Characterization of acid-stable glucose isomerase from Streptomyces sp., and development of single-step processes for high-fructose corn sweetener (HFCS) production

The glucose isomerase from Streptomyces olivaceoviridis E-86 was purified by chromatographic procedures, showing one single protein band in the SDS-PAGE. The enzyme had high acid stability, and there was no loss in enzyme activity at pH 5.0 after incubation at 60 degrees C for 30 hr. The enzyme had sufficients activity at 60 degrees C, pH 5.5, (which is the reaction condition for a single-step process with a glucoamylase from A. niger), and at 58 degrees C, pH 6.0, (condition with a glucoamylase from R. niveus). By using this acid-stable glucose isomerase, a single-step process to produce high-fructose corn sweetener (HFCS) from liquefied starch was formed without any reductant or other reagents for enzyme stabilization. The HFCS produced was about fifty percent fructose and less than 1.5% unknown oligosaccharides.     

Protracted Administration of L-Asparaginase in Maintenance Phase Is the Risk Factor for Hyperglycemia in Older Patients with Pediatric Acute Lymphoblastic Leukemia

Although L-asparaginase related hyperglycemia is well known adverse event, it is not studied whether the profile of this adverse event is affected by intensification of L-asparaginase administration. Here, we analyzed the profile of L-asparaginase related hyperglycemia in a 1,176 patients with pediatric acute lymphoblastic leukemia treated according to the Japan Association of Childhood Leukemia Study ALL-02 protocol using protracted L-asparaginase administration in maintenance phase. We determined that a total of 75 L-asparaginase related hyperglycemia events occurred in 69 patients. Although 17 events (17/1176, 1.4%) developed in induction phase, which was lower incidence than those (10–15%) in previous reports, 45 events developed during the maintenance phase with protracted L-asparaginase administration. Multivariate analysis showed that older age at onset (≥10 years) was a sole independent risk factor for L-asparaginase-related hyperglycemia (P<0.01), especially in maintenance phase. Contrary to the previous reports, obesity was not associated with L-asparaginase-related hyperglycemia. These findings suggest that protracted administration of L-asparaginase is the risk factor for hyperglycemia when treating adolescent and young adult acute lymphoblastic leukemia patients.     

Characterization of the rRNA locus of Pfiesteria piscicida and development of standard and quantitative PCR-based detection assays targeted to the nontranscribed spacer

Pfiesteria piscicida is a heterotrophic dinoflagellate widely distributed along the middle Atlantic shore of the United States and associated with fish kills in the Neuse River (North Carolina) and the Chesapeake Bay (Maryland and Virginia). We constructed a genomic DNA library from clonally cultured P. piscicida and characterized the nontranscribed spacer (NTS), small subunit, internal transcribed spacer 1 (ITS1), 5.8S region, ITS2, and large subunit of the rRNA gene cluster. Based on the P. piscicida ribosomal DNA sequence, we developed a PCR-based detection assay that targets the NTS. The assay specificity was assessed by testing clonal P. piscicida and Pfiesteria shumwayae, 35 additional dinoflagellate species, and algal prey (Rhodomonas sp.). Only P. piscicida and nine presumptive P. piscicida isolates tested positive. All PCR-positive products yielded identical sequences for P. piscicida, suggesting that the PCR-based assay is species specific. The assay can detect a single P. piscicida zoospore in 1 ml of water, 10 resting cysts in 1 g of sediment, or 10 fg of P. piscicida DNA in 1 micro g of heterologous DNA. An internal standard for the PCR assay was constructed to identify potential false-negative results in testing of environmental sediment and water samples and as a competitor for the development of a quantitative competitive PCR assay format. The specificities of both qualitative and quantitative PCR assay formats were validated with >200 environmental samples, and the assays provide simple, rapid, and accurate methods for the assessment of P. piscicida in water and sediments.     

Regulation of the Gts1p level by the ubiquitination system to maintain metabolic oscillations in the continuous culture of yeast

Yeast cells exhibit sustained ultradian oscillations of energy metabolism in coupling with cell cycle and stress resistance oscillations in continuous culture. We have reported that the rhythmic expression of Gts1p is important for the maintenance of ultradian rhythms. Structurally, Gts1p contains sequence motifs similar to N-degron and the ubiquitin association domain, raising the possibility that the Gts1p level is regulated by degradation via ubiquitination. When the lysine residue at the putative ubiquitination site of the N-degron was substituted with arginine, both the protein level and half-life of mutant Gts1p increased. During continuous culture, the protein level of the mutant Gts1p was elevated and did not fluctuate, leading to the disappearance of metabolic oscillation within a day. Furthermore, using three Gts1ps containing mutations in the ubiquitin association domain, we showed that the lower the binding activity of the mutant Gts1ps for polyubiquitin in vitro, the higher the protein level in vivo. Expression of the mutant Gts1ps in the continuous culture resulted in an increase in Gts1p and early loss of the oscillation. Therefore, Gts1p is degraded through conjugation with ubiquitin, and the UBA domain promoted the degradation of ubiquitinated Gts1p, causing a fluctuation in protein level, which is required for the maintenance of metabolic oscillations.     

The amino-terminal half of Sendai virus C protein is not responsible for either counteracting the antiviral action of interferons or down-regulating viral RNA synthesis

The Sendai virus C proteins, C', C, Y1, and Y2, are a nested set of independently initiated carboxy-coterminal proteins translated from a reading frame overlapping the P frame on the P mRNA. The C proteins are extremely versatile and have been shown to counteract the antiviral action of interferons (IFNs), to down-regulate viral RNA synthesis, and to promote virus assembly. Using the stable cell lines expressing the C, Y1, Y2, or truncated C protein, we investigated the region responsible for anti-IFN action and for down-regulating viral RNA synthesis. Truncation from the amino terminus to the middle of the C protein maintained the inhibition of the signal transduction of IFNs, the formation of IFN-stimulated gene factor 3 (ISGF3) complex, the generation of the anti-vesicular stomatitis virus state, and the synthesis of viral RNA, but further truncation resulted in the simultaneous loss of all of these inhibitory activities. A relatively small truncation from the carboxy terminus also abolished all of these inhibitory activities. These data indicated that the activities of the C protein to counteract the antiviral action of IFNs and to down-regulate viral RNA synthesis were not encoded within a region of at least 98 amino acids in its amino-terminal half.     

Germplasm conservation of the tropical forest trees,Cedrela odorata L.,Guazuma crinita Mart., andJacaranda mimosaefolia D. Don., by shoot tip encapsulation in calcium-alginate and storage at 12–25°C

Germplasm conservation of the tropical forest trees,Cedrela odorata L.,Guazuma crinita Mart., andJacaranda mimosaefolia D. Don., at above-freezing temperatures following alginate-bead encapsulation was attempted. Shoot tips excised from in vitro plantlets were encapsulated in calcium-alginate beads and stored on different substrates at 12, 20, and 25 °C. Percent viability when encapsulated shoot tips were stored on substrate containing only water solidified with 1% (wt/vol) agar was 80% after 12 months at 12°C forC. odorata, 90% after 12 months at 25°C forG. crinita, and 70% after 6 months at 20°C forJ. mimosaefolia.Abbreviations BAP 6-Benzylaminopurine - KIN 6-Furfurylaminopurine     

Determination of glucosylceramide contents in crop tissues and by-products from their processing

Glucosylceramides were surveyed in crop tissues and by-products from their processing. Apple pulp contained the highest amount (0.94 mg g(-1)) of glucosylceramide and relatively less sterylglucoside, which is the major contaminant of partially purified glucosylceramide. Glucosylceramide from apple pulp was principally composed of 2-hydroxypalmitic acid as the fatty acid, 4-hydroxy-cis-8-sphingenine as the sphingoid base, and glucose as the hexose, similar to those of commercial preparations isolated from rice bran or wheat germ. Apple pulp may be an alternative source for the commercial production of glucosylceramides.     

Vasorelaxant effects of macrocyclic bis(bibenzyls) from liverworts

Vasorelaxant effects of a series of bis(bibenzyls) from liverworts such as Marchantia polymorpha and Marchantia paleacea on rat aorta demonstrated that they relaxed phenylephrine (PE)-induced contractions, which may be mediated through the increased release of NO from endothelial cells as well as opening of K(+) channels, and inhibition of Ca(2+) influx through voltage-dependent Ca(2+) channels (VDCs) and/or receptor-operated Ca(2+) channels (ROCs). Structure-activity relationship based on their structures was discussed. The presence of two aromatic rings which can be connected through two atoms bridge spacer may play an important role for vasorelaxant effect.     

Effects of the core lipid on the energetics of binding of ApoA-I to model lipoprotein particles of different sizes

Interaction of apolipoproteins (apo) with lipid surfaces plays crucial roles in lipoprotein metabolism and cholesterol homeostasis. To elucidate the thermodynamics of binding of apoA-I to lipid, we used lipid emulsions composed of triolein (TO) and egg phosphatidylcholine (PC) as lipoprotein models. Determination of the level of binding of wild-type (WT) apoA-I and some deletion mutants to large (120 nm diameter; LEM) and small (35 nm diameter; SEM) emulsions indicated that N-terminal (residues 44-65) and C-terminal (residues 190-243 and 223-243) deletions have large effects on lipid interaction, whereas deletion of the central region (residues 123-166) has little effect. Substitution of amino acids at either L230 or L230, L233, and Y236 with proline residues also decreases the level of binding, indicating that an alpha-helix conformation in this C-terminal region is required for efficient lipid binding. Calorimetry showed that binding of WT apoA-I to SEM generates endothermic heat (DeltaH approximately 30 kcal/mol) in contrast to the exothermic heat (ca. -85 kcal/mol) generated upon binding to LEM and egg PC small unilamellar vesicles (SUV). This exothermic heat arises from an approximately 25% increase in alpha-helix content, and it drives the binding of apoA-I to LEM and SUV. There is a similar increase in alpha-helix content of apoA-I upon binding to either SEM or SUV, but the binding of apoA-I to SEM is an entropy-driven process. These results suggest that the presence of a core triglyceride modifies the highly curved SEM surface packing and thereby the thermodynamics of apoA-I binding in a manner that compensates for the exothermic heat generated by alpha-helix formation.