Isolation and Characterization of a Cytokinin-Binding Protein from the Water-Soluble Fraction of Tobacco Leaves

Cytokinin-binding protein (CBPI) was purified from the watersoluble fraction of tobacco leaves by successive chromatographyon benzyladenine-linked (BA-linked) Sepharose 4B, TSK-Gel G3000SWXL,t-zeatin-linked Sepharose 6B and TSK-Gel G3000SWXL. CBPI wasobtained as a monomer with a molecular weight of 31 kDa. Ithas one cytokinin-binding site, which shows a high affinityfor BA (Kd=1.1x10^–7 M) and other cytokinins. Biologicallyactive cytokinins competed with BA for binding to this protein,while biologically inactive analogues of adenine did not. Inall cases, cytokinin-binding activity was assayed by equilibriumdialysis. ¹ Present address: National Institute for Basic Biology, Okazaki,444 Japan.     

Changes in Levels of Heme a, Protoheme and Protochlorophyll(ide) in Submerged Rice Seedlings after Exposure to Air

Rice (Oryza sativa L. cv. Yamabiko) seedlings germinated underwater for 5 days contained small amounts of heme a and protohemebut no protochlorophyll(ide) [Pchl(ide)]. Levels of hemes andPchl(ide) increased rapidly upon transfer to air. When expressedin terms of fresh weight of tissue, hemes reached the levelsin aerobic controls after 24 h of contact with air, but Pchl(ide)did not. A comparison of the increases during 24-h adaptationto air in levels of heme a and Pchl(ide), which are specificto mitochondria and plastids, respectively, suggested that thedevelopment of mitochondria preceded that of plastids. The rateof synthesis of 5-aminolevulinic acid (ALA) was low in submergedseedlings, as compared to the rate in aerobic controls, butit increased during air adaptation. The sum of the amounts ofheme a, protoheme and Pchl(ide) increased in parallel with theamount of porphyrins, equivalent to the amount of ALA synthesizedduring the experimental period. When submerged seedlings thathad been pretreated with levulinic acid were exposed to air,no Pchl(ide) was formed. In contrast, Pchl(ide) accumulatedunder water when submerged seedlings were fed with ALA. Theseresults indicate that the synthesis of ALA, the limiting stepin the synthesis of Pchl(ide), is repressed under hypoxic conditions. ¹ Present address: KRI International, Inc., Kyoto Research Park17, Chudoji Minami-machi, Shimogyo-ku, Kyoto, 600 Japan. ² Present address: Research Institute for Bioresources, OkayamaUniversity, Kurashiki, 710 Japan.     

Analyses of beta-1 syntrophin, syndecan 2 and gem GTPase as candidates for chicken muscular dystrophy.

Despite intensive studies of muscular dystrophy of chicken, the responsible gene has not yet been identified. Our recent studies mapped the genetic locus for abnormal muscle (AM) of chicken with muscular dystrophy to chromosome 2q using the Kobe University (KU) resource family, and revealed the chromosome region where the AM gene is located has conserved synteny to human chromosome 8q11-24.3, where the beta-1 syntrophin (SNTB1), syndecan 2 (SDC2) and Gem GTPase (GEM) genes are located. It is reasonable to assume those genes might be candidates for the AM gene. In this study, we cloned and sequenced the chicken SNTB1, SDC2 and GEM genes, and identified sequence polymorphisms between parents of the resource family. The polymorphisms were genotyped to place these genes on the chicken linkage map. The AM gene of chromosome 2q was mapped 130 cM from the distal end, and closely linked to calbindin 1 (CALB1). SNTB1 and SDC2 genes were mapped 88.5 cM distal and 27.6 cM distal from the AM gene, while the GEM gene was mapped 18.5 cM distal from the AM gene and 9.1 cM proximal from SDC2. Orthologues of SNTB1, SDC2 and GEM were syntenic to human chromosome 8q. SNTB1, SDC2 and GEM did not correspond to the AM gene locus, suggesting it is unlikely they are related to chicken muscular dystrophy. However, this result also suggests that the genes located in the proximal region of the CALB1 gene on human chromosome 8q are possible candidates for this disease.     

Identification and characterization of ispA, a Shigella flexneri chromosomal gene essential for normal in vivo cell division and intercellular spreading

The virulent phenotype of Shigella requires loci on the chromosome as well as on the large virulence plasmid, and is regulated via a complex web of interactions amongst various chromosomal and large plasmid genes. To further investigate the role of chromosomal loci in virulence, we performed random Tn 10 mutagenesis in Shigella flexneri YSH6000T, and isolated an avirulent mutant (V3404) incapable of spreading throughout an epithelial cell monolayer. Although V3404 initially spread intercellularly at the same rate as the wild-type, it gradually slowed down and ceased spreading as a result of increasing defects in cell division, leading to the formation of long filamentous bacteria lacking septa, trapped within cells. In addition, the mutation affected the ability of V3404 to polymerize actin, a prerequisite for intra- and inter-cellular spreading ability. Sequencing of Tn 10 -flanking DNA revealed that the mutated gene, designated ispA (intracellular septation), was equivalent to a previously sequenced but uncharacterised gene of Escherichia coli located between trp and tonB . Using E. coli sequence data, we cloned the ispA gene from the YSH6000T chromosome and found that it complemented the V3404 mutation. Nucleotide sequencing and in vitro expression experiments revealed that ispA coded for a small (21 kDa), very hydrophobic protein. These results thus show that ispA is an essential virulence gene affecting several functions of the virulence process.     

Application of leaf extract causes repetitive action potentials inBiophytum sensitivum

A wound stimulus evoked a number of repetivite action potentials in the leaf ofBiophytum sensitivum. When the cut end of a leaf was immersed in a leaf extract, the resulting repetitive action potentials continue for a long time. These repetitive action potentials disappeared immediately when the leaf extract, which contains a proposed stimulant, was removed and the cut end was washed with water.     

Over-production of Thermus protease in dense culture of Escherichia coli using two carbon sources

Escherichia coli JM109(DE3) harboring expression plasmid pkAQNÆC30, which carries the Thermus protease aqualysin I (AQI) gene, was cultivated with glucose as a sole carbon source. The final cell concentration was over 15 g dry weight/l and the amount of AQI produced reached approximately 130 kU/ml broth. Moreover, by using two carbon sources, glucose and glycerol, the production yield was increased to over 200 kU AQI/ml, while suppressing the formation of inhibitory acetic acid.     

Mouse {beta}-galactoside {alpha}2,3-sialyltransferases: comparison of in vitro substrate specificities and tissue specific expression

Four types of ß-galactoside     

Optimal strategy of plant antiherbivore defense: Implications for apparency and resource-availability theories

Plants produce chemicals as methods against animal herbivory. Such chemical defenses are classified into two major categories: (i) quantitative defenses with massive production of indigestible substances; and (ii) qualitative defenses with production of poisonous substances. A mathematical model was developed that identified factors that favored the evolution of quantitative defenses. Selecting an annual plant for simplicity, the allocation of photosynthetic production between growth substances and defense substances was considered. If the plant invests more in defense substances, it can protect itself more efficiently from herbivory but with a reduced growth rate. If it invests more in growth substances, the contrary holds. Using Pontoryagin's maximum principle, the following results were obtained: (i) the plant should conduct quantitative defenses when the growth rate (G), reflecting resource-availability, is low and the growth period (T) is long as well; (ii) if the plant invests in quantitative defenses, the optimal proportion of defense substances (χ^*) should be higher asG is smaller, but it is independent ofT; and (iii) the value of χ^* is not monotone for the effectiveness of defense substance (A), but has a maximum at an intermediate value ofA. Predictions of the model partly supported both Feeny's apparency theory, claiming that apparent plants or their parts for herbivores should quantitatively defend themselves, and Coley's resource-availability theory, claiming that plants with rich resources should invest in growth rather than defense.     

Electron-microscope cytochemistry of acetylcholine-like cation by means of low-temperature "ionic fixation"

A fresh preparation of frog neuromuscle was fixed at low temperatures (0 degree-4 degrees C) by means of an "ionic-fixation" procedure which is based on the precipitation of quaternary ammonium cations, such as choline and acetylcholine, with molybdic or tungstic heteropolyanions. A low temperature was used to slow down drastically the biological processus of vesicular exocytosis so that ionic fixation, the speed of which is only slightly influenced by temperature variation, could be performed efficiently. In addition to the conventional point-like precipitate in the synaptic vesicle which is considered to be vesicular acetylcholine, numerous spot-like precipitates were observed in the synaptic cleft. Most of these were contiguous to the active zone, and some were in a paired form and corresponded to the double rows of the synaptic vesicles in contact with active zones. It is concluded that these spot-like precipitates were acetylcholine-like cations of the synaptic vesicles which had been discharged into the synaptic cleft by exocytosis and captured by the ionic fixation procedure. The results are discussed in relation to the vesicular and non-vesicular hypothesis of acetylcholine release.