Characterization and purification of a calcium-sensitive ATP diphosphohydrolase from pig pancreas

An ATP diphosphohydrolase (EC 3.6.1.5) from the pancreas of the pig has been characterized and purified. The enzyme which has an optimum pH between 8 and 9 is specific for diphospho- and triphosphonucleosides. The Km values for ADP and ATP are 7.4 and 7.3 x 10(-4) M, respectively, and the purified enzyme has specific activities of 13 and 15.2 mumol of Pi/min/m of protein, respectively. It requires calcium or magnesium ions and it is insensitive to ATPase inhibitors, namely oligomycin, ouabain, and ruthenium red, and to levamisole, an inhibitor of alkaline phosphatase. Denaturation experiments, by heat and trypsin treatments, indicated that only one enzyme is involved. This is confirmed by the solubilization and purification process and by polyacrylamide gel electrophoresis. A 270-fold purification was obtained by centrifugation and successive column chromatography on Sepharose 4B and Affi-Gel blue. It is a glycoprotein with a molecular weight of 65,000 as estimated by polyacrylamide gel electrophoresis.     

Members of the Arabidopsis FAE1-like 3-ketoacyl-CoA synthase gene family substitute for the Elop proteins of Saccharomyces cerevisiae

Several 3-keto-synthases have been studied, including the soluble fatty acid synthases, those involved in polyketide synthesis, and the FAE1-like 3-ketoacyl-CoA synthases. All of these condensing enzymes have a common ancestor and an enzymatic mechanism that involves a catalytic triad consisting of Cys, His, and His/Asn. In contrast to the FAE1-like family of enzymes that mediate plant microsomal fatty acid elongation, the condensation step of elongation in animals and in fungi appears to be mediated by the Elop homologs. Curiously these proteins bear no resemblance to the well characterized 3-keto-synthases. There are three ELO genes in yeast that encode the homologous Elo1p, Elo2p, and Elo3p proteins. Elo2p and Elo3p are required for synthesis of the very long-chain fatty acids, and mutants lacking both Elo2p and Elo3p are inviable confirming that the very long-chain fatty acids are essential for cellular functions. In this study we show that heterologous expression of several Arabidopsis FAE1-like genes rescues the lethality of an elo2Deltaelo3Delta yeast mutant. We further demonstrate that FAE1 acts in conjunction with the 3-keto and trans-2,3-enoyl reductases of the elongase system. These studies indicate that even though the plant-specific FAE1 family of condensing enzymes evolved independently of the Elop family of condensing enzymes, they utilize the same reductases and presumably dehydratase that the Elop proteins rely upon.     

The sunflower plastidial ω3-fatty acid desaturase (HaFAD7) contains the signalling determinants required for targeting to, and retention in, the endoplasmic reticulum membrane in yeast but requires co-expressed ferredoxin for activity

Although plant plastidial ω3-desaturases are closely related to microsomal desaturases, heterologous expression in yeast of the Helianthus annuus FAD7 ω3-desaturase showed low activity in contrast to similar expression of microsomal FAD3 ω3-desaturases. However, the removal of the plastidial transit peptide and the incorporation of a KKNL motif to the C-terminus of HaFAD7 increased the activity by 10-fold compared to the native protein. N-terminal fusion of transmembrane-domains from either the yeast microsomal ELO3, (a type III signal anchor domain), or FAE1, an endoplasmic reticulum membrane anchoring domain, resulted in moderate increases in enzyme activity (5- and 7-fold, respectively), suggesting that the first, most hydrophobic transmembrane domain of HaFAD7 is sufficient to direct targeting to, and insertion into, the endoplasmic reticulum membrane. Furthermore, fusing a hemagglutinin (HA) epitope tag upstream of an endogenous C-terminal KEK motif resulted in a significant loss of activity compared to the un-tagged construct, indicating that the endogenous KEK C-terminal di-lysine motif is capable of directing in yeast the ER-retention of this normally plastidial-located protein. Western blotting analysis of constructs with internal HA epitope revealed that in whole cell extracts, with the exception of the one bound to C-terminal, it did not display a reduced level of protein accumulation. Whilst ferredoxin was shown to be required for HaFAD7 activity in yeast, it appears not necessary for protein stability and accumulation of this plastidial desaturase in the endoplasmic reticulum.     

Scabies and Bacterial Superinfection among American Samoan Children, 2011–2012

BackgroundScabies, a highly pruritic and contagious mite infestation of the skin, is endemic among tropical regions and causes a substantial proportion of skin disease among lower-income countries. Delayed treatment can lead to bacterial superinfection, and treatment of close contacts is necessary to prevent reinfestation. We describe scabies incidence and superinfection among children in American Samoa (AS) to support scabies control recommendations.Conclusions/SignificanceScabies and its sequelae cause substantial morbidity among AS children. Bacterial superinfection prevalence and frequent reinfestations highlight the importance of diagnosing scabies and early treatment of patients and close contacts. Investigating why certain AS counties have a lower scabies incidence might help guide recommendations for improving scabies control among counties with a higher incidence. We recommend interventions targeting infants and young children who have frequent close family contact.     

Identity of epibiotic bacteria on symbiontid euglenozoans in O2-depleted marine sediments: evidence for symbiont and host co-evolution

A distinct subgroup of euglenozoans, referred to as the ‘Symbiontida,'' has been described from oxygen-depleted and sulfidic marine environments. By definition, all members of this group carry epibionts that are intimately associated with underlying mitochondrion-derived organelles beneath the surface of the hosts. We have used molecular phylogenetic and ultrastructural evidence to identify the rod-shaped epibionts of the two members of this group, Calkinsia aureus and B.bacati, hand-picked from the sediments of two separate oxygen-depleted, sulfidic environments. We identify their epibionts as closely related sulfur or sulfide-oxidizing members of the epsilon proteobacteria. The epsilon proteobacteria generally have a significant role in deep-sea habitats as primary colonizers, primary producers and/or in symbiotic associations. The epibionts likely fulfill a role in detoxifying the immediate surrounding environment for these two different hosts. The nearly identical rod-shaped epibionts on these two symbiontid hosts provides evidence for a co-evolutionary history between these two sets of partners. This hypothesis is supported by congruent tree topologies inferred from 18S and 16S rDNA from the hosts and bacterial epibionts, respectively. The eukaryotic hosts likely serve as a motile substrate that delivers the epibionts to the ideal locations with respect to the oxic/anoxic interface, whereby their growth rates can be maximized, perhaps also allowing the host to cultivate a food source. Because symbiontid isolates and additional small subunit rDNA gene sequences from this clade have now been recovered from many locations worldwide, the Symbiontida are likely more widespread and diverse than presently known.     

Pathways of secretion in the exocrine pancreas: the status of resting secretion

In the last decade the concept of two distinct pathways of secretion in the exocrine pancreas has slowly emerged. According to this concept, one pathway is involved in stimulated (regulated) conditions and another under resting (constitutive) conditions. This hypothesis was elaborated at first from the comparison of the specific radioactivities of secretory proteins released by the gland under resting and stimulated conditions. Analysis of the protein composition of the juice released under these two physiological conditions further supported that hypothesis. More recent studies compared the kinetic of accumulation of newly synthesized proteins in zymogen granule and their release in the gland lumen. The latter results are in agreement with a model in which secretory proteins are channelled in two separate pathways, one regulated, and one constitutive. Essentially, the constitutive pathway would correspond to a paragranular route in which the proteins would be immediately secreted instead of being stored in zymogen granules. In addition, some of the proteins released in the juice under "resting" conditions are associated to microvesicles. The term "microvesicular secretion" is used to designate that type of secretion.     

A convenient method for the ATPase assay.

A new method for the determination of inorganic phosphorus released in ATPase assay has been evaluated. The method is based on the reduction of a phosphomolybdate complex by Elon in a copper acetate buffer. In contrast to current methods, there is no interference by ATP with color development. There is also less or no interference by other compounds usually present in ATPase assay media. The method is simple, sensitive, and reproducible.     

Guidelines for indications for coronary revascularization in The Netherlands

In 1996 the Minister of Public Health, Welfare and Sports in The Netherlands published a 'Planning Decree Special Interventions in the Heart'. She requested from the professional organizations guidelines for the indications for interventions in the heart. A working group was formed with representatives from the Dutch professional organizations for cardiology and thoracic surgery, to address this issue for patients with coronary artery disease. The working group confirmed the need to discuss all patients who are considered for either elective or emergency revascularization during a multidisciplinary consultation in (or with) one of the specialized Dutch hospitals. During this meeting of the 'heart team', at least one interventional cardiologist and one thoracic surgeon should be present. There are three possible outcomes of the heart team's consultations for each patient: drug therapy only ('conservative management'), coronary surgery or catheter intervention. For each case, the team should indicate the expected benefit, the risk of the intervention, the urgency and the estimated waiting time. The guidelines presented in this paper address these issues for three patient categories: stable angina pectoris, unstable angina pectoris and acute myocardial infarction.     

Isolation of symbiotically expressed genes from the dinoflagellate symbiont of the solitary radiolarian Thalassicolla nucleata

Symbiotic associations are fundamental to the survival of many organisms on Earth. The ability of the symbiont to perform key biochemical functions often allows the host to occupy environments that it would otherwise find inhospitable. This can have profound impacts upon the diversification and distribution of the host. Cellular organelles (chloroplasts and mitochondria) represent the final stages of integration of endosymbionts. These organelles were of critical importance to the evolution and success of eukaryotic lineages on our planet because they allowed the host cells to harness light energy and to thrive in the presence of oxygen. The marine photosymbiotic associations that we study represent an earlier stage in the process of symbiont integration-one in which the photobiont can still be removed from the host and exist on its own. These systems are of interest to us for two reasons. First, they are ecologically important in the marine environment where they occur. These organisms form zones of photosynthetic production in oceanic regions typically low in nutrients. Second, investigation of these interactions may shed light on the molecular and evolutionary mechanisms involved in the integration of cells and their genomes.