NAADP+ is an agonist of the human P2Y11 purinergic receptor

Nicotinic acid adenine dinucleotide phosphate (NAADP+) is an intracellular second messenger releasing Ca2+ from intracellular stores in different cell types. In addition, it is also active in triggering [Ca2+](i) increase when applied extracellularly and various underlying mechanisms have been proposed. Here, we used hP2Y(11)-transfected 1321N1 astrocytoma cells to unequivocally establish whether extracellular NAADP+ is an agonist of the P2Y(11) receptor, as previously reported for beta-NAD+ [I. Moreschi, S. Bruzzone, R.A. Nicholas, et al., Extracellular NAD+ is an agonist of the human P2Y11 purinergic receptor in human granulocytes, J. Biol. Chem. 281 (2006) 31419-31429]. Extracellular NAADP+ triggered a concentration-dependent two-step elevation of [Ca2+](i) in 1321N1-hP2Y(11) cells, but not in wild-type 1321N1 cells, secondary to the intracellular production of IP(3), cAMP and cyclic ADP-ribose (cADPR). Specifically, the transient [Ca2+](i) rise proved to be related to IP(3) overproduction and to consequent Ca2+ mobilization, while the sustained [Ca2+](i) elevation was caused by the cAMP/ADP-ribosyl cyclase (ADPRC)/cADPR signalling cascade and by influx of extracellular Ca2+. In human granulocytes, endogenous P2Y(11) proved to be responsible for the NAADP+-induced cell activation (as demonstrated by the use of NF157, a selective and potent inhibitor of P2Y(11)), unveiling a role of NAADP+ as a pro-inflammatory cytokine. In conclusion, we provide unequivocal evidence for the activation of a member of the P2Y receptor subfamily by NAADP+.     

The sample processing time interval as an influential factor in flow cytometry analysis of lymphocyte subsets

The objective of this paper is to propose a protocol to analyze blood samples in yellow fever 17DD vaccinated which developed serious adverse events. We investigated whether or not the time between sample collection and sample processing could interfere in lymphocyte subset percentage, for it is often impossible to analyze blood samples immediately after collection due to transport delay from collection places to the flow cytometry facility. CD4+CD38+ T, CD8+CD38+ T, CD3+ T, CD19+ B lymphocyte subsets were analyzed by flow cytometry in nine healthy volunteers immediately after blood collection and after intervals of 24 and 48 h. The whole blood lysis method and gradient sedimentation by Histopaque were applied to isolate peripheral blood mononuclear cells for flow cytometry analyses. With the lysis method, there was no significant change in lymphocyte subset percentage between the two time intervals (24 and 48 h). In contrast, when blood samples were processed by Histopaque gradient sedimentation, time intervals for sample processing influenced the percentage in T lymphocyte subsets but not in B cells. From the results obtained, we could conclude that the whole blood lysis method is more appropriate than gradient sedimentation by Histopaque for immunophenotyping of blood samples collected after serious adverse events, due to less variation in the lymphocyte subset levels with respect to the time factor.     

Effect of hyperosmotic shock on phosphoenolpyruvate carboxykinase gene expression and gluconeogenic activity in the crab muscle

Chasmagnathus granulata phosphoenolpyruvate carboxykinase (PEPCK) cDNA from jaw muscle was cloned and sequenced, showing a specific domain to bind phosphoenolpyruvate in addition to the kinase-1 and kinase-2 motifs to bind guanosine triphosphate (GTP) and Mg(2+), respectively, specific for all PEPCKs. In the kinase-1 motifs the GK was changed to RK. The first 19 amino acids of the putative enzyme contain hydrophobic amino acids and hydroxylated residues specific to a mitochondrial type signal. The PEPCK is expressed in hepatopancreas, muscles, nervous system, heart, and gills. Hyperosmotic stress for 24 h increased the PEPCK mRNA level, gluconeogenic and PEPCK activities in muscle.     

Cytosolic and plastoglobule-targeted carotenoid dioxygenases from Crocus sativus are both involved in beta-ionone release

Saffron, the processed stigma of Crocus sativus, is characterized by the presence of several apocarotenoids that contribute to the color, flavor, and aroma of the spice. However, little is known about the synthesis of aroma compounds during the development of the C. sativus stigma. The developing stigma is nearly odorless, but before and at anthesis, the aromatic compound beta-ionone becomes the principal norisoprenoid volatile in the stigma. In this study, four carotenoid cleavage dioxygenase (CCD) genes, CsCCD1a, CsCCD1b, CsCCD4a, and CsCCD4b, were isolated from C. sativus. Expression analysis showed that CsCCD1a was constitutively expressed, CsCCD1b was unique to the stigma tissue, but only CsCCD4a and -b had expression patterns consistent with the highest levels of beta-carotene and emission of beta-ionone derived during the stigma development. The CsCCD4 enzymes were localized in plastids and more specifically were present in the plastoglobules. The enzymatic activities of CsCCD1a, CsCCD1b, and CsCCD4 enzymes were determined by Escherichia coli expression, and subsequent analysis of the volatile products was generated by GC/MS. The four CCDs fell in two phylogenetically divergent dioxygenase classes, but all could cleave beta-carotene at the 9,10(9',10') positions to yield beta-ionone. The data obtained suggest that all four C. sativus CCD enzymes may contribute in different ways to the production of beta-ionone. In addition, the location and precise timing of beta-ionone synthesis, together with its known activity as a fragrance and insect attractant, suggest that this volatile may have a role in Crocus pollination.     

Agroinjection of tomato fruits. A tool for rapid functional analysis of transgenes directly in fruit

Transient expression of foreign genes in plant tissues is a valuable tool for plant biotechnology. To shorten the time for gene functional analysis in fruits, we developed a transient methodology that could be applied to tomato (Solanum lycopersicum cv Micro Tom) fruits. It was found that injection of Agrobacterium cultures through the fruit stylar apex resulted in complete fruit infiltration. This infiltration method, named fruit agroinjection, rendered high levels of 35S Cauliflower mosaic virus-driven beta-glucuronidase and yellow fluorescence protein transient expression in the fruit, with higher expression levels around the placenta and moderate levels in the pericarp. Usefulness of fruit agroinjection was assayed in three case studies: (1) the heat shock regulation of an Arabidopsis (Arabidopsis thaliana) promoter, (2) the production of recombinant IgA antibodies as an example of molecular farming, and (3) the virus-induced gene silencing of the carotene biosynthesis pathway. In all three instances, this technology was shown to be efficient as a tool for fast transgene expression in fruits.     

Knowledge and use of medicinal plants by local specialists in an region of Atlantic Forest in the state of Pernambuco (Northeastern Brazil)

This paper investigates the wealth of medicinal plants used by the Apatani tribe of Arunachal Pradesh. Apatani have traditionally settled in seven villages in the Ziro valley of Lower Subansiri district of Arunachal Pradesh in the Eastern Himalayan region of India. The present study has resulted in the documentation of 158 medicinal plant species used by the Apatani group of villages. These medicinal plant species were distributed across 73 families and 124 genera. Asteraceae was the most dominant family (19 species, 11 genera) of medicinal plants, followed by Zingiberaceae, Solanaceae, Lamiaceae and Araceae. For curing ailments, the use of aboveground plant parts was higher (80%) than the belowground plant parts in the Apatani group of villages. Of the aboveground plant parts, leaf was used in the majority of cases (56 species), followed by fruit. Different belowground plant forms such as root, tuber, rhizome, bulb and pseudo-bulb were used by Apatani as a medicine. About 52 types of ailments were cured by using these 158 medicinal plant species. The results of this study are further discussed in the changing socio-economic contexts.     

Identification of the catalytic nucleophile of the family 29 alpha-L-fucosidase from Sulfolobus solfataricus via chemical rescue of an inactive mutant

We have recently reported that a functional alpha-L-fucosidase could be expressed by a single insertional mutation in the region of overlap between the ORFs SSO11867 and SSO3060 of the hyperthermophilic Archaeon Sulfolobus solfataricus [Cobucci-Ponzano et al. J. Biol. Chem. (2003) 278, 14622-14631]. This enzyme, belonging to glycoside hydrolase family 29 (GH29), showed micromolar specificity for p-nitrophenyl-alpha-L-fucoside (pNp-Fuc) and promoted transfucosylation reactions by following a reaction mechanism in which the products retained the anomeric configuration of the substrate. The active site residues in GH29 enzymes are still unknown. We describe here the identification of the catalytic nucleophile of the reaction in the alpha-L-fucosidase from S. solfataricus by reactivation with sodium azide of the mutant Asp242Gly that shows a 10(3)-fold activity reduction on pNp-Fuc. The detailed stereochemical analysis of the fucosyl-azide produced by the mutant reactivated on pNp-Fuc revealed its inverted (beta-fucosyl azide) configuration compared with the substrate. This allows for the first time the unambiguous assignment of Asp242, and its homologous residues, as the nucleophilic catalytic residues of GH29 alpha-L-fucosidases. This is the first time that this approach is used for alpha-L-glycosidases, widening the applicability of this method.     

ABCG transporters export cutin precursors for the formation of the plant cuticle

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