Structural analysis of a Lotus japonicus genome. IV. Sequence features and mapping of seventy-three TAC clones which cover the 7.5 mb regions of the genome.

Using the sequence information of expressed sequences tags (ESTs), cDNAs and genes from Lotus japonicus and other legumes, 73 TAC (transformation-competent artificial chromosomes) clones were selected from a genomic library of L. japonicus accession MG-20, and their nucleotide sequences were determined. The length of the DNA sequenced in this study was 7,455,959 bp, and the total length of the DNA regions sequenced so far is 26,167,443 bp together with the nucleotide sequences of 183 TAC clones previously reported. By similarity searches against the sequences in protein and EST databases and prediction by computer programs, a total of 699 potential protein-encoding genes with known or predicted functions, 163 gene segments and 267 pseudogenes were assigned to the newly sequenced regions. Based oil the nucleotide sequences of the clones, simple sequence repeat length polymorphism (SSLP) or derived cleaved amplified polymorphic sequence (dCAPS) markers were generated, and each clone was located onto the linkage map of two accessions of L. japonicus, Gifu B-129 and Miyakojima MG-20. The sequence data, gene information and mapping information are available through the World Wide Web at http://www.kazusa.or.jp/lotus/.     

Effects of prey density, prey instar, and patch size on the development of the predatory mosquito, Toxorhynchites towadensis

Effects of prey density, prey instar, and patch size on the development of the predatory mosquito larva, Toxorhynchites towadensis, were investigated in the laboratory. Survivors of T. towadensis showed different developmental patterns in relation to prey age structure. All predatory larvae in containers with only second instar prey developed into the third instar. However, in several containers with fourth instar prey, mortality of predators was observed. During the third instar, no predatory larva died, but both prey density and prey instar significantly affected the survival of predators during their fourth instar. Large prey size promoted large predator adults, and predatory larvae which grew up in small surface containers responded by developing to large sizes than those in large containers. Larval developmental time of the predators differed in each treatment. During first and second instars, faster predator development was observed in containers with small surface areas and containing young prey individuals. However, when development was enhanced by the presence of old prey individuals, no surface effect was observed. The fastest predator development was observed with prey of mixed instars and high density. This study suggests that a small surface container containing prey of mixed instars and high density is suitable for development of predators.     

Effects of space flight on GLUT-4 content in rat plantaris muscle

 The effects of 14 days of space flight on the glucose transporter protein (GLUT-4) were studied in the plantaris muscle of growing 9-week-old, male Sprague Dawley rats. The rats were randomly separated into five groups: pre-flight vivarium ground controls (PF-VC) sacrificed approximately 2 h after launch; flight groups sacrificed either approximately 5 h (F-R0) or 9 days (F-R9) after the return from space; and synchronous ground controls (SC-R0 and SC-R9) sacrificed at the same time as the respective flight groups. The flight groups F-R0 and F-R9 were exposed to micro-gravity for 14 days in the Spacelab module located in the cargo bay of the shuttle transport system – 58 of the manned Space Shuttle for the NASA mission named ”Spacelab Life Sciences 2”. Body weight and plantaris weight of SC-R0 and F-R0 were significantly higher than those of PF-VC. Neither body weight nor plantaris muscle weight in either group had changed 9 days after the return from space. As a result, body weight and plantaris muscle weight did not differ between the flight and synchronous control groups at any of the time points investigated. The GLUT-4 content (cpm/μg membrane protein) in the plantaris muscle did not show any significant change in response to 14 days of space flight or 9 days after return. Similarly, citrate synthase activity did not change during the course of the space flight or the recovery period. These results suggest that 14 days of space flight does not affect muscle mass or GLUT-4 content of the fast-twitch plantaris muscle in the rat. Received: 25 March 1997 / Accepted: 18 August 1997     

Regulatory Role of the GTP-Binding Protein, Go, in the Mechanism of Exocytosis in Adrenal Chromaffin Cells

To elucidate the possible involvement of GTP-binding proteins (G proteins) in the mechanism of exocytosis, we studied effects of pertussis toxin (PTX), guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S), and antibodies against the G proteins (Gi and G(o)) on the secretory function of bovine adrenal chromaffin cells. Pretreatment of chromaffin cells with PTX resulted in an increase in acetylcholine-evoked catecholamine release. High K(+)-, histamine-, or gamma-aminobutyric acid-evoked catecholamine release was also potentiated by PTX pretreatment. The concentration of extracellular Ca2+ required for maximal release by 10(-4) M acetylcholine was decreased significantly in PTX-treated cells. In digitonin-permeabilized cells, PTX pretreatment resulted in a decrease of the half-maximal concentration (Km) of Ca2+ required for exocytosis with no significant change in the maximal stimulation (Vmax). Exposure of permeabilized cells to GTP-gamma-S (a nonhydrolyzable GTP analogue) inhibited Ca(2+)-dependent exocytosis by reducing the affinity for Ca2+. The effects of PTX pretreatment were mimicked by treatment of permeabilized cells with polyclonal antibodies selective for the alpha subunit of the PTX-sensitive G protein, G(o). Treatment with similar antibodies against the alpha subunit of Gi had no effect. These findings suggest that G(o) directly controls the Ca(2+)-triggered process in the machinery of exocytosis by lowering the affinity of the unknown target for Ca2+.     

NovelN-linked oligo-mannose type oligosaccharides containing an α-d-galactofuranosyl linkage found in α-d-galactosidase fromAspergillus niger

Structures of oligosaccharides fromAspergillus niger -d-galactosidase [EC 3.2.1.22] were studied. Purified -d-galactosidase was treated withN-glycosidase F, and six kinds of oligosaccharides were isolated by gel chromatography and anion-exchange chromatography. The structures of the oligosaccharides were determined by¹H-NMR and compositional analysis to be Man₅GlcNAc₂, Man₆GlcNAc₂, Man₉GlcNAc₂, GlcMan₉GlcNAc₂, GalMan₄GlcNAc₂ and GalMan₅GlcNAc₂. From mild acid hydrolysis, methylation analysis and ROESY spectral analysis, it was ascertained that the galactosyl residue in two oligosaccharides was in the furanose form and was bound to mannose at the nonreducing end with an 1–2 linkage (GalfMan₄GlcNAc₂ and GalfMan₅GlcNAc₂).     

Pineal-dependent locomotor activity of lamprey,Lampetra japonica,measured in relation to LD cycle and circadian rhythmicity

Summary Locomotor activity of the river lamprey, Lampetra japonica, was investigated under a light-dark (LD 1212) cycle and under continuous dark conditions. Intact lampreys were entrained to the light:dark cycle. They were active mainly in the early half of the dark period and inactive in light period. The light:dark entrainment continued in 72.7% of lampreys after the removal of bilateral eyes, but additional pinealectomy made the entrainment disappear in all lampreys. When lampreys were pinealectomized with their eyes intact, light: dark entrainment was abolished in most cases. The results indicate that the pineal organ of the lamprey is a photoreceptive organ responsible for synchronizing locomotor activity to LD cycle. Under continuous dark conditions, the locomotor activity began to free-run with a period of 21.3 ± 0.9 h (mean ± SD, n = 53). This circadian rhythmicity was not affected by the removal of lateral eyes but was abolished by pinealectomy. The pineal organ appears to function as an oscillator, or as one of the oscillators, for the circadian locomotor rhythm of lampreys.Abbreviations DD continuous dark - LD light:dark     

Alkaloid production by plants regenerated from cultured cells of Datura innoxia

Cellular aggregates in Datura innoxia suspension cultures give rise to large numbers of shoots when such aggregates are cultured in the light on an auxin-free agar medium supplemented with kinetin. These shoots form roots on a kinetin-free medium to develop into complete plants. Most of the regenerated plants are diploid, and the frequencies of ancuploid or polyploid plants are much lower than might be expected from the distribution of chromosome number in the cultured cells. During root ditterentiation and plant development, scopolamine synthesis is initiated and there is a progressive increase in the alkaloid content. Consequently, the general pattern of alkaloid composition is restored to a normal state in the majority of the regenerated plants including aneuploid or polyploids. Nevertheless, some of the plants show an abnormal expression in alkaloid metabolism, such as the complete hydrolysis of scopolamine in the dried leaves.     

Effect of Polymyxin on the Bacteriophage Receptors of the Cell Walls of Gram-Negative Bacteria

Treatment of gram-negative bacteria with lethal doses of polymyxin B and colistin resulted in the formation of projections of the outer layer of the cell wall. Phages T3, T4, and T7, which use wall lipopolysaccharide as receptors, were specifically prevented from adsorbing to Escherichia coli B cells treated with polymyxin, whereas phages T1, T2, T5, and T6 were not. In the systems of phage P22C-Salmonella typhimurium LT2 and phage C21-S. typhimurium variant SL1069, the phage were prevented from adsorbing to the host cell treated with the antibiotics. Electron microscopic observations show that phage T2 adsorbed irreversibly to the normal smooth surface between the projections on the outer layer caused by the drug treatment. These results indicate that lipopolysaccharide is affected by polymyxin functionally and morphologically, but lipoprotein is not. The purified lipopolysaccharide showed a ribbon-like structure when viewed face on and showed trilamellar structure when viewed edge on. The lipopolysaccharide from E. coli B was irreversibly adsorbed by phages T3, T4, and T7, but not phage T2. Often, phage T4 adsorbed to both sides of the lipopolysaccharide strand at comparable distances. Phage P22C adsorbed through the spikes of the tail-plates to the lipopolysaccharide from S. typhimurium LT2. Lipopolysaccharide which was treated with low doses of the drug (2.5 to 6.25 mug of polymyxin B per ml to 100 mug of lipopolysaccharide per ml) turned into the coiled form and was partially broken down into short segments with coiled form. The loosely coiled lipopolysaccharide retains both its function as the receptor and its trilamellar structure. Treatment with high doses of the drug (12.5 to 25 mug of polymyxin B per ml to 100 mug of lipopolysaccharide per ml) caused the collapse of the trilamellar structure of the strand. These collapsed lipopolysaccharides became flat and fused with each other, making an amorphous mass, and finally they were broken into small collapsed fragments.