In vivo testing of a tobacco plastid DNA segment for guide RNA function in psbL editing

A C-to-U RNA editing event creates a functional initiation codon for translation of the psbL mRNA in tobacco plastids. Small trans-acting guide RNAs (gRNAs) have been shown to be involved in editing site selection in kinetoplastid mitochondria. A computer search of the tobacco plastid genome (ptDNA) identified such a putative gRNA, a 14-nucleotide sequence motif that is complementary to the psbL mRNA, including the A nucleotide required to direct the C-to-U change. The critical A nucleotide of the putative gRNA gene was changed to G by plastid transformation. We report here that the introduced mutation did not abolish psbL editing. Since no other region of the plastid genome contains significant complementarity to the psbL editing site we suggest that, if gRNAs serve as trans-acting factors for plastid psbL mRNA editing, they either have only a limited complementarity to the editing site, or are encoded in the nuclear genome.     

<Emphasis Type="Italic">Sacculospora felinovii</Emphasis>, a novel arbuscular mycorrhizal fungal species (Glomeromycota) from dunes on the west coast of India

During an arbuscular mycorrhiza fungal spore survey on a primary coastal sand-dune system in Goa on the west coast of India, entrophosporoid spores tightly covered with a dense hyphal mantle were recovered. When intact, the spores, at first sight, seemed to be identical in morphology to those of Sacculospora baltica (originally described as Entrophospora baltica) extracted from Polish maritime sand dunes and, to date, the sole member of the recently described genus Sacculospora in the new family Sacculosporaceae, phylum Glomeromycota. Later detailed morphological studies indicated that both fungi produce two-walled spores but the structure and phenotypic features of components of the outer spore wall in the novel fungus differ considerably from those of S. baltica. Differences between the fungi were subsequently confirmed in the phylogenetic analysis of SSU–ITS–LSU nrDNA sequences. Consequently, we describe the novel species as Sacculospora felinovii sp. nov.     

Introduction of a heterologous editing site into the tobacco plastid genome: the lack of RNA editing leads to a mutant phenotype.

The psbF mRNA is edited in spinach plastids by a C to U conversion, changing a serine to a conserved phenylalanine codon. In tobacco at this position a phenylalanine codon is present at the DNA level, and the psbF mRNA here is not edited. To test if the psbF editing capacity is evolutionarily conserved, the tobacco psbF gene was modified to match the corresponding spinach sequence. The endogenous tobacco gene was replaced with the modified copy using biolistic transformation. We report here that the heterologous editing site remains unmodified in transplastomic tobacco plants. The lack of editing is associated with slower growth, lowered chlorophyll content and high chlorophyll fluorescence, a phenotype characteristic of photosynthetic mutants. This finding confirms that the editing of the psbF mRNA is an essential processing step for protein function and thus provides direct proof for the biological significance of plant organellar RNA editing. Given that a mutant phenotype is associated with the lack of editing, it seems likely that the evolutionary loss of the site-specific capacity for psbF editing was preceded by the mutation that eliminated the editing requirement.     

The preparation of some bromodeoxy- and dibromodideoxy-pentonolactones

Brief reaction of d-lyxono-1,4-lactone (1) with hydrogen bromide in acetic acid (HBA) yields 2-bromo-2-deoxy-d-xylono-1,4-lactone (2), and a similar treatment of d-ribono-1,4-lactone (8) gives 2-bromo-2-deoxy-d-arabinono-1,4-lactone (12). On longer reaction with HBA, 1 is converted into 2,5-dibromo-2,5-dideoxy-d-xylono-1,4-lactone, whereas 8 forms a mixture of 2,5-dibromolactones. Reduction of 2 and 12 gives 2-bromo-2-deoxy-d-xylose and -d-arabinose, respectively. On hydrogenolysis, 2 and 12 are converted into 2-deoxy-d-threo- and 2-deoxy-d-erythro-pentono-1,4-lactone, respectively. The 2,5-dibromolactones can be selectively hydrogenolysed to 5-bromo-2,5-dideoxy-d-pentono-1,4-lactones.     

Mapping of Schistosomiasis and Soil-Transmitted Helminths in Namibia: The First Large-Scale Protocol to Formally Include Rapid Diagnostic Tests

Background

Namibia is now ready to begin mass drug administration of praziquantel and albendazole against schistosomiasis and soil-transmitted helminths, respectively. Although historical data identifies areas of transmission of these neglected tropical diseases (NTDs), there is a need to update epidemiological data. For this reason, Namibia adopted a new protocol for mapping of schistosomiasis and geohelminths, formally integrating rapid diagnostic tests (RDTs) for infections and morbidity. In this article, we explain the protocol in detail, and introduce the concept of ‘mapping resolution’, as well as present results and treatment recommendations for northern Namibia.

Methods/Findings/Interpretation

This new protocol allowed a large sample to be surveyed (N = 17 896 children from 299 schools) at relatively low cost (7 USD per person mapped) and very quickly (28 working days). All children were analysed by RDTs, but only a sub-sample was also diagnosed by light microscopy. Overall prevalence of schistosomiasis in the surveyed areas was 9.0%, highly associated with poorer access to potable water (OR = 1.5, P<0.001) and defective (OR = 1.2, P<0.001) or absent sanitation infrastructure (OR = 2.0, P<0.001). Overall prevalence of geohelminths, more particularly hookworm infection, was 12.2%, highly associated with presence of faecal occult blood (OR = 1.9, P<0.001). Prevalence maps were produced and hot spots identified to better guide the national programme in drug administration, as well as targeted improvements in water, sanitation and hygiene. The RDTs employed (circulating cathodic antigen and microhaematuria for Schistosoma mansoni and S. haematobium, respectively) performed well, with sensitivities above 80% and specificities above 95%.

Conclusion/Significance

This protocol is cost-effective and sensitive to budget limitations and the potential economic and logistical strains placed on the national Ministries of Health. Here we present a high resolution map of disease prevalence levels, and treatment regimens are recommended.     

Role of the streptokinase alpha-domain in the interactions of streptokinase with plasminogen and plasmin

The role of the streptokinase (SK) alpha-domain in plasminogen (Pg) and plasmin (Pm) interactions was investigated in quantitative binding studies employing active site fluorescein-labeled [Glu]Pg, [Lys]Pg, and [Lys]Pm, and the SK truncation mutants, SK-(55-414), SK-(70-414), and SK-(152-414). Lysine binding site (LBS)-dependent and -independent binding were resolved from the effects of the lysine analog, 6-aminohexanoic acid. The mutants bound indistinguishably, consistent with unfolding of the alpha-domain on deletion of SK-(1-54). The affinity of SK for [Glu]Pg was LBS-independent, and although [Lys]Pg affinity was enhanced 13-fold by LBS interactions, the LBS-independent free energy contributions were indistinguishable. alpha-Domain truncation reduced the affinity of SK for [Glu]Pg 2-7-fold and [Lys]Pg 相似文献   

Neuroplastin-65 and a mimetic peptide derived from its homophilic binding site modulate neuritogenesis and neuronal plasticity

Neuroplastin-65 (Np65) is a brain-specific cell adhesion molecule belonging to the immunoglobulin superfamily. Homophilic trans-interaction of Np65 mediates adhesion between cells and modulates synaptic plasticity. This interaction solely occurs through the first immunoglobulin (Ig) module of Np65, but the exact binding mechanism has not yet been elucidated. In this study, we identify the homophilic binding motif of Np65 and show that a synthetic peptide modeled after this motif, termed enplastin, binds to Np65. We demonstrate that both Np65- and enplastin-induced intracellular signaling depends on fibroblast growth factor receptor, p38 mitogen-activated protein kinase, Ca(2+) /calmodulin-dependent protein kinase, and cytoplasmic Ca(2+) concentration. In addition, we show that interference with Np65 homophilic binding by enplastin has an inhibitory effect on Np65-mediated neurite outgrowth in vitro and on the initial phase of spatial learning in rats.     

Antithrombin III Utah: proline-407 to leucine mutation in a highly conserved region near the inhibitor reactive site

A dysfunctional antithrombin III (ATIII) gene encoding a qualitatively and quantitatively abnormal anticoagulant molecule is responsible for hereditary thrombosis in a Utah kindred [Bock et al. (1985) Am. J. Hum. Genet. 37, 32-41]. Nucleotide sequencing of the entire protein-encoding portion of the cloned ATIII-Utah gene revealed a C to T transitional mutation which converts proline-407 to leucine. Proline-407 is located 14 amino acids C-terminal to the reactive site arginine of ATIII in a core region of the molecule that has been highly conserved during evolution of the serine protease inhibitor (serpin) gene family. The location of this proline in the crystal structure of the homologous serpin alpha 1-antitrypsin suggests that the leucine substitution in ATIII-Utah may interfere with correct folding of the mutant gene product, leading to its rapid turnover and the low antithrombin levels observed in patient plasmas. The Pro-407 to Leu mutation does not interfere with binding of antithrombin III to heparin. Patient antithrombin III, isolated by affinity chromatography on heparin-Sepharose, was reacted with purified thrombin. ATIII encoded by the patient's normal gene formed protease-inhibitor complexes with thrombin, whereas the product of the ATIII-Utah gene did not. The Pro-407 to Leu mutation destroys a restriction site for the enzyme StuI, permitting rapid diagnosis of affected members of the Utah kindred by Southern blotting of genomic DNA.