Transaldolase/glucose-6-phosphate isomerase bifunctional enzyme and ribulokinase as factors to increase xylitol production from D-arabitol in Gluconobacter oxydans

Xylitol production from D-arabitol by the membrane and soluble fractions of Gluconobacter oxydans was investigated. Two proteins in the soluble fraction were found to have the ability to increase xylitol production. Both of these xylitol-increasing factors were purified, and on the basis of their NH(2)-terminal amino acid sequences the genes encoding both of the factors were cloned. Expression of the cloned genes in Escherichia coli showed that one of the xylitol-increasing factors is the bifunctional enzyme transaldolase/glucose-6-phosphate isomerase, and the other is ribulokinase. Using membrane and soluble fractions of G. oxydans, 3.8 g/l of xylitol were produced from 10 g/l D-arabitol after incubation for 40 h, and addition of purified recombinant transaldolase/glucose-6-phosphate isomerase or ribulokinase increased xylitol to 5.4 g/l respectively, confirming the identity of the xylitol-increasing factors.     

Regulation of type 1 protein phosphatase/inhibitor-2 complex by glycogen synthase kinase-3beta in intact cells

Inhibitor 2 (I-2) is a ubiquitous regulator of type 1 protein phosphatase (PP1). Previous in vitro studies suggested that its inhibitory activity towards PP1 is regulated by phosphorylation at Thr72 by glycogen synthase kinase-3beta (GSK-3beta), and at Ser86, Ser120, and Ser121 by casein kinase 2 (CK2). Here we report that GSK-3beta expressed in COS-7 cells phosphorylates wild-type I-2 but not an I-2 mutant carrying a T to A substitution at residue 72, showing that GSK-3beta phosphorylates I-2 at T72 in vivo as well. Co-immunoprecipitation study demonstrated that HA-GSK-3beta and I-2-FLAG co-exist in a same complex in the intact cells, but they do not bind directly. It is noteworthy that co-expression of Myc-PP1C significantly increased co-precipitation of HA-GSK-3beta with I-2-FLAG, showing a complex formation of HA-GSK-3beta/Myc-PP1C / I-2-FLAG in vivo. Further studies using a GSK-3beta kinase-dead mutant and LiCl, an inhibitor of GSK-3beta, showed that the enzyme activity of GSK-3beta is required for co-precipitation. IP-Western study using several I-2 mutants substituted at phosphorylation sites (T72, S86, S120, and S121) suggested that phosphorylation of I-2 by CK2 is also involved in enhancement of association between GSK-3beta and I-2 in vivo. This study is the first demonstration that GSK-3beta associates with PP1C/I-2 complex and phosphorylates I-2 at T72 in the intact cells.     

Impaired glucagon secretory responses in mice lacking the type 1 sulfonylurea receptor

Pancreatic alpha-cells, like beta-cells, express ATP-sensitive K(+) (K(ATP)) channels. To determine the physiological role of K(ATP) channels in alpha-cells, we examined glucagon secretion in mice lacking the type 1 sulfonylurea receptor (Sur1). Plasma glucagon levels, which were increased in wild-type mice after an overnight fast, did not change in Sur1 null mice. Pancreas perfusion studies showed that Sur1 null pancreata lacked glucagon secretory responses to hypoglycemia and to synergistic stimulation by arginine. Pancreatic alpha-cells isolated from wild-type animals exhibited oscillations of intracellular free Ca(2+) concentration ([Ca(2+)](i)) in the absence of glucose that became quiescent when the glucose concentration was increased. In contrast, Sur1 null alpha-cells showed continuous oscillations in [Ca(2+)](i) regardless of the glucose concentration. These findings indicate that K(ATP) channels in alpha-cells play a key role in regulating glucagon secretion, thereby adding to the paradox of how mice that lack K(ATP) channels maintain euglycemia.     

Molecular characterization of a novel salt-inducible gene for an OSBP (oxysterol-binding protein)-homologue from soybean

Oxysterol-binding protein (OSBP) and its homologues constitute a protein family in many eukaryotes from yeast to humans, which are involved in cellular lipid metabolism, vesicle transport and signal transduction. In this study, we characterized a novel salt-inducible gene for an OSBP-homologue from soybean (Glycine max [L.] Merr.). The soybean OSBP-homologous gene, denoted as G. max OSBP (GmOSBP), encoded a 789 aa putative protein with two characteristic domains; the pleckstrin homology (PH) domain and the ligand-binding (LB) domain, in the N- and C-terminus, respectively. The GmOSBP-PH domain showed localization into/around the nucleus in a transient subcellular localization assay. The phylogenetic relationship of the GmOSBP-LB domain to those in other OSBP-homologues suggested that GmOSBP might bind a lipid molecule(s) different from the ligand-candidates found for the human/yeast OSBP-homologues. The GmOSBP gene was constitutively transcribed in all of the soybean organs examined--root, stem and trifoliate leaf--at low levels and was highly induced in all these organs by high-salt stress (300 mM NaCl). Interestingly, gene expression of GmOSBP was also markedly induced in the senesced soybean cotyledon, which contains high levels of a variety of cellular lipids utilized for energy for germination and as membrane components. Therefore, we suggest that GmOSBP may be involved in some physiological reactions for stress-response and cotyledon senescence in the soybean.     

RT-CaCCO process: an improved CaCCO process for rice straw by its incorporation with a step of lime pretreatment at room temperature

We improved the CaCCO process for rice straw by its incorporation with a step of lime pretreatment at room temperature (RT). We firstly optimized the RT-lime pretreatment for the lignocellulosic part. When the ratio of lime/dry-biomass was 0.2 (w/w), the RT lime-pretreatment for 7-d resulted in an effect on the enzymatic saccharification of cellulose and xylan equivalent to that of the pretreatment at 120°C for 1h. Sucrose, starch and β-1,3-1,4-glucan, which could be often detected in rice straw, were mostly stable under the RT-lime pretreatment condition. Then, the pretreatment condition in the conventional CaCCO process was modified by the adaptation of the optimized RT lime-pretreatment, resulting in significantly better carbohydrate recoveries via enzymatic saccharification than those of the CaCCO process (120°C for 1 h). Thus, the improved CaCCO process (the RT-CaCCO process) could preserve/pretreat the feedstock at RT in a wet form with minimum loss of carbohydrates.     

The Piperaceae Amides I: Structure of Pipercide,A New Insecticidal Amide from Piper nigrum L.

It was evidenced that mutagenic principles in tryptophan pyrolysate, 3-amino-1,4-dimethyl-5H pyrido(4,3-b) indole and 3-amino-1-methyl-5H pyrido(4,3-b) indole (abbreviated as Trp-P-1 and Trp-P-2, respectively) bind to DNA without activation by rat liver microsomes. The bindings of Trp-P-1 and Trp-P-2 were not random and did not introduce strand scissions into DNA. Trp-P-1 bound more easily than Trp-P-2. The bindings of these mutagenic principles to DNA were concluded by using negatively superhelical simian virus 40 (SV40) DNA from following experimental data. (1) The intensity of ethidium bromide (EtBr)-DNA fluorescence by illumination with UV light and the electrophoretic mobility of superhelical DNA in agarose gel decreased as a function of the amounts of Trp-P-1 and Trp-P-2. (2) In vitro RNA synthesis catalyzed by Escherichia coli DNA-dependent RNA polymerase and nick-translation catalyzed by Escherichia coli DNA polymerase I (Kornberg enzyme) were inhibited significantly on DNA treated with Trp-P-1 and Trp-P-2. (3) The negative superhelicity of SV40 DNA introduces unpaired regions into DNA. These regions can be cleaved by single-strand-specific S1 endonuclease to generate unit length linear duplex molecules. It was found that this S1-sensitivity of DNA decreased by treatment with Trp-P-1. (4) The cleavage patterns of Trp-P-1 treated DNA with five restriction endonucleases were investigated. The protection of the cleavage site by the drug was observed against HincII, HindIII and EcoRII, whereas not against HaeIII and HinfI. These results show that the binding of Trp-P-1 to DNA is not random. Identical results were also obtained in Trp-P-2.

However, the bindings of Trp-P-1 and Trp-P-2 were not so tight, and phenol extraction of the complex dissociated these drugs from DNA.     

Characteristics of microbial fermentation and potential digestibility of fiber in the hindgut of dugongs (Dugong dugon)

The number of microorganisms in the hindgut of dugongs (Dugong dugon) were estimated and their in vitro volatile fatty acid (VFA) production and degradation of eelgrass measured. Scanning electron microscopy showed that some rod bacteria attached to the surface of plant tissue degraded and eroded the cell walls. Number of starch-, lactate-, cellobiose-, pectin-, xylan- and cellulose-utilizing bacteria, sulfate-reducing bacteria and methane-producing bacteria were estimated at 10⁹ ～ 10¹⁰ colony forming units g^?1. Microorganisms degraded the cellulose and noncellulolytic components of the eelgrass, and about 47.3% of dry matter was degraded after 36?h in vitro incubation. The total VFA concentration was 10.5?mmol?dL^?1 at 36?h incubation, which included 55.7?mol% acetate, 18.0?mol% n-butyrate and 15.1?mol% propionate. The gas composition of in vitro fermentation was 68.4% carbon dioxide, 22.2% methane and 9.4% hydrogen.     

Phylogenetic analysis of caprellid and corophioid amphipods (Crustacea) based on the 18S rRNA gene, with special emphasis on the phylogenetic position of Phtisicidae

Members of the amphipod suborder Caprellidea exhibit degenerated abdomens and pereopods 3 and 4. Some genera of Podoceridae (Gammaridea, Corophioidea) such as Dulichia also show reduced abdomens and pereopods and thus are generally regarded as a sister group of the Caprellidea. In addition, one of the caprellid families, the Caprogammaridae, exhibits abdominal segments that are similar to those of the podocerids, as well as rudimentary pereopods 3 and 4, which are more consistent with those of other caprellids. Therefore, an evolutionary scheme has been suggested on the basis of the gradual degeneration of the pereopods and abdomen: [Dulichia, (caprogammarids, caprellids)]. However, the Phtisicidae (Caprellidea) contradict this hypothesis because they exhibit well-developed pereopods 3 and 4, along with degenerated abdomens. Therefore, previous studies have suggested that the Phtisicidae and other caprellids may be polyphyletic. We examined the phylogenetic position of the Phtisicidae and other caprellid amphipods, using 18S rRNA gene sequence data. The results strongly indicate that the Phtisicidae and other caprellid families form a monophyletic clade. However, a close phylogenetic relationship among Dulichia (Corophioidea) and taxa belonging to the Caprellidea was not definitively supported. This study is the first to use molecular data to investigate the phylogenetic relationships among the Caprellidea.     

Fatty Acids as Inhibitors of Microbial Cell Wall Synthesis

The Maillard reaction of DNA with ketoses was investigated. Several days of incubation of d-fructose 6-phosphate with deoxyribonucleotides or with polymer DNA in an aqueous buffer resulted in the formation of chromophores and fluorophores. Aminoguanidine and sodium cvanoborohydride inhibited the formation of fluorophores. Transition metal ions such as Cu²⁺, Fe³⁺, Fe²⁺, or Mn^{2 +} promoted the formation of chromophores and fluorophores. Metal-chelating agents such as DETAPAC, citrate, and Desferal inhibited the formation of fluorophores. Superoxide dismutase and catalase also inhibited the formation of fluorophores. The transition metal ion-catalyzed autoxidation of d-fructose 6-phosphate or of the Heyns rearrangement products were to be partially involved in the glycation of DNA and subsequent formation of chromophores and of fluorophores.