Nucleoside triphosphate synthesis catalysed by adenylate kinase is ADP dependent

Adenylate kinase (Adk) that catalyses the synthesis of ADP from ATP and AMP has also been shown to perform an ATP dependent phosphorylation of ribo- and deoxynucleoside diphosphates to their corresponding nucleoside triphosphate; ATP+(d)NDP<-->ADP+(d)NTP. This reaction, suggested to occur by the transfer of the gamma-phosphoryl from ATP to the nucleoside diphosphate, is overall similar to that normally carried out by nucleoside diphosphate kinase (Ndk). Accordingly, Adk was proposed to be responsible for residual Ndk-like activity measured in a mutant strain of Escherichia coli, where the ndk gene was disrupted. We present data supporting a mechanism for the synthesis of nucleoside triphosphates by Adk that unlike the previously suggested mechanism mentioned above are in complete agreement with the current knowledge about the Adk enzyme and its various catalytic properties. We propose that nucleoside triphosphate synthesis occurs by beta-phosphoryl transfer from ADP to any bound nucleoside diphosphate. Our results point to the fact that the proposed Ndk-like mechanism of Adk originated from an erroneous interpretation of data, in that contamination of ATP preparations with AMP and ADP was not taken into account. Our results also address the proposed role of Adk in restoring a normal growth rate of mutant strains of E. coli lacking Ndk. These mutant strains apparently, in spite of a mutator phenotype, are able to synthesise nucleoside triphosphates by alternative pathways to maintain the same growth rate as the wildtype.     

Pregnane X receptor-agonists down-regulate hepatic ATP-binding cassette transporter A1 and scavenger receptor class B type I

Pregnane X receptor (PXR) is the molecular target for a wide variety of endogenous and xenobiotic compounds. It regulates the expression of genes central to the detoxification (cytochrome P-450 enzymes) and excretion (xenobiotic transporters) of potentially harmful compounds. The aim of the present investigation was to determine the role of PXR in regulation of high-density lipoprotein (HDL) cholesterol metabolism by studying its impact on ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type I (SR-BI) expression in hepatocytes. ABCA1 and SR-BI are major factors in the exchange of cholesterol between cells and HDL. Expression analyses were performed using Western blotting and quantitative real time RT-PCR. Luciferase reporter gene assays were used to measure promoter activities. Total cholesterol was measured enzymatically after lipid extraction (Folch's method). The expression of ABCA1 and SR-BI was inhibited by the PXR activators rifampicin and lithocholic acid (LCA) in HepG2 cells and pregnenolone 16alpha-carbonitrile (PCN) in primary rat hepatocytes. Thus, PXR appears to be a regulator of hepatic cholesterol transport by inhibiting genes central to cholesterol uptake (SR-BI) and efflux (ABCA1).     

Septicaemia associated with an Aerococcus viridans infection in immunodeficient mice

This report describes a case series of septicaemia caused by infection with Aerococcus viridans in immunodeficient NOD/LtSz-Prkdc(scid) (NOD/SCID) mice. During a period of 3 weeks more than 40 animals died or became ill with clinical signs of ruffled coat, weight loss, laboured breeding, and distended abdomen. At necropsy it was found that the animals displayed symptoms of sepsis with widespread abscesses in the liver, heart, lungs or pyogenic peritonitis. A Gram-positive coccus was isolated in pure culture from the abscesses or peritoneum from affected animals. According to phenotypic and phylogenetic characterization, the isolate was identified as A. viridans. This is the first report of a spontaneous outbreak of septicaemia caused by A. viridans infection in immunodeficient laboratory mice and we conclude that A. viridans should be considered as a pathogen in immunodeficient mice.     

Genetic and environmental influences of surfactant protein D serum levels

The collectin surfactant protein D (SP-D) is an important component of the pulmonary innate immune system, but SP-D is also present on extrapulmonary epithelial surfaces and in serum, where it has been used as a biomarker for pulmonary disease states. In this study, we investigate the mechanisms defining the constitutional serum level of SP-D and determine the magnitude of the genetic contribution to serum SP-D in the adult population. Recent studies have demonstrated that serum SP-D concentrations in children are genetically determined and that a single nucleotide polymorphism (SNP) located in the NH(2)-terminal region (Met11Thr) of the mature protein is significantly associated with the serum SP-D levels. A classic twin study was performed on a twin population including 1,476 self-reported healthy adults. The serum SP-D levels increased with male sex, age, and smoking status. The intraclass correlation was significantly higher for monozygotic (MZ) twin pairs than for dizygotic (DZ) twin pairs. Serum SP-D variance was influenced by nonshared environmental effects and additive genetic effects. Multivariate analysis of MZ and DZ covariance matrixes showed significant genetic correlation among serum SP-D and metabolic variables. The Met11Thr variant explained a significant part of the heritability indicating that serum SP-D variance could be decomposed into non-shared environmental effects (e(2) = 0.19), additive genetic effects (h(2) = 0.42), and the effect of the Met11Thr variations (q(2) = 0.39).     

FYCO1 is a Rab7 effector that binds to LC3 and PI3P to mediate microtubule plus end–directed vesicle transport

Autophagy is the main eukaryotic degradation pathway for long-lived proteins, protein aggregates, and cytosolic organelles. Although the protein machinery involved in the biogenesis of autophagic vesicles is well described, very little is known about the mechanism of cytosolic transport of autophagosomes. In this study, we have identified an adaptor protein complex, formed by the two autophagic membrane-associated proteins LC3 and Rab7 and the novel FYVE and coiled-coil (CC) domain–containing protein FYCO1, that promotes microtubule (MT) plus end–directed transport of autophagic vesicles. We have characterized the LC3-, Rab7-, and phosphatidylinositol-3-phosphate–binding domains in FYCO1 and mapped part of the CC region essential for MT plus end–directed transport. We also propose a mechanism for selective autophagosomal membrane recruitment of FYCO1.     

Nucleocytoplasmic Shuttling of p62/SQSTM1 and Its Role in Recruitment of Nuclear Polyubiquitinated Proteins to Promyelocytic Leukemia Bodies

p62, also known as sequestosome1 (SQSTM1), A170, or ZIP, is a multifunctional protein implicated in several signal transduction pathways. p62 is induced by various forms of cellular stress, is degraded by autophagy, and acts as a cargo receptor for autophagic degradation of ubiquitinated targets. It is also suggested to shuttle ubiquitinated proteins for proteasomal degradation. p62 is commonly found in cytosolic protein inclusions in patients with protein aggregopathies, it is up-regulated in several forms of human tumors, and mutations in the gene are linked to classical adult onset Paget disease of the bone. To this end, p62 has generally been considered to be a cytosolic protein, and little attention has been paid to possible nuclear roles of this protein. Here, we present evidence that p62 shuttles continuously between nuclear and cytosolic compartments at a high rate. The protein is also found in nuclear promyelocytic leukemia bodies. We show that p62 contains two nuclear localization signals and a nuclear export signal. Our data suggest that the nucleocytoplasmic shuttling of p62 is modulated by phosphorylations at or near the most important nuclear localization signal, NLS2. The aggregation of p62 in cytosolic bodies also regulates the transport of p62 between the compartments. We found p62 to be essential for accumulation of polyubiquitinated proteins in promyelocytic leukemia bodies upon inhibition of nuclear protein export. Furthermore, p62 contributed to the assembly of proteasome-containing degradative compartments in the vicinity of nuclear aggregates containing polyglutamine-expanded Ataxin1Q84 and to the degradation of Ataxin1Q84.     

Phototransduction genes are up-regulated in a global gene expression study of Drosophila melanogaster selected for heat resistance

The genetic architecture underlying heat resistance remains partly unclear despite the well-documented involvement of heat shock proteins (Hsps). It was previously shown that factors besides Hsps are likely to play an important role for heat resistance. In this study, gene expression arrays were used to make replicate measurements of gene expression before and up to 64 hours after a mild heat stress treatment, in flies selected for heat resistance and unselected control flies, to identify genes differentially expressed in heat resistance-selected flies. We found 108 genes up-regulated and 10 down-regulated using the Affymetrix gene expression platform. Among the up-regulated genes, a substantial number are involved in the phototransduction process. Another group of genes up-regulated in selected flies is characterized by also responding to heat shock treatment several hours after peak induction of known Hsps revert to nonstress levels. These findings suggest phototransduction genes to be critically involved in heat resistance, and support a role for components of the phototransduction process in stress-sensing mechanisms. In addition, the results suggest yet-uncharacterized genes responding to heat stress several hours after treatment to be involved in heat stress resistance. These findings mark an important increase in the understanding of heat resistance.     

Genetic regulation of fluxes: iron homeostasis of Escherichia coli

Iron is an essential trace-element for most organisms. However, because high concentration of free intracellular iron is cytotoxic, cells have developed complex regulatory networks that keep free intracellular iron concentration at optimal range, allowing the incorporation of the metal into iron-using enzymes and minimizing damage to the cell. We built a mathematical model of the network that controls iron uptake and usage in the bacterium Escherichia coli to explore the dynamics of iron flow. We simulate the effect of sudden decrease or increase in the extracellular iron level on intracellular iron distribution. Based on the results of simulations we discuss the possible roles of the small RNA RyhB and the Fe–S cluster assembly systems in the optimal redistribution of iron flows. We suggest that Fe–S cluster assembly is crucial to prevent the accumulation of toxic levels of free intracellular iron when the environment suddenly becomes iron rich.     

Viral and bacterial diseases of Atlantic cod Gadus morhua, their prophylaxis and treatment: a review

This review summarises the state of knowledge of both viral and bacterial diseases of Atlantic cod Gadus morhua, and their diagnosis, prophylaxis and treatment. The most important losses have been at the larval and juvenile stages, and vibriosis has long been the most important bacterial disease in cod, with Listonella (Vibrio) anguillarum dominant among pathogenic isolates. Vaccination of cod against pathogens such as L. anguillarum and Aeromonas salmonicida clearly demonstrates that the cod immune system possesses an effective memory and appropriate mechanisms sufficient for protection, at least against some diseases. Well-known viruses such as the nodavirus that causes viral encephalopathy and retinopathy (VER), infectious pancreatic necrosis virus (IPNV) and viral haemorrhagic septicaemia virus (VHSV) have been isolated from Atlantic cod and can be a potential problem under intensive rearing conditions. No commercial vaccines against nodavirus are currently available, whereas vaccines against IPNV infections based upon inactivated virus as well as IPNV recombinant antigens are available. A number of investigations of the pharmacokinetic properties of antibacterial agents in cod and their efficacy in treating bacterial infections have been reviewed.