Enhancement of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production in the transgenic Arabidopsis thaliana by the in vitro evolved highly active mutants of polyhydroxyalkanoate (PHA) synthase from Aeromonas caviae

In this study, the enhancement of photosynthetic PHA production was achieved using the highly active mutants of PHA synthase created by the in vitro evolutionally techniques. The wild-type and mutated PHA synthase genes from Aeromonas caviae were introduced into Arabidopsis thaliana together with the NADPH-dependent acetoacetyl-CoA reductase gene from Ralstonia eutropha. Expression of the highly active mutated PHA synthase genes, N149S and D171G, led to an 8-10-fold increase in PHA content in the T1 transgenic Arabidopsis, compared to plants harboring the wild-type PHA synthase gene. In homozygous T2 progenies, PHA content was further increased up to 6.1 mg/g cell dry weight. GC/MS analysis of the purified PHA from the transformants revealed that these PHAs were poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] copolymers consisting of 0.2-0.8 mol % 3HV. The monomer composition of the P(3HB-co-3HV) copolymers synthesized by the wild-type and mutated PHA synthases reflected the substrate specificities observed in Escherichia coli. These results indicate that in vitro evolved PHA synthases can enhance the productivity of PHA and regulate the monomer composition in transgenic plants.     

Studies toward a conjugate vaccine for anthrax. Synthesis and characterization of anthrose [4,6-dideoxy-4-(3-hydroxy-3-methylbutanamido)-2-O-methyl-D-glucopyranose] and its methyl glycosides

The key step in the first chemical synthesis of anthrose (16) and its methyl alpha- (6) and beta-glycoside (22) was inversion of configuration at C-2 in triflates 10, 2, and 18, respectively, obtained from the common intermediate, methyl 4-azido-3-O-benzyl-4,6-dideoxy-alpha-D-mannopyranoside (1). To prepare methyl alpha-anthroside (6), methylation at O-2 of the gluco product 3, obtained from 2, was followed by hydrogenation/hydrogenolysis of the formed 2-methyl ether 4, to simultaneously remove the protecting benzyl group and reduce the azido function. Subsequent N-acylation of the formed amine 5 with 3-hydroxy-3-methylbutyric acid gave the target methyl alpha-glycoside 6. Synthesis of methyl beta-anthroside (22) comprised the same sequence of reactions, starting from the known methyl 4-azido-3-O-benzyl-4,6-dideoxy-beta-D-mannopyranoside (17), which was prepared from 1. In the synthesis of anthrose (16), 1-thio-beta-glucoside 11, obtained from 1 through 10, was methylated at O-2, and the azido function in the resulting benzylated 1-thioglycoside 12 was selectively reduced to give amine 13. After N-acylation with 3-hydroxy-3-methylbutyric acid, 1-thioglycoside 14 was hydrolyzed to give the corresponding reducing sugar, aldol 15, which was debenzylated to afford anthrose.     

Synthesis of the beta anomer of the spacer-equipped tetrasaccharide side chain of the major glycoprotein of the Bacillus anthracis exosporium

The beta glycoside of the tetrasaccharide sequence beta-Ant-(1-->3)-alpha-l-Rhap-(1-->3)-alpha-l-Rhap-(1-->2)-l-Rhap, whose aglycon allows conjugation to proteins, was synthesized for the first time. A stepwise synthetic approach was applied with thioglycosides as glycosyl donors, and the beta anomer of the compound was obtained equipped with a spacer group whose further transformation allows conjugation to suitable carriers. To synthesize the beta-anthrosyl linkage with high stereoselectivity, a linker-equipped rhamnotriose derivative was glycosylated with ethyl 4-azido-3-O-benzyl-2-O-bromoacetyl-4,6-dideoxy-1-thio-beta-d-glucopyranoside. Further functionalization of the tetrasaccharide thus obtained, followed by deprotection, gave the target substance.     

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.     

Immunogenicity of synthetic saccharide fragments of Vibrio cholerae O1 (Ogawa and Inaba) bound to Exotoxin A

Recombinant exotoxin A (rEPA) from Pseudomonas aeruginosa conjugated to Vibrio cholerae O1 serotype-specific polysaccharides (mono-, di- and hexasaccharide) were immunogenic in mice. Monosaccharide conjugates boosted the humoral responses to the hexasaccharide conjugates. Prior exposure to purified Ogawa lipopolysaccharide (LPS) enabled contra-serotype hexasaccharide conjugates to boost the vibriocidal response, but Inaba LPS did not prime for an enhanced vibriocidal response by a contra-serotype conjugate. Prior exposure to the carrier, and priming B cells with the LPS of either serotype, resulted in enhanced vibriocidal titers if the Ogawa hexasaccharides were used, but a diminished response to the Inaba LPS. These studies demonstrate that the 'functional' B cell epitopes on the LPS differ from those of the neoglycoconjugates and that the order of immunization and the serotype of the boosting conjugate can influence the epitope specificity and function of the antisera.     

Effects of peripheral cannabinoid receptor ligands on motility and polarization in neutrophil-like HL60 cells and human neutrophils

The possible role of the peripheral cannabinoid receptor (CB2) in neutrophil migration was investigated by using human promyelocytic HL60 cells differentiated into neutrophil-like cells and human neutrophils isolated from whole blood. Cell surface expression of CB2 on HL60 cells, on neutrophil-like HL60 cells, and on human neutrophils was confirmed by flow cytometry. Upon stimulation with either of the CB2 ligands JWH015 and 2-arachidonoylglycerol (2-AG), neutrophil-like HL60 cells rapidly extended and retracted one or more pseudopods containing F-actin in different directions instead of developing front/rear polarity typically exhibited by migrating leukocytes. Activity of the Rho-GTPase RhoA decreased in response to CB2 stimulation, whereas Rac1, Rac2, and Cdc42 activity increased. Moreover, treatment of cells with RhoA-dependent protein kinase (p160-ROCK) inhibitor Y27632 yielded cytoskeletal organization similar to that of CB2-stimulated cells. In human neutrophils, neither JWH015 nor 2-AG induced motility or morphologic alterations. However, pretreatment of neutrophils with these ligands disrupted N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced front/rear polarization and migration and also substantially suppressed fMLP-induced RhoA activity. These results suggest that CB2 might play a role in regulating excessive inflammatory response by controlling RhoA activation, thereby suppressing neutrophil migration.