New aspects concerning to the characterization and the relationship with the immune response in vivo of the spiny lobster Panulirus argus nitric oxide synthase.

Nitric oxide (NO) is a short-lived radical generated by nitric oxide synthases (NOS). NO is involved in a variety of functions in invertebrates, including host defense. In a previous study, we isolated and sequenced for the first time the NOS gene from hemocytes of Panulirus argus, demonstrating the inducibility of this enzyme by lipopolysaccharide (LPS) in vitro. In the present work, lobster hemocytes and gills exposed to Escherichia coli O55:B5 LPS showed an increase in both NOS activity and NOS gene expression in vivo. This response was dose and time dependent. The 3D NOS structure was predicted by comparative modeling showing the oxygenase and reductase domains. These domains contain the conserved binding motifs of NOS already found in a variety of organisms. The 3D structure prediction analysis allowed the selection of a fragment of 666bp that was cloned and subsequently expressed in E. coli BL21, in which a recombinant product of around 31KDa was obtained. Hyperimmune serum obtained from immunized rabbits was tested and employed to specifically detect the recombinant polypeptide or the endogenous NOS from lobster hemocytes by western blot and immunofluorescence. This study contributes to enlarge the existing knowledge related to NOS structure and NOS participation in the immune response in lobsters. The evaluation of an antibody capable to recognize NOS from lobsters constitutes a novel and interesting tool for the implementation of further studies on NOS functions in crustaceans.

NOA36/ZNF330 is a conserved cystein-rich protein with proapoptotic activity in human cells.

Translocations of regulator proteins from or to the mitochondria are key events in apoptosis regulation. NOA36/ZNF330 is a highly evolutionary conserved protein with a characteristic cystein-rich domain. In this work we address its mitochondrial localization and we demonstrate that a blockage of endogenous NOA36/ZNF330 expression by small-interfering RNA (siRNA) reduced apoptotic response to etoposide (ETO), camptothecin (CPT) and staurosporine (STS) but not to CH11 anti-Fas antibody or tumour-necrosis-factor-related apoptosis-inducing ligand (TRAIL) in HeLa cells. In contrast, when ectopically expressed in the cytoplasm, NOA36/ZNF330 induces apoptotic cell death. We also found that the domain responsible for this proapoptotic activity is located its cystein-rich region. We propose that NOA36/ZNF330 is translocated from the mitochondria to the cytoplasm when apoptosis is induced and that it contributes to cytochrome c release.

Stemphylium Leaf Blight of Garlic (Allium sativum ) in Spain: Taxonomy and In Vitro Fungicide Response.

The most serious aerial disease of garlic is leaf blight caused by Stemphylium spp. Geographical variation in the causal agent of this disease is indicated. Stemphylium vesicarium has been reported in Spain, whereas S. solani is the most prevalent species recorded in China. In this study, Stemphylium isolates were obtained from symptomatic garlic plants sampled from the main Spanish production areas. Sequence data for the ITS1-5.8S-ITS2 region enabled assignation of the isolates to the Pleospora herbarum complex and clearly distinguished the isolates from S. solani. Conidial morphology of the isolates corresponded to that of S. vesicarium and clearly discriminated them from S. alfalfae and S. herbarum on the basis of the size and septation pattern of mature conidia. Conidial morphology as well as conidial length, width and length:width ratio also allowed the Spanish isolates to be distinguished from S. botryosum and S. herbarum. Control of leaf blight of garlic is not well established. Few studies are available regarding the effectiveness of chemical treatments to reduce Stemphylium spp. incidence on garlic. The effectiveness of nine fungicides of different chemical groups to reduce Stemphylium mycelial growth in vitro was tested. Boscalid + pyraclostrobin (group name, succinate dehydrogenase inhibitors + quinone outside inhibitors), iprodione (dicar-boximide), and prochloraz (demethylation inhibitors) were highly effective at reducing mycelial growth in S. vesicarium with EC50 values less than 5 ppm. In general, the effectiveness of the fungicide was enhanced with increasing dosage.

NOA36 protein contains a highly conserved nucleolar localization signal capable of directing functional proteins to the nucleolus, in mammalian cells.

NOA36/ZNF330 is an evolutionarily well-preserved protein present in the nucleolus and mitochondria of mammalian cells. We have previously reported that the pro-apoptotic activity of this protein is mediated by a characteristic cysteine-rich domain. We now demonstrate that the nucleolar localization of NOA36 is due to a highly-conserved nucleolar localization signal (NoLS) present in residues 1-33. This NoLS is a sequence containing three clusters of two or three basic amino acids. We fused the amino terminal of NOA36 to eGFP in order to characterize this putative NoLS. We show that a cluster of three lysine residues at positions 3 to 5 within this sequence is critical for the nucleolar localization. We also demonstrate that the sequence as found in human is capable of directing eGFP to the nucleolus in several mammal, fish and insect cells. Moreover, this NoLS is capable of specifically directing the cytosolic yeast enzyme polyphosphatase to the target of the nucleolus of HeLa cells, wherein its enzymatic activity was detected. This NoLS could therefore serve as a very useful tool as a nucleolar marker and for directing particular proteins to the nucleolus in distant animal species.

Corynebacterium macginleyi isolated from urine in a patient with a permanent bladder catheter.

An 82-y-old male patient with a neurogenic bladder and vesical stones presented with a urinary tract infection caused by Corynebacterium macginleyi. This is the first case of isolation of C. macginleyi from a non-conjunctival specimen. The patient recovered fully with antimicrobial treatment.