Changes of intracranial pressure during head-down tilt in anesthetized and conscious rabbits

Effects of head-down tilt on intracranial pressure were studied in anesthetized and conscious rabbits. Adult Japanese white rabbits of both sexes, weighing 2.5-3.5 kg, were used in the experiments. Experiment 1. Animals were anesthetized with pentobarbital, and ICP was monitored through a catheter inserted into the subarachnoid space. ICP elevated immediately after the onset of 45 degrees HDT and gradually reduced toward the baseline level in the next 8 hours. Experiment 2. Each rabbit was exposed to 45 degrees HDT for 24 hours and the ICP was measured through a catheter which had been implanted 7 days before. In the conscious rabbits, ICP increased about 4 mmHg after the onset of 45 degrees HDT, further increased gradually to the peak at 11 hours of HDT, and then started to return to the baseline. These results suggest that the time course of the change in ICP during HDT is considerably different between anesthetized and conscious rabbits.     

Isolation and characterization of a halotolerant <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">subtilis</Emphasis> BBK-1 which produces three kinds of lipopeptides: bacillomycin L,plipastatin, and surfactin

Twenty-three halotolerant and biosurfactant producing strains were collected from salty conditions in central Thailand. One of the strains designated BBK-1 produced the biosurfactants with the highest activity. BBK-1 was isolated from fermented foods and was identified as B. subtilis based on its physiological characteristics and 16S rRNA gene sequence. We show that the strain grows in media containing NaCl up to 16% (w/v) and produces biosurfactants in NaCl up to 8%. We found that B. subtilis BBK-1 produces three kinds of surface-active lipopeptides simultaneously. By their respective molecular weights and amino acid compositions, it is indicated that these lipopeptides are bacillomycin L, plipastatin, and surfactin. In order to analyze the production mechanism of lipopeptides further in the strain, a generally important biosynthetic gene encoding 4'-phosphopantetheinyl transferase was cloned and sequenced. The gene existed in a single copy in the genome and the deduced amino acid sequence was almost identical to that of Lpa-14 from B. subtilis strain RB14, which co-produces iturin A and surfactin.     

Differential recognition of tyrosine-based basolateral signals by AP-1B subunit mu1B in polarized epithelial cells

To investigate the importance of tyrosine recognition by the AP-1B clathrin adaptor subunit mu1B for basolateral sorting of integral membrane proteins in polarized epithelial cells, we have produced and characterized a mutant form of mu1B. The mutant (M-mu1B) contains alanine substitutions of each of the four conserved residues, which in the AP-2 adaptor subunit micro2 are critical for interacting with tyrosine-based endocytosis signals. We show M-mu1B is defective for tyrosine binding in vitro, but is nevertheless incorporated into AP-1 complexes in transfected cells. Using LLC-PK1 cells expressing either wild type or M-mu1B, we find that there is inefficient basolateral expression of membrane proteins whose basolateral targeting signals share critical tyrosines with signals for endocytosis. In contrast, membrane proteins whose basolateral targeting signals are distinct from their endocytosis signals (transferrin and low-density lipoprotein receptors) accumulate at the basolateral domain normally, although in a manner that is strictly dependent on mu1B or M-mu1B expression. Our results suggest that mu1B interacts with different classes of basolateral targeting signals in distinct ways.     

Gene Transfer and Cloning of Flanking Chromosomal Regions Using the Medaka Fish Tol2 Transposable Element

For the ultimate purpose of developing genetic tools using the medaka fish Tol2 transposable element, we examined whether it can transfer a marker gene into the fish genome and also be applied for cloning of chromosomal regions adjacent to insertion points. An internal region of Tol2 was removed and replaced with the green fluorescent protein (GFP) gene and a bacterial plasmid replication origin. This modified Tol2 clone was microinjected into fertilized eggs together with messenger RNA for the Tol2 transposase. The GFP gene was found to be integrated into chromosomes and transmitted to subsequent generations. Restriction enzyme digestion of genomic DNA of a transformant fish, followed by ligation and introduction into bacteria, produced a plasmid containing the entire element and flanking chromosomal regions. Sequencing analysis of this clone demonstrated transposition of the element in the germline of the first generation. Thus, the basic requirements for a gene transfer vector and gene tagging system were fulfilled. Received July 30, 2001; accepted October 4, 2001     

Glucose promotes pancreatic islet beta-cell survival through a PI 3-kinase/Akt-signaling pathway

The concentration of glucose in plasma is an important determinant of pancreatic beta-cell mass, whereas the relative contributions of hypertrophy, proliferation, and cell survival to this process are unclear. Glucose results in depolarization and subsequent calcium influx into islet beta-cells. Because depolarization and calcium (Ca(2+)) influx promote survival of neuronal cells, we hypothesized that glucose might alter survival of islet beta-cells through a similar mechanism. In the present studies, cultured mouse islet beta-cells showed a threefold decrease in apoptosis under conditions of 15 mM glucose compared with 2 mM glucose (P < 0.05). MIN6 insulinoma cells incubated in 25 mM glucose for 24 h showed a threefold decrease in apoptosis compared with cells in 5 mM glucose (1.7 +/- 0.2 vs. 6.3 +/- 1%, respectively, P < 0.001). High glucose (25 mM) enhanced survival-required depolarization and Ca(2+) influx and was blocked by phosphatidylinositol (PI) 3-kinase inhibitors. Glucose activation of the protein kinase Akt was demonstrated in both insulinoma cells and cultured mouse islets by means of an antibody specific for Ser(473) phospho-Akt and by an in vitro Akt kinase assay. Akt phosphorylation was dependent on PI 3-kinase but not on MAPK. Transfection of insulinoma cells with an Akt kinase-dead plasmid (Akt-K179M) resulted in loss of glucose-mediated protection, whereas transfection with a constitutively active Akt enhanced survival in glucose-deprived insulinoma cells. The results of these studies defined a novel pathway for glucose-mediated activation of a PI 3-kinase/Akt survival-signaling pathway in islet beta-cells. This pathway may provide important targets for therapeutic intervention.     

Sources of dissolved methane in Lake Biwa

The spatial distribution and seasonal variation in the concentration and carbon isotopic composition of dissolved methane in a river–lake ecosystem were studied in Lake Biwa, Japan, and its tributary rivers. Methane concentrations in all subsystems examined were supersaturated with respect to the atmosphere. The epilimnion showed higher concentrations of dissolved methane than the hypolimnion in the pelagic zone. Peak methane concentrations were observed at the thermocline. The largest amount of methane in the pelagic water column was recorded at the end of a stagnant period, at which the bottom water of the sublittoral zone (30m in depth) exhibited increased methane concentration. Transect observation of dissolved methane revealed three methane peaks at different water depths in the lake, and river water and the sediments in littoral and sublittoral zones were suggested to be the corresponding sources. Water at the river mouth was replete with dissolved oxygen but also contained a high concentration of methane. The present results suggest that river water and littoral sediment are potential sources of dissolved methane in Lake Biwa, and other sources, such as internal waves, are responsible for increased methane in the pelagic zone at the end of stagnant periods. Carbon stable isotope analysis indicated that there were different sources of dissolved methane, although it was difficult to identify the origins due to high variation of the isotopic composition of methane from different sources.     

Purification and properties of a heat-stable inulin fructotransferase from Arthrobacter ureafaciens

An inulin fructotransferase producing difructose dianhydride I (EC 2.4.1.200) was purified from Arthrobacter ureafaciens A51-1. It had maximum activity at pH 5.5 and 45 °C, and was stable up to 80 °C. This is the highest thermal stability for this enzyme reported to date. The molecular mass was estimated to be 38000 by SDS-PAGE, and 61000 by gel filtration. It was therefore estimated to be a dimer.     

Tumor necrosis factor-alpha from bone marrow-derived cells is not essential for the expression of adhesion molecules in lipopolysaccharide-induced nasal inflammation

Both the role and source of tumor necrosis factor (TNF-alpha) in lipopolysaccharide (LPS)-induced nasal inflammation were investigated using TNF-alpha gene deficient (TNF-alpha -/-) mice and chimeric mice that are TNF-alpha gene deficient only in bone marrow-derived cells. In the present study, intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) mRNA expression levels in the nasal mucosa were significantly decreased following intranasal instillation of LPS in TNF-alpha gene deficient mice compared to those in wild type mice. In contrast, ICAM-1 and VCAM-1 mRNA expressions were not significantly decreased although TNF-alpha mRNA expression was dramatically decreased in TNF-alpha gene deficient bone marrow-transplanted-chimeric (TNF-alpha -/--->+/+) mice compared to those in wild type bone marrow-transplanted-control (TNF-alpha +/+-->+/+) mice. These results indicate that the elevation of TNF-alpha mRNA in the nasal mucosa is mainly originated from bone marrow-derived cells. However, even low expression of TNF-alpha at local inflammation sites is sufficient to induce the expression of adhesion molecules in acute LPS-induced experimental rhinitis.     

Geographic variation and diversity of the cytochrome b gene in Japanese wild populations of medaka, Oryzias latipes

We conducted a polymerase chain reaction--restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome b gene to elucidate the detailed genetic population structure of Japanese wild populations of medaka, Oryzias latipes. The analysis of 1,225 specimens collected from 303 sites identified 67 mitotypes. Subsequently we determined the nucleotide sequences of the complete cytochrome b gene (1141-bp) to clarify the phylogenetic relationships among mitotypes. The phylogenetic tree based on nucleotide sequences indicated three major clades (A, B and C) that differed by 11.3-11.8%, corresponding to three clusters previously identified by RFLP analysis of entire mitochondrial DNAs. The geographic distribution of mitotypes in clades A and B was fully concordant with the Northern and Southern Populations defined by allozymes. Clade A could be subdivided into three subclades and clade B into eleven, with sequence divergences among subclades of 1.3-5.8%. Each distribution of mitotypes in subclades roughly corresponded to that of mtDNA haplotypes in subclusters previously identified. Mitotypes in clade C were found only in the Kanto district. The phylogenetic relationships and the estimated divergence times suggest that three Japanese clades originated from a common ancestor and were separated during the Pliocene, and that the regional differentiation of subclades was closely connected with the geological history of the Quaternary. This study has also demonstrated the possibility of artificial disturbance of natural distribution especially in the Kanto district and the superior efficacy of PCR-RFLP analysis as a simple method for detecting genetic variation and artificial gene flow of medaka.