids were also found in cellular lipids of Sphingobacterium multivorum ATCC 33613(T), Sphingobacterium mizutaii ATCC 33299(T), Sphingobacterium faecium IFO 15299(T), Sphingobacterium thalpophilum ATCC 43320(T), and Sphingobacterium antarcticum ATCC 51969(T). To our knowledge, the existence of CerPM-1 is a novel sphingophospholipid through eukaryotic and prokaryotic cells.     

Photoproduction of Hydrogen by the Marine Heterocystous Cyanobacterium Anabaena Species TU37-1 Under a Nitrogen Atmosphere

Hydrogen production rates by Anabaena sp. strain TU37-1 obtained after an initial 1-day incubation period were approximately 70 to 80 and 3 to 9 µmol (mg chl)^–1 h^–1 under argon and nitrogen atmospheres, respectively. Hydrogen production under argon was not enhanced by addition of carbon dioxide, but was enhanced to some extent under nitrogen by increasing the initial carbon dioxide concentration. Rates of hydrogen and oxygen production during the initial 7-hour period were 15 and 220 µmol (mg chl)^–1 h^–1, respectively, in vessels with 18.5% initial carbon dioxide. Hydrogen production under nitrogen was enhanced by addition of carbon monoxide (1%). The rate obtained from the initial 1-day incubation period was about 40 µmol (mg chl)^–1 h^–1, which corresponded to about 60% of that under argon. On the basis of these observations, a possible strategy for hydrogen production by nitrogen-fixing cyanobacteria under nitrogen in the presence of carbon monoxide is indicated.     

Affinity of isoflavonoids for lipid bilayers evaluated with liposomal systems

Three parameters, i.e., the proportion of the amount incorporated into the liposomes, the partition coefficient in a system of n-octanol/phosphate buffered saline, and the retention times by HPLC, were measured to determine the lipophilicity of isoflavonoids. The presence of a hydroxyl group at 5-position of the A-ring and a methoxyl group at 4'-position of the B-ring in the isoflavonoid structure increased the three parameters. The localization of isoflavonoids in lipid bilayers was investigated by a liposome system with fluorescent probes. The location of the isoflavonoid depended on its structure. The cytotoxicity of isoflavonoids was investigated by a colony-formation assay with Chinese hamster lung fibroblast V79 cells. The structure-activity relationship of the cytotoxic activity partly reflected those of the three parameters. This suggests that the biological activities of isoflavonoids in vitro could be attributable to their affinity for lipid components in the cases where the estrogen receptors have no role.     

Genetic and ultrastructural demonstration of strong reversibility in human mesenchymal stem cell

We examined human bone marrow mesenchymal stem cells by applying real-time quantitative polymerase chain reaction (PCR) (RT-PCR) technology and electron-microscopic techniques. Our RT-PCR demonstrated that the values of peroxisome proliferation-activated receptor gamma2 (PPARgamma2) and lipoprotein lipase (LPL) mRNA dramatically increased according to adipogenic stimulation. The expressions of both PPARgamma2 and LPL mRNA were significantly reduced ( P<0.01) and almost disappeared after stimulation had ceased. The expressions of both genes, however, increased again by stimulation even though the cells were in a dedifferentiated state for a month. In the ultrastructural study, over 80% of the cells proceeded into morphologically well-developed adipocytes at the 12th day of induction/maintenance which were packed with lipid droplets and clusters. In the next process these lipid products were excreted from the cell bodies and the peripheral small parts containing numerous droplets were torn from the greater parts, which stuck tightly to each other and adhered to culture dishes. Adipocytes were not detected in the culture media during the final stage. The total cell number was equal to and over 90% of the cells dedifferentiated into fibroblast-like stem cells during the final maintenance period of 1 month. Furthermore the dedifferentiated cells quickly differentiated again into adipocytes by stimulation even if they were quiescent for 1 month. Thus we conclude that mesenchymal stem cells have strong reversibility from both the genetic and morphological points of view.     

Elevated interleukin-9 receptor expression and response to interleukins-9 and -7 in thymocytes during radiation-induced T-cell lymphomagenesis in B6C3F1 mice

Dysregulation of cytokine receptor expression and responsiveness to cytokines is hypothesized to play an important role in the development and expansion of preneoplastic cells or progression of neoplastic cells during the early and late stages of leukemogenesis. To determine the crucial changes in initiated cells that confer significant growth during the early stage of radiation-induced lymphomagenesis, we examined both the expression of receptors for thymus-derived cytokines and thymocyte response to cytokines before the onset of T cell lymphomas in B6C3F1 mice after split-dose irradiation. After irradiation, thymic T cell subsets underwent delayed regeneration consisting of two phases as determined by receptor expression. The first phase occurred within 1 week post-irradiation and was accompanied by transient expansion of T cell subsets strongly expressing receptor genes for IL-1, IL-2, IL-6, IL-7, IL-15, and TNF alpha. The second phase occurred 12 weeks after irradiation and was characterized by increased expression of IL-9R alpha. Thymocytes from non-irradiated control mice were unresponsive to IL-9. However, IL-9 acted synergistically with IL-7 and PHA to stimulate the proliferation of irradiated cells during the second post-irradiation phase. Moreover, these cells showed hyper-responsiveness to IL-7 or PHA alone compared to age-matched non-irradiated control thymocytes. These results suggest that the unusual expression of IL-9 receptors and/or increased responsiveness of thymocytes to cytokines are key processes in the development of radiation-induced T cell lymphomas.     

Simultaneous determination of barbiturates in human biological fluids by direct immersion solid-phase microextraction and gas chromatography-mass spectrometry

Simultaneous determination of seven barbiturates in human whole blood and urine by combining direct immersion solid-phase microextraction (DI-SPME) with gas chromatography-mass spectrometry (GC-MS) is presented. The main parameters affecting the DI-SPME process, such as SPME fibers, salt additives, pHs, extraction temperatures and immersion times were optimized for simultaneous determination of the drugs. The extraction efficiencies were 0.0180-0.988 and 0.0156-2.76% for whole blood and urine, respectively. The regression equations of the drugs showed excellent linearity for both samples; the correlation coefficients (r(2)) were 0.994-0.999. The detection limits for whole blood were 0.05-1 microg x ml(-1), and those for urine 0.01-0.6 microg x ml(-1). Actual quantitation could be made for pentobarbital in whole blood and urine obtained from volunteers, who had been orally administered a therapeutic dose of the drug. The DI-SPME/GC-MS procedure for barbiturates established in this study is simple and sensitive enough to be adopted in the fields of clinical and forensic toxicology.     

Characterization of gamma-terpinene synthase from Citrus unshiu (Satsuma mandarin)

Gamma-terpinene is a monoterpene and a major component of essential oils made from citrus fruits and shows strong antioxidant activity in various assay systems. Plant gamma-terpinene synthase is a member of the monoterpene cyclase family, which produces a specific monoterpene through cyclization of geranyl diphosphate (GPP), but the monoterpene cyclases have not been fully characterized. It is necessary to prepare large amounts of gamma-terpinene synthase from Citrus unshiu (Satsuma mandarin) for the characterization, on this purpose we expressed the protein in Escherichia coli (E. coli) cells. As most monoterpene synthases have plastid-targeting signals, a gene lacking these signals was prepared and functionally expressed in E. coli cells harboring extra copies of the argU gene. The purified enzyme was incubated with GPP and the main product was confirmed to be gamma-terpinene by GC/MS.     

Octanoate reduces very low-density lipoprotein secretion by decreasing the synthesis of apolipoprotein B in primary cultures of chicken hepatocytes

Fatty acids of varying lengths and saturation differentially affect plasma apolipoprotein B (apoB) levels. To identify the mechanisms underlying the effect of octanoate on very low-density lipoprotein (VLDL) secretion, chicken primary hepatocytes were incubated with either fatty acid-bovine serum albumin (BSA) complexes or BSA alone. Addition of octanoate to culture medium significantly reduced VLDL-triacylglycerol (TG), VLDL-cholesterol and apoB secretion from hepatocytes compared to both control cultures with BSA only and palmitate treatments, but did not modulate intracellular TG accumulation. However, no differences in cellular microsomal triglyceride transfer protein levels were observed in the cultures with saturated fatty acid. In pulse-chase studies, octanoate treatment resulted in reduced apoB-100 synthesis, in agreement with its promotion of secretion. This characteristic effect of octanoate was confirmed by addition of a protease inhibitor, N-acetyl-leucyl-leucyl-norleucinal (ALLN), to hepatocyte cultures. Analysis showed that the level of apoB mRNA was lower in cultures supplemented with octanoate than in the control cultures, but no significant changes were observed in the levels of apolipoprotein A-I, fatty acid synthase and 3-hydroxy-3-methylglutaryl-CoA reductase mRNA as a result of octanoate treatment. Time-course studies indicate that a 50% reduction in apoB mRNA levels requires 12 h of incubation with octanoate. We conclude that octanoate reduced VLDL secretion by the specific down-regulation of apoB gene expression and impairment of subsequent synthesis of apoB, not by the modulation of intracellular apoB degradation, which is known to be a major regulatory target of VLDL secretion of other fatty acids.