Oligoribonuclease is a common downstream target of lithium-induced pAp accumulation in Escherichia coli and human cells

We identified Oligoribonuclease (Orn), an essential Escherichia coli protein and the only exonuclease degrading small ribonucleotides (5mer to 2mer) and its human homologue, small fragment nuclease (Sfn), in a screen for proteins that are potentially regulated by 3′-phosphoadenosine 5′-phosphate (pAp). We show that both enzymes are sensitive to micromolar amounts of pAp in vitro. We also demonstrate that Orn can degrade short DNA oligos in addition to its activity on RNA oligos, similar to what was documented for Sfn. pAp was shown to accumulate as a result of inhibition of the pAp-degrading enzyme by lithium, widely used to treat bipolar disorder, thus its regulatory targets are of significant medical interest. CysQ, the E.coli pAp-phosphatase is strongly inhibited by lithium and calcium in vitro and is a main target of lithium toxicity in vivo. Our findings point to remarkable conservation of the connection between sulfur- and RNA metabolism between E.coli and humans.     

<Emphasis Type="Italic">Poseidonocella pacifica</Emphasis> gen. nov., sp. nov. and <Emphasis Type="Italic">Poseidonocella sedimentorum</Emphasis> sp. nov., novel alphaproteobacteria from the shallow sandy sediments of the Sea of Japan

The taxonomic study of two Gram-negative, aerobic, non-pigmented bacteria KMM 9010^T and KMM 9023^T isolated from a sandy sediment sample collected from the Sea of Japan seashore was performed. On the basis of the nearly complete 16S rRNA gene sequences, strains KMM 9010^T and KMM 9023^T clustered with the Roseobacter lineage (class Alphaproteobacteria) forming a distinct phylogenetic line adjacent to the genus Donghicola. Novel strains shared the highest sequence similarity of 96.4% to each other and lower than 96.1% similarities to other validly named genera of the class Alphaproteobacteria. In both strains, ubiquinone Q-10 was found to be the major respiratory quinone; phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidic acid, and an unknown aminolipid were the major polar lipids and C_18:1ω7c and 11-methyl C_18:1ω7c were predominant fatty acids. The DNA G+C content was 60.5 mol% (KMM 9010^T) and 65.4 mol% (KMM 9023^T). Based on phenotypic properties and phylogenetic evidence, strains KMM 9010^T and KMM 9023^T should be classified as two novel species in a new genus, Poseidonocella gen. nov., with Poseidonocella pacifica sp. nov., the type species with the type strain KMM 9010^T (= NRIC 0794^T = JCM 17310^T), and Poseidonocella sedimentorum sp. nov. as the second species with the type strain KMM 9023^T (= NRIC 0796^T = JCM 17311^T).     

CNS penetration of intrathecal-lumbar idursulfase in the monkey, dog and mouse: implications for neurological outcomes of lysosomal storage disorder

A major challenge for the treatment of many central nervous system (CNS) disorders is the lack of convenient and effective methods for delivering biological agents to the brain. Mucopolysaccharidosis II (Hunter syndrome) is a rare inherited lysosomal storage disorder resulting from a deficiency of iduronate-2-sulfatase (I2S). I2S is a large, highly glycosylated enzyme. Intravenous administration is not likely to be an effective therapy for disease-related neurological outcomes that require enzyme access to the brain cells, in particular neurons and oligodendrocytes. We demonstrate that intracerebroventricular and lumbar intrathecal administration of recombinant I2S in dogs and nonhuman primates resulted in widespread enzyme distribution in the brain parenchyma, including remarkable deposition in the lysosomes of both neurons and oligodendrocytes. Lumbar intrathecal administration also resulted in enzyme delivery to the spinal cord, whereas little enzyme was detected there after intraventricular administration. Mucopolysaccharidosis II model is available in mice. Lumbar administration of recombinant I2S to enzyme deficient animals reduced the storage of glycosaminoglycans in both superficial and deep brain tissues, with concurrent morphological improvements. The observed patterns of enzyme transport from cerebrospinal fluid to the CNS tissues and the resultant biological activity (a) warrant further investigation of intrathecal delivery of I2S via lumbar catheter as an experimental treatment for the neurological symptoms of Hunter syndrome and (b) may have broader implications for CNS treatment with biopharmaceuticals.     

Negative Selection Assay Based on Stimulation of T Cell Receptor Transgenic Thymocytes with Peptide-MHC Tetramers

Thymocyte negative selection is a requirement for the development of self tolerance. Although it is possible to assay the induction of cell death in thymocytes in vitro using antibody cross-linking, this stimulus is much stronger than the normal range of T cell receptor ligands that could be encountered during normal development. Signaling in thymocytes is finely balanced between positive and negative selection stimuli, where a negative selecting ligand can be only marginally higher affinity than a positive selecting ligand. We have therefore developed an assay for the induction of negative selection that can distinguish such cases, and that is amenable to high-throughput analysis. The assay is based on the induction of activated caspase 3 in thymocytes expressing a defined T cell receptor, after stimulation with MHC-peptide tetramers in vitro for 24 hours or less.     

Distinct Distribution of Ectopically Expressed Histone Variants H2A.Bbd and MacroH2A in Open and Closed Chromatin Domains

Background

It becomes increasingly evident that nuclesomes are far from being identical to each other. This nucleosome diversity is due partially to the existence of histone variants encoded by separate genes. Among the known histone variants the less characterized are H2A.Bbd and different forms of macroH2A. This is especially true in the case of H2A.Bbd as there are still no commercially available antibodies specific to H2A.Bbd that can be used for chromatin immunoprecipitation (ChIP).

Methods

We have generated HeLa S3 cell lines stably expressing epitope-tagged versions of macroH2A1.1, H2A.Bbd or canonical H2A and analyzed genomic distribution of the tagged histones using ChIP-on-chip technique.

Results

The presence of histone H2A variants macroH2A1.1 and H2A.Bbd has been analyzed in the chromatin of several segments of human chromosomes 11, 16 and X that have been chosen for their different gene densities and chromatin status. Chromatin immunoprecipitation (ChIP) followed by hybridization with custom NimbleGene genomic microarrays demonstrated that in open chromatin domains containing tissue-specific along with housekeeping genes, the H2A.Bbd variant was preferentially associated with the body of a subset of transcribed genes. The macroH2A1.1 variant was virtually absent from some genes and underrepresented in others. In contrast, in closed chromatin domains which contain only tissue-specific genes inactive in HeLa S3 cells, both macroH2A1.1 and H2A.Bbd histone variants were present and often colocalized.

Conclusions

Genomic distribution of macro H2A and H2A.Bbd does not follow any simple rule and is drastically different in open and closed genomic domains.     

Semi-continuum electrostatic calculations of redox potentials in photosystem I

The midpoint redox potentials (E(m)) of all cofactors in photosystem I from Synechococcus elongatus as well as of the iron-sulfur (Fe(4)S(4)) clusters in two soluble ferredoxins from Azotobacter vinelandii and Clostridium acidiurici were calculated within the framework of a semi-continuum dielectric approach. The widely used treatment of proteins as uniform media with single dielectric permittivity is oversimplified, particularly, because permanent charges are considered both as a source for intraprotein electric field and as a part of dielectric polarizability. Our approach overcomes this inconsistency by using two dielectric constants: optical epsilon(o)=2.5 for permanent charges pre-existing in crystal structure, and static epsilon(s) for newly formed charges. We also take into account a substantial dielectric heterogeneity of photosystem I revealed by photoelectric measurements and a liquid junction potential correction for E(m) values of relevant redox cofactors measured in aprotic solvents. We show that calculations based on a single permittivity have the discrepancy with experimental data larger than 0.7 V, whereas E(m) values calculated within our approach fall in the range of experimental estimates. The electrostatic analysis combined with quantum chemistry calculations shows that (i) the energy decrease upon chlorophyll dimerization is essential for the downhill mode of primary charge separation between the special pair P(700) and the primary acceptor A(0); (ii) the primary donor is apparently P(700) but not a pair of accessory chlorophylls; (iii) the electron transfer from the A branch quinone Q(A) to the iron-sulfur cluster F(X) is most probably downhill, whereas that from the B branch quinone Q(B) to F(X) is essentially downhill.     

Trafficking and intracellular ATPase activity of human ecto-nucleotidase NTPDase3 and the effect of ER-targeted NTPDase3 on protein folding

Ecto-nucleoside triphosphate diphosphohydrolases, NTPDase1 (CD39) and NTPDase3, are integral plasma membrane proteins that hydrolyze extracellular nucleotides, thereby modulating the function of purinergic receptors. During processing in the secretory pathway, the active sites of ecto-nucleotidases are located in the lumen of vesicular compartments, thus raising the question whether the ecto-nucleotidases affect the ATP-dependent processes in these compartments, including protein folding in the endoplasmic reticulum (ER). It has been reported (J. Biol. Chem. (2001) 276, 41518-41525) that CD39 is not active until it reaches the plasma membrane, suggesting that terminal glycosylation in Golgi is critical for its activity. To investigate the subcellular location and the mechanism of ecto-nucleotidase activation, we expressed human NTPDase3 in COS-1 cells and blocked the secretory transport with monensin or brefeldin A, or by targeting to ER with a signal peptide. Cell surface biotinylation, sensitivity to glycosidases, and fluorescence microscopy analyses suggest that, in contrast to the previous report on CD39, NTPDase3 becomes catalytically active in the ER or in the ER-Golgi intermediate compartment, and that terminal glycosylation in Golgi is not essential for activity. Moreover, ER-targeted NTPDase3, but not wild-type NTPDase3 or ER-targeted inactive G221A mutant, significantly diminished the folding efficiency and the transport to the plasma membrane of coexpressed CD39 used as a reporter protein. These data suggest that ER-targeted NTPDase3 significantly depletes ATP in ER, whereas wild-type NTPDase3 is likely to acquire ATPase activity in a post-ER, but pre-Golgi, compartment, thus avoiding unproductive ATP hydrolysis and interference with protein folding in the ER. ER-targeted NTPDase3 may be a useful experimental tool to study the effects of ER ATP depletion on ER function under normal and stress conditions.