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, anticancer activity and apoptosis inducing ability of bisindole linked pyrrolo[2,1-c][1,4]benzodiazepine conjugates

A series of bisindole-pyrrolobenzodiazepine conjugates (5a-f) linked through different alkane spacers was prepared and evaluated for their anticancer activity. All compounds exhibited significant anticancer potency and the most potent compounds 5b and 5e were taken up for detailed studies on MCF-7 cell line. Cell cycle effects were examined apart from investigating the inhibition of tubulin polymerization for compounds 2a, 2b, 5b and 5e at 2μM. FACS analysis showed that at higher concentrations (4 and 8μM) there was an increase of sub-G1 phase cells and decrease of G2/M phase cells, thus indicating that compounds 5b and 5e are effective in causing apoptosis in MCF-7 cells. It was also observed that compounds 5b and 5e showed the down regulation of histone deacetylase protein levels such as HDAC1, 2, 3, 8 and increase in the levels of p21, followed by apoptotic cell death. The apoptotic nature of these compounds was further evidenced by increased expression of cleaved-PARP and active caspase-7 in MCF-7 cells.     

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.     

Preparation of glycoconjugates by dialkyl squarate chemistry revisited

The methyl 6-hydroxyhexanoyl glycoside of lactose was treated with each of 1,2-diaminoethane or hydrazine hydrate, and the corresponding amino amide 4 and acyl hydrazide 13, were treated with each of squaric acid dimethyl, diethyl, dibutyl, and didecyl esters. The monoesters were conjugated to bovine serum albumin (BSA) at different concentrations of hapten using 0.05 and 0.5M pH 9 borate buffer. Maximum loading was achieved faster, and the conjugation efficiency was higher, when the conjugation was conducted at higher concentrations of both hapten and buffer. Conjugations involving haptens 14-17 prepared from hydrazide 13 were generally slower and less efficient than those with compounds 5-8, which were made from amino amide 4. Maintaining pH 9 during conjugation was found to be the most important factor in ensuring that the conjugation was a fast, highly efficient, and reproducible process. When the pH of the conjugation mixture fell during the reaction, resulting in decreased reaction rate or even termination of the conjugation process, the normal course of the conjugation process could be restored by addition of buffer salts. Hydrolysis studies with monoesters formed from amino amide 4 under conjugation conditions showed that decyl ester 8 was the most stable and that the methyl compound 5 was the one most readily hydrolyzed. The stability of monoesters prepared from hydrazide 13 was similar and comparable to the decyl ester prepared from 4. No definite advantage was found for the use of any of the four dialkyl squarate reagents (methyl-, ethyl-, butyl-, and decyl-) for conversion of carbohydrate derivatives to species amenable for conjugation. Nevertheless, dimethyl squarate seemed to be the most convenient reagent because it is a crystalline, easy to handle, and commercially available material with very good reactivity.