Production of β-xylosidase from <Emphasis Type="Italic">Trichoderma asperellum</Emphasis> KIF125 and its application in efficient hydrolysis of pretreated rice straw with fungal cellulase

On-site cellulase and hemicellulase production is a promising way to reduce enzyme cost in the commercialization of the lignocellulose-to-ethanol process. A hemicellulase-producing fungal strain suitable for on-site enzyme production was selected from cultures prepared using wet disc-milling rice straw (WDM-RS) and identified as Trichoderma asperellum KIF125. KIF125 hemicellulase showed uniquely high abundance of β-xylosidase in the xylanolytic enzyme system compared to other fungal hemicellulase preparations. Supplementation of Talaromyces cellulolyticus cellulase with KIF125 hemicellulase was more effective than that with the hemicellulases from other fungal sources in reducing the total enzyme loading for the improvement of xylose yield in the hydrolysis of ball-milling RS, due to its high β-xylosidase dominance. β-Xylosidase in KIF125 hemicellulase was purified and classified as a glycosyl hydrolase family 3 enzyme with relatively high specificity for xylobiose. The production of KIF125 β-xylosidase in the fermentor was estimated as 118 U/g-WDM-RS (2350 U/L culture) at 48 h. These results demonstrate that KIF125 is promising as a practical hemicellulase source to combine with on-site cellulase production using T. cellulolyticus.     

Characterization of a GDP-D-mannose 3',5'-epimerase from rice

The enzymatic characterization of GDP-d-mannose 3',5'-epimerase (GME), a key enzyme in the biosynthesis of vitamin C in plants is described. The GME gene (Genbank Accession No. AB193582) in rice was cloned, and expressed as a fusion protein in Escherichia coli. Reaction products from GDP-d-mannose, as produced by GME catalysis, were separated by recycling HPLC on an ODS column, and were determined to be GDP-l-galactose and GDP-l-gulose, based on their NMR spectra and sugar analysis. The reaction catalyzed by GME was inhibited by GDP, and was strongly accelerated by NAD(+) in contrast to the case of GME from Arabidopsis thaliana. This difference in the effect of NAD(+) on GME activity can be attributed to the NAD binding domain which is conserved in the rice gene, but not in the Arabidopsis thaliana gene. The apparent K(m) and k(cat) were determined to be 1.20x10(-5)M and 0.127s(-1), respectively, in the presence of 20microM NAD(+). The fractions of GDP-d-mannose, GDP-l-galactose and GDP-l-gulose, at equilibrium, were approximately 0.75, 0.20 and 0.05, respectively.     

Effects of detergents on the secondary structures of prion protein peptides as studied by CD spectroscopy.

Pathogenic prion proteins (PrP(Sc)) are thought to be produced by alpha-helical to beta-sheet conformational changes in the normal cellular prion proteins (PrP(C)) located solely in the caveolar compartments. In order to inquire into the possible conformational changes due to the influences of hydrophobic environments within caveolae, the secondary structures of prion protein peptides were studied in various kinds of detergents by CD spectra. The peptides studied were PrP(129-154) and PrP(192-213); the former is supposed to assume beta-sheets and the latter alpha-helices, in PrP(Sc). The secondary structure analyses for the CD spectra revealed that in buffer solutions, both PrP(129-154) and PrP(192-213) mainly adopted random-coils (approximately 60%), followed by beta-sheets (30%-40%). PrP(129-154) showed no changes in the secondary structures even in various kinds of detergents such as octyl-beta-D-glucopyranoside (OG), octy-beta-D-maltopyranoside (OM). sodium dodecyl sulfate (SDS), Zwittergent 3-14 (ZW) and dodecylphosphocholine (DPC). In contrast, PrP(192-213) changed its secondary structure depending on the concentration of the detergents. SDS, ZW, OG and OM increased the alpha-helical content, and decreased the beta-sheet and random-coil contents. DPC also increased the alpha-helical content, but to a lesser extent than did SDS, ZW, OG or OM. These results indicate that PrP(129-154) has a propensity to adopt predominantly beta-sheets. On the other hand, PrP(192-213) has a rather fickle propensity and varies its secondary structure depending on the environmental conditions. It is considered that the hydrophobic environments provided by these detergents may mimic those provided by gangliosides in caveolae, the head groups of which consist of oligosaccharide chains containing sialic acids. It is concluded that PrP(C) could be converted into a nascent PrP(Sc) having a transient PrP(Sc) like structureunder the hydrophobic environments produced by gangliosides.     

Hamster PIWI proteins bind to piRNAs with stage-specific size variations during oocyte maturation

In animal gonads, transposable elements are actively repressed to preserve genome integrity through the PIWI-interacting RNA (piRNA) pathway. In mice, piRNAs are abundantly expressed in male germ cells, and form effector complexes with three distinct PIWIs. The depletion of individual Piwi genes causes male-specific sterility with no discernible phenotype in female mice. Unlike mice, most other mammals have four PIWI genes, some of which are expressed in the ovary. Here, purification of PIWI complexes from oocytes of the golden hamster revealed that the size of the PIWIL1-associated piRNAs changed during oocyte maturation. In contrast, PIWIL3, an ovary-specific PIWI in most mammals, associates with short piRNAs only in metaphase II oocytes, which coincides with intense phosphorylation of the protein. An improved high-quality genome assembly and annotation revealed that PIWIL1- and PIWIL3-associated piRNAs appear to share the 5′-ends of common piRNA precursors and are mostly derived from unannotated sequences with a diminished contribution from TE-derived sequences, most of which correspond to endogenous retroviruses. Our findings show the complex and dynamic nature of biogenesis of piRNAs in hamster oocytes, and together with the new genome sequence generated, serve as the foundation for developing useful models to study the piRNA pathway in mammalian oocytes.     

Postischemic Cerebral Lipid Peroxidation In Vitro: Modification by Dietary Vitamin E

Using an in vitro system, we studied the effect of postischemic reoxygenation on cerebral lipid peroxidation in relation to the dietary intake of vitamin E (VE) in rats. Homogenates prepared from VE-deficient, -normal, and -supplemented brains, which were previously rendered ischemic for 30 min by decapitation, were incubated under air or nitrogen gas for 60 min. The extent of peroxidation in brain tissue was estimated by a thiobarbituric acid (TBA) test and by diene conjugation in total lipid extracts. The brain levels of alpha-tocopherol and of total and free fatty acids (FAs) were also determined. Aerobic incubation increased TBA reactants in all dietary groups; the effect was largest in the VE-deficient group, intermediate in the VE-normal group, and smallest in the VE-supplemented group. In contrast, nitrogen incubation did not alter the basal levels of TBA reactants except for a small rise associated with VE deficiency. Conjugated dienes changed in parallel with TBA reactants. alpha-Tocopherol decreased after aerobic incubation and also, to a lesser degree, after nitrogen incubation in each dietary group. Only in the reoxygenated samples of the VE-deficient group was there a significant fall in total polyunsaturated FAs. The levels of free FAs continuously increased throughout ischemia and subsequent incubation. However, the level of free polyunsaturated FAs was similar after aerobic and nitrogen incubation in each dietary group, and was not affected by VE. Thus, cerebral reoxygenation after ischemia propagates peroxidative reactions within esterified polyunsaturated FAs. The modification by VE of reoxygenation-induced lipid peroxidation suggests free radical mediation.     

Cooperative Interception of Neuronal Apoptosis by BCL-2 and BAG-1 Expression: Prevention of Caspase Activation and Reduced Production of Reactive Oxygen Species

Abstract: Neuronally differentiated PC12 cells undergo synchronous apoptosis when deprived of nerve growth factor (NGF). Here we show that NGF withdrawal induces actinomycin D- and cycloheximide-sensitive caspase (ICE-like) activity. The peptide inhibitor of caspase activity, N -acetyl-Asp-Glu-Val-Asp-aldehyde, was more potent than acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone in preventing NGF withdrawal-induced apoptosis, suggesting an important role for caspase-3 (CPP32)-like proteases. We observed a peak of reactive oxygen species (ROS) 6 h after NGF withdrawal. ROS appear to be required for apoptosis, because cell death is prevented by the free radical spin trap, N-tert -butyl-α-phenylnitrone, and the antioxidant, N -acetylcysteine. ROS production was blocked by actinomycin D, cycloheximide, and caspase protease inhibitors, suggesting that ROS generation is downstream of new mRNA and protein synthesis and activation of caspases. Forced expression of either BCL-2 or the BCL-2-binding protein BAG-1 blocked NGF withdrawal-induced apoptosis, activation of caspases, and ROS generation, showing that they function upstream of caspases. Coexpression of BCL-2 and BAG-1 was more protective than expression of either protein alone.     

Production and characterization of ligninolytic enzymes ofBjerkandera adusta grown on wood meal/wheat bran culture and production of these enzymes using a rotary-solid fermenter

Manganese peroxidase (MnP) and lignin peroxidase (LiP) were produced by growing a white-rot fungusBjerkandera adusta statically, on a wood meal/wheat bran culture in flasks. MnP and LiP reached their maximum activity after 6 and 19 days of inoculation, respectively. Both MnP and LiP are thought to be important enzymes in lignin biodegradation byB. adusta. Ion exchange chromatography showed thatB. adusta produced a single LiP and a single MnP enzyme in wood meal/wheat bran culture. These enzymes were separated and characterized. The molecular weight of MnP was 46,500 with a pl of 3.9. The molecular weight of LiP was estimated to be 47,000 with a pl of 3.5. Spectral analysis demonstrated that both enzymes are heme proteins. Production of these enzymes was also achieved using a rotarysolid culture fermenter. MnP, LiP and veratryl alcohol oxidase were produced byB. adusta in the fermenter.     

Persistent High Numbers of Clostridium perfringens in the Intestines of Japanese Aged Adults

TSN agar was applicable for enumeration of Clostridium perfringens in fecal samples of adults but not in those of infants. It was demonstrated using TSN agar that some healthy aged adults had persistently carried C. perfringens at levels ranging from 10⁷ to 10⁹, while some others ranged from 10³ to 10⁶ per ml volume of fecal sample although all of these adults had the same diets. In the test for agglutinability of isolates of C. perfringens collected from two elderly adults, a younger adult and a baby, it was demonstrated that most of the isolates obtained from an aged adult of high levels for 19 months belonged the same serotype, while rapid alteration of serotypes could be observed in three other persons with high or low levels. In spite of as many as 10⁹ C. perfringens per ml of feces, no trace of α-toxin could be detected in the fecal samples. In in vitro tests, fecal suspension suppressed the production of α-toxin although it allowed the organism to grow sufficiently.     

Dystrophin in rod spherules; submembranous dense regions facing bipolar cell processes

 It is known that the retina contains the protein dystrophin in the ribbon synapse, but the ultrastructural analysis is not yet fully elucidated. Our previous study reported that dystrophin is localized under the rod cell membranes in rat retinas. In the present study, we have investigated the relationship between dystrophin-rich regions of rod cell membranes and other neuronal processes in mouse retinas with a monoclonal antibody raised against the human dystrophin C-terminus. Immunoblotting, immunofluorescence stainings, and immunoelectron microscopy were employed. Immunoblotting analysis indicated that mouse retinas possessed some of the dystrophin isoforms of approximately 260 kDa, 140 kDa, and 70 kDa molecular weight. Confocal images showed a punctate appearance in the outer plexiform layer, as previously described. Immunoelectron microscopy showed that dystrophin immunoreactive products were always observed at submembranous dense regions of the rod spherule abutting bipolar processes. These results suggest that retinal dystrophin may be closely involved in signal transmission from rods to bipolar cells. Accepted: 7 May 1997