Expression of the plant sulphite reductase in cell suspension cultures from Catharanthus roseus L.

Sulphur-heterotrophic growth exhibited a dual response to the expression of sulphate-assimilating enzymes. The level of ATP-sulphurylase (EC 2.7.7.4) appeared repressed while sulphite reductase (EC 1.8.7.1) and O-acetyl-l-serine sulphhydrylase (EC 4.2.99.8) were derepressed and coordinated in their occurrence. The capability of the cells to reduce adenylylphosphosulphate or 3-phospho adenylylphosphosulphate to cysteine coincided with the activity of sulphite reductase. The expression of these reducing steps lacked correlation with the regulation of ATP-sulphurylase.Abbreviations APS adenylylphosphosulphate - MVH reduced methylviologen - OAS O-acetyl-l-serine - PAPS 3-phospho adenylylphosulphate     

Development of cell surface saccharides on embryonic pancreatic cells

Using a battery of seven lectin-ferritin conjugates as probes for cell surface glycoconjugates, we have studied the pattern of plasmalemmal differentiation of cells in the embryonic rat pancreas from day 15 in utero to the early postpartum stage. Our results indicate that differentiation of plasmalemmal glycoconjugates on acinar, endocrine, and centroacinar cells is temporally correlated with development and is unique for each cell type, as indicated by lectin-ferritin binding. Specifically, (a) expression of adult cell surface saccharide phenotype can be detected on presumptive acinar cells as early as 15 d in utero, as indicated by soybean agglutinin binding, and precedes development of intracellular organelles characteristic of mature acinar cells; (b) maturation of the plasmalemma of acinar cells is reached after intracellular cytodifferentiation is completed, as indicated by appearance of Con A and fucoselectin binding sites only at day 19 of development; conversely, maturation of the endocrine cell plasmalemma is accompanied by "loss" (masking) of ricinus communis II agglutinin receptors; and (c) binding sites for fucose lectins and for soybean agglutinin are absent on endocrine and centroacinar cells at all stages examined. We conclude that acinar, centroacinar, and endocrine cells develop from a common progenitor cell(s) whose plasmalemmal carbohydrate composition resembles most closely that of the adult centroacinar cell. Finally, appearance of acinar lumina beginning at approximately 17 d in utero is accompanied by differenetiation of apical and basolateral plasmalemmal domains of epithelial cells, as indicated by enhanced binding of several lectin-ferritin conjugates to the apical plasmalemmal, a pattern that persists from this stage through adult life.     

Age-Specific Mortality During the 1918 Influenza Pandemic: Unravelling the Mystery of High Young Adult Mortality

The worldwide spread of a novel influenza A (H1N1) virus in 2009 showed that influenza remains a significant health threat, even for individuals in the prime of life. This paper focuses on the unusually high young adult mortality observed during the Spanish flu pandemic of 1918. Using historical records from Canada and the U.S., we report a peak of mortality at the exact age of 28 during the pandemic and argue that this increased mortality resulted from an early life exposure to influenza during the previous Russian flu pandemic of 1889–90. We posit that in specific instances, development of immunological memory to an influenza virus strain in early life may lead to a dysregulated immune response to antigenically novel strains encountered in later life, thereby increasing the risk of death. Exposure during critical periods of development could also create holes in the T cell repertoire and impair fetal maturation in general, thereby increasing mortality from infectious diseases later in life. Knowledge of the age-pattern of susceptibility to mortality from influenza could improve crisis management during future influenza pandemics.

“The war is over – and I must go” Egon Schiele, 1890–1918.

     

Molecular and catalytic properties of Arabidopsis thaliana adenylyl sulfate (APS)-kinase

A cDNA clone (Atakn1) from Arabidopsis thaliana encoding APS-kinase (EC 2.7.1.25) was investigated for structural and catalytic properties of the gene product. Recombinant his10-AtAkn1 formed PAPS at a Vmax of 7.35 U x mg(-1). The Km for APS was 0.14 microM and for ATP 147 microM. APS caused a severe substrate inhibition (K(i) 4.5 microM). The type of inhibition is uncompetitive with respect to MgATP. High ionic strength and reducing thiols stabilized the enzyme activity. Plant APS-kinase is regulated in vitro by the redox charge with thioredoxin as essential activator. Mutagenesis of a serine in S182C and S182F presumed to be involved in the transfer of the phosphoryl group had no effect upon catalytic activity. Using a yeast two-hybrid system with AtAkn1 as bait, an interacting clone was detected from a cDNA library of A. thaliana cv. Columbia that codes for an APS-kinase iso-form (Atakn2). Complementation of APS-kinase-deficient Saccharomyces cerevisiae met14 showed that AtAkn2 is functionally active as APS-kinase. It was immunologically related to AtAkn1 and presumably represents a plastidal iso-form of the plant APS-kinase gene family.     

The cover of living and dead corals from airborne remote sensing

Trends in coral cover are widely used to indicate the health of coral reefs but are costly to obtain from field survey over large areas. In situ studies of reflected spectra at the coral surface show that living and recently dead colonies can be distinguished. Here, we investigate whether such spectral differences can be detected using an airborne remote sensing instrument. The Compact Airborne Spectrographic Imager (Itres Research Ltd, Canada) was flown in two configurations: 10 spectral bands with 1-m² pixels and 6 spectral bands with 0.25-m² pixels. First, we show that an instrument with 10 spectral bands possesses adequate spectral resolution to distinguish living Porites, living Pocillopora spp., partially dead Porites, recently dead Porites (total colony mortality within 6 months), old dead (>6 months) Porites, Halimeda spp., and coralline red algae when there is no water column to confuse spectra. All substrata were distinguished using fourth-order spectral derivatives around 538 nm and 562 nm. Then, at a shallow site (Tivaru) at Rangiroa Atoll, Tuamotu Archipelago (French Polynesia), we show that live and dead coral can be distinguished from the air to a depth of at least 4 m using first- and fourth-order spectral derivatives between 562–580 nm. However, partially dead and recently dead Porites colonies could not be distinguished from an airborne platform. Spectral differences among substrata are then exploited to predict the cover of reef substrata in ten 25-m² plots at nearby Motu Nuhi (max depth 8 m). The actual cover in these plots was determined in situ using quadrats with a 0.01-m² grid. Considerable disparity occurred between field and image-based measures of substrate cover within individual 25-m² quadrats. At this small scale, disparity, measured as the absolute difference in cover between field and remote-sensing methods, reached 25% in some substrata but was always less than 10% for living coral (99% of which consisted of Porites spp.). At the scale of the reef (all ten 25-m² quadrats), however, disparities in percent cover between imagery and field data were less than 10% for all substrata and extremely low for some classes (e.g. <3% for living Porites, recently dead Porites and Halimeda). The least accurately estimated substrata were sand and coralline red algae, which were overestimated by absolute values 7.9% and 6.6%, respectively. The precision of sampling was similar for field and remote-sensing methods: field methods required 19 plots to detect a 10% difference in coral cover among three reefs with a statistical power of 95%. Remote-sensing methods required 21 plots. However, it took 1 h to acquire imagery over 92,500 m² of reef, which represents 3,700 plots of 25 m² each, compared with 3 days to survey 10 such plots underwater. There were no significant differences in accuracy between 1-m² and 0.25-m² image resolutions, suggesting that the advantage of using smaller pixels is offset by reduced spectral information and an increase in noise (noise was observed to be 1.6–1.8 times greater in 0.25-m² pixels). We show that airborne remote sensing can be used to monitor coral and algal cover over large areas, providing that water is shallow and clear, and that brown fleshy macroalgae are scarce, that depth is known independently (e.g. from sonar survey).     

Gene transfer to pre-hematopoietic and committed hematopoietic precursors in the early mouse Yolk Sac: a comparative study between <Emphasis Type="Italic">in situ</Emphasis> electroporation and retroviral transduction

Background  

Hematopoietic development in vertebrate embryos results from the sequential contribution of two pools of precursors independently generated. While intra-embryonic precursors harbour the features of hematopoietic stem cells (HSC), precursors formed earlier in the yolk sac (YS) display limited differentiation and self-renewal potentials. The mechanisms leading to the generation of the precursors in both sites are still largely unknown, as are the molecular basis underlying their different potential. A possible approach to assess the role of candidate genes is to transfer or modulate their expression/activity in both sites. We thus designed and compared transduction protocols to target either native extra-embryonic precursors, or hematopoietic precursors.     

European society of intensive care medicine study of therapeutic hypothermia (32-35°C) for intracranial pressure reduction after traumatic brain injury (the Eurotherm3235Trial)

Background

Severe malaria remains a major cause of global morbidity and mortality. Despite the use of potent anti-parasitic agents, the mortality rate in severe malaria remains high. Adjunctive therapies that target the underlying pathophysiology of severe malaria may further reduce morbidity and mortality. Endothelial activation plays a central role in the pathogenesis of severe malaria, of which angiopoietin-2 (Ang-2) has recently been shown to function as a key regulator. Nitric oxide (NO) is a major inhibitor of Ang-2 release from endothelium and has been shown to decrease endothelial inflammation and reduce the adhesion of parasitized erythrocytes. Low-flow inhaled nitric oxide (iNO) gas is a US FDA-approved treatment for hypoxic respiratory failure in neonates.

Methods/Design

This prospective, parallel arm, randomized, placebo-controlled, blinded clinical trial compares adjunctive continuous inhaled nitric oxide at 80 ppm to placebo (both arms receiving standard anti-malarial therapy), among Ugandan children aged 1-10 years of age with severe malaria. The primary endpoint is the longitudinal change in Ang-2, an objective and quantitative biomarker of malaria severity, which will be analysed using a mixed-effects linear model. Secondary endpoints include mortality, recovery time, parasite clearance and neurocognitive sequelae.

Discussion

Noteworthy aspects of this trial design include its efficient sample size supported by a computer simulation study to evaluate statistical power, meticulous attention to complex ethical issues in a cross-cultural setting, and innovative strategies for safety monitoring and blinding to treatment allocation in a resource-constrained setting in sub-Saharan Africa.

Trial Registration

ClinicalTrials.gov Identifier: NCT01255215     

Characterization and reconstitution of a 4Fe-4S adenylyl sulfate/phosphoadenylyl sulfate reductase from Bacillus subtilis

CysH1 from Bacillus subtilis encodes a 3'-phospho/adenosine-phosphosulfate-sulfonucleotide reductase (SNR) of 27 kDa. Recombinant B. subtilis SNR is a homodimer, which is bispecific and reduces adenylylsulfate (APS) and 3'-phosphoadenylylsulfate (PAPS) alike with thioredoxin 1 or with glutaredoxin 1 as reductants. The enzyme has a higher affinity for PAPS (K(m)PAPS 6.4 microm Trx-saturating, 10.7 microm Grx-saturating) than for APS (K(m) APS 28.7 microm Trx-saturating, 105 microm Grx-saturating) at a V(max) ranging from 280 to 780 nmol sulfite mg(-1) min(-1). The catalytic efficiency with PAPS as substrate is higher by a factor of 10 (K(cat)/K(m) 2.7 x 10(4)-3.6 x 10(4) liter mol(-1) s(-1). B. subtilis SNR contains one 4Fe-4S cluster per polypeptide chain. SNR activity and color were lost rapidly upon exposure to air or upon dilution. M?ssbauer and absorption spectroscopy revealed that the enzyme contained a 4Fe-4S cluster when isolated, but degradation of the 4Fe-4S cluster produced an inactive intermediate with spectral properties of a 2Fe-2S cluster. Activity and spectral properties of the 4Fe-4S cluster were restored by preincubation of SNR with the iron-sulfur cluster-assembling proteins IscA1 and IscS. Reconstitution of the 4Fe-4S cluster of SNR did not affect the reductive capacity for PAPS or APS. The interconversion of the clusters is thought to serve as oxygen-sensitive switch that suppresses SO(3) formation under aerobiosis.     

The distribution and evolutionary history of the PRP8 intein

Background  

We recently described a mini-intein in the PRP8 gene of a strain of the basidiomycete Cryptococcus neoformans, an important fungal pathogen of humans. This was the second described intein in the nuclear genome of any eukaryote; the first nuclear encoded intein was found in the VMA gene of several saccharomycete yeasts. The evolution of eukaryote inteins is not well understood. In this report we describe additional PRP8 inteins (bringing the total of these to over 20). We compare and contrast the phylogenetic distribution and evolutionary history of the PRP8 intein and the saccharomycete VMA intein, in order to derive a broader understanding of eukaryote intein evolution. It has been suggested that eukaryote inteins undergo horizontal transfer and the present analysis explores this proposal.