Isolation, optimization, and partial purification of amylase from Chrysosporium asperatum by submerged fermentation

A potent fungus for amylase production, Chrysosporium asperatum, was isolated from among 30 different cultures obtained from wood samples collected in the Junagadh forest, India. All of the isolated cultures were screened for their ability to produce amylase by submerged fermentation. Among the selected cultures, C. asperatum (Class Euascomycetes; Onygenales; Onygenaceae) gave maximum amylase production. In all of the different media tested, potato starch was found to be a good substrate for production of amylase enzyme at 30 degrees C and pH 5.0. Production of enzyme reached the maximum when a combination of starch and 2% xylose, and organic nitrogen (1% yeast extract) and ammonium sulfate were used as carbon and nitrogen sources, respectively. There was no significant effect of metal ions on enzyme activity. The enzyme was relatively stable at 50 degrees C for 20 min, and no inhibitory effect of Ca+2 ions on amylase production was observed.     

Evaluation of immunostimulatory and growth promoting effect of seed fractions of Achyranthes aspera in common carp Cyprinus carpio and identification of active constituents

Immunostimulatory and growth promoting properties of Achyranthes aspera seeds were studied with larvae of common carp Cyprinus carpio. Four experimental diets were prepared using raw (D1) and alcohol (D2), petroleum ether (D3) and 50% aqueous alcohol (D4) extracts of A. aspera seeds. Diet without seed served as control (D5). Fish were fed with test/control diet for 30 days and then immunized with 10 μl of c-RBC. Blood samples were collected 7 days after immunization. Survival (93 ± 3%) of fish was significantly (P < 0.05) higher in D1 diet fed group compared to others. Highest specific growth rate was found in fish fed with diet D2. Significantly (P < 0.05) higher levels of serum protein and albumin were found in D1 and D3 compared to others. Highest serum globulin level was found in D1, which was followed by D3, D2, D4 and D5. Hemagglutination titer level was 5-18 folds higher in diet D3 fed fish compared to others. SGOT and SGPT levels were significantly (P < 0.05) higher in control group compared to the treated groups. Myeloperoxidase activity was significantly (P < 0.05) higher in D1 (2.513 ± 0.27 λ 450 nm) and D3 (2.38 ± 0.07 λ 450 nm) diets fed groups compared to others. The best performance of fish was found in raw A. aspera seeds incorporated diet fed group and the active constituents were identified as ecdysterone and two essential fatty acids linolenic acid and oleic acid.     

Seasonal adaptations of the tuberous roots of Ranunculus asiaticus to desiccation and resurrection by changes in cell structure and protein content

The annual developmental cycle of tuberous roots of Ranunculus asiaticus was studied with respect to structure and content of their cells, to understand how these roots are adapted to desiccation, high temperature and rehydration. Light microscopy, histochemical analysis, and protein analyses by SDS-PAGE were employed at eight stages of annual root development. During growth and maturation of the roots, cortical cells increased in size and their cell walls accumulated pectin materials in a distinct layer to the inside of the primary walls, with pits between adjoining cells. The number of starch granules and protein bodies also increased within the cells. Several discrete proteins accumulated. Following quiescence and rehydration of the roots there was a loss of starch and proteins from the cells, and cell walls decreased in thickness. The resurrection geophyte R. asiaticus possesses desiccation-tolerant annual roots. They store carbon and nitrogen reserves within their cells, and pectin within the walls to support growth of the plant following summer quiescence and rehydration.     

Molecular cloning and characterization of an almond 9-hydroperoxide lyase, a new CYP74 targeted to lipid bodies

Oxylipin metabolism represents one of many defence mechanisms employed by plants. It begins with the oxygenation of polyunsaturated fatty acids by lipoxygenases to form fatty acid hydroperoxides that are substrates for several enzymes, including specialized cytochrome P450s known as CYP74s. The targeting of a new CYP74, a 9-hydroperoxide lyase (HPL) from almonds, to the endomembrane system and lipid bodies, both as enzyme activity in almond seeds and as GFP fusions transiently expressed in tobacco protoplasts, is described. Such association of a CYP74 with lipid bodies has not been reported previously. Also described are the properties of a 9-HPL gene, the developmental regulation of its expression, the production and characterization of recombinant 9-HPL in Escherichia coli, and the developmental correlation between gene expression, enzyme activity, and the appearance of volatile C9 aldehydes from HPL action.     

Protein kinase C-theta is an early survival factor required for differentiation of effector CD8+ T cells

CD8(+) T cells are crucial for host defense against invading pathogens and malignancies. However, relatively little is known about intracellular signaling events that control the genetic program of their activation and differentiation. Using CD8(+) T cells from TCR-transgenic mice crossed to protein kinase C-theta (PKCtheta)-deficient mice, we report that PKCtheta is not required for Ag-induced CD8(+) T cell proliferation, but is important for T cell survival and differentiation into functional, cytokine-producing CTLs. Ag-stimulated PKCtheta(-/-) T cells underwent accelerated apoptosis associated with deregulated expression of Bcl-2 family proteins and displayed reduced activation of ERKs and JNKs. Some defects in the function of PKCtheta(-/-) T cells (poor survival and reduced Bcl-2 and Bcl-x(L) expression, CTL activity, and IFN-gamma expression) were partially or fully restored by coculture with wild-type T cells or by addition of exogenous IL-2, whereas others (increased Bim(EL) expression and TNF-alpha production) were not. These findings indicate that PKCtheta, although not essential for initial Ag-induced proliferation, nevertheless plays an important role in promoting and extending T cell survival, thereby enabling the complete genetic program of effector CD8(+) differentiation. The requirement for PKCtheta in different types of T cell-dependent responses may, therefore, depend on the overall strength of signaling by the TCR and costimulatory receptors and may reflect, in addition to its previously established role in activation, an important, hitherto unappreciated, role in T cell survival.     

The Adenomatous Polyposis Coli-associated Guanine Nucleotide Exchange Factor Asef Is Involved in Angiogenesis

Mutation of the tumor suppressor adenomatous polyposis coli (APC) is a key early event in the development of most colorectal tumors. APC promotes degradation of β-catenin and thereby negatively regulates Wnt signaling, whereas mutated APCs present in colorectal tumor cells are defective in this activity. APC also stimulates the activity of the guanine nucleotide exchange factor Asef and regulates cell morphology and migration. Truncated mutant APCs constitutively activate Asef and induce aberrant migration of colorectal tumor cells. Furthermore, we have recently found that Asef and APC function downstream of hepatocyte growth factor and phosphatidylinositol 3-kinase. We show here that Asef is required for basic fibroblast growth factor- and vascular endothelial growth factor-induced endothelial cell migration. We further demonstrate that Asef is required for basic fibroblast growth factor- and vascular endothelial growth factor-induced microvessel formation. Furthermore, we show that the growth as well as vascularity of subcutaneously implanted tumors are markedly impaired in Asef^−/− mice compared with wild-type mice. Thus, Asef plays a critical role in tumor angiogenesis and may be a promising target for cancer chemotherapy.     

The synaptic vesicle SNARE neuronal Synaptobrevin promotes endolysosomal degradation and prevents neurodegeneration

Soluble NSF attachment protein receptors (SNAREs) are the core proteins in membrane fusion. The neuron-specific synaptic v-SNARE n-syb (neuronal Synaptobrevin) plays a key role during synaptic vesicle exocytosis. In this paper, we report that loss of n-syb caused slow neurodegeneration independent of its role in neurotransmitter release in adult Drosophila melanogaster photoreceptor neurons. In addition to synaptic vesicles, n-Syb localized to endosomal vesicles. Loss of n-syb lead to endosomal accumulations, transmembrane protein degradation defects, and a secondary increase in autophagy. Our evidence suggests a primary defect of impaired delivery of vesicles that contain degradation proteins, including the acidification-activated Cathepsin proteases and the neuron-specific proton pump and V0 adenosine triphosphatase component V100. Overexpressing V100 partially rescued n-syb-dependent degeneration through an acidification-independent endosomal sorting mechanism. Collectively, these findings reveal a role for n-Syb in a neuron-specific sort-and-degrade mechanism that protects neurons from degeneration. Our findings further shed light on which intraneuronal compartments exhibit increased or decreased neurotoxicity.     

N-arachidonoyl glycine induces macrophage apoptosis via GPR18

N-arachidonoyl glycine (NAGly), a member of lipoamino acids, was reported to exhibit anti-inflammatory effects in experimental ear edema or peritonitis. However the underlying mechanisms have not been clarified so far. In this study, we attempt to investigate the effects of NAGly on macrophages, including the relevant signaling pathways. NAGly potently induced apoptosis in mouse macrophage-derived cell line, RAW264.7. Pretreatment with inhibitors for MEK and p38 MAPK prevented the apoptosis induced by NAGly, although NAGly activated ERK1/2, p38 MAPK and JNK. Further, we focused on implication of GPR18, one of the orphan G protein-coupled receptors, because NAGly has been reported as a candidate ligand for GPR18. Pretreatment with pertussis toxin or siRNA to knock down the expression of GPR18 significantly attenuated the apoptosis induced by NAGly. In mouse peritoneal macrophages, the expression of GPR18 mRNA was elevated in proinflammatory stimulated macrophages but not in anti-inflammatory stimulated macrophages; consistently, NAGly remarkably reduced cell viability of the former, as compared to the latter. These results suggest that NAGly might be involved in function of macrophages through GPR18.     

Synthesis of spacer-equipped di-, tri-, and the tetrasaccharide fragments of the deacetylated O-PS1 of Citrobacter gillenii O9a,9b, and a related pentasaccharide

The title rhamnooligosaccharides [alpha-D-Rhap4NAc-(1-->3)-alpha-D-Rhap4NAc-1-O-(CH(2))(5)COOMe, alpha-D-Rhap4NAc-(1-->3)-alpha-D-Rhap4NAc-(1-->3)-alpha-D-Rhap4NAc-1-O-(CH(2))(5)COOMe, alpha-D-Rhap4NAc-(1-->2)-alpha-D-Rhap4NAc-(1-->3)-alpha-D-Rhap4NAc-(1-->3)-alpha-D-Rhap4NAc-1-O-(CH(2))(5)COOMe, and alpha-D-Rhap4NAc-(1-->3)-alpha-D-Rhap4NAc-(1-->2)-alpha-D-Rhap4NAc-(1-->3)-alpha-D-Rhap4NAc-(1-->3)-alpha-D-Rhap4NAc-1-O-(CH(2))(5)COOMe] were synthesized in a stepwise fashion from 5-methoxycarbonylpentyl 4-azido-4,6-dideoxy-2-O-benzyl-alpha-D-mannopyranoside and orthogonally protected 1-thioglycoside glycosyl donors. The amorphous, final products were fully characterized as corresponding per-O-acetyl derivatives.