Inhibition of poly(ADP-ribose)polymerase does not affect the recombination events in CHO xrs6 and wild type cells

Activation of poly (ADP-ribose) polymerase -1 (PARP-1) is an early DNA damage response event that, together with phosphorylation of p53, prompts various cellular functions important in the maintenance of the genome stability. In mammalian cells, DSB are repaired by nonhomologous end-joining (NHEJ) and by homologous recombination (HR). To investigate the role of PARP-1 in HR, CHO-K1 wild type and xrs-6 mutant cell line were transfected with pLrec plasmids which carry two nonfunctional copies of the β-galactosidase (lacZ) gene in a tandem array. In result of HR they can give rise to a functional copy of β-galactosidase. To test whether PARP-1 affects the frequency of spontaneous and induced recombination repair, we treated CHO-K1 and xrs6 clones carrying chromosomally integrated pLrec with the PARP-1 inhibitor 3-aminobenzamide (3AB). Our results show that the spontaneous homologous intrachromosomal recombination frequency between the two lacZ copies was almost two orders of magnitude higher in xrs6 cells than in CHO-K1 cells, but that it was not affected by 3AB treatment. Induction of DNA damage by irradiation or electroporation of restriction enzymes did not significantly increase the recombination frequency. Furthermore, in both the cell lines, the effect of PARP-1 inhibition on DSB repair was examined using the neutral comet assay. There was no effect of 3AB treatment on DSB rejoining after 10 Gy irradiation. The results presented support the conclusion that PARP-1 is not directly involved in HR.     

Molecular-Beacon Multiplex Real-Time PCR Assay for Detection of Vibrio cholerae

A multiplex real-time PCR assay was developed using molecular beacons for the detection of Vibrio cholerae by targeting four important virulence and regulatory genes. The specificity and sensitivity of this assay, when tested with pure culture and spiked environmental water samples, were high, surpassing those of currently published PCR assays for the detection of this organism.     

The Arabidopsis elch mutant reveals functions of an ESCRT component in cytokinesis

Recently, an alternative route to the proteasomal protein-degradation pathway was discovered that specifically targets transmembrane proteins marked with a single ubiquitin to the endosomal multivesicular body (MVB) and, subsequently, to the vacuole (yeast) or lysosome (animals), where they are degraded by proteases. Vps23p/TSG101 is a key component of the ESCRT I-III machinery in yeast and animals that recognizes mono-ubiquitylated proteins and sorts them into the MVB. Here, we report that the Arabidopsis ELCH (ELC) gene encodes a Vps23p/TSG101 homolog, and that homologs of all known ESCRT I-III components are present in the Arabidopsis genome. As with its animal and yeast counterparts, ELC binds ubiquitin and localizes to endosomes. Gel-filtration experiments indicate that ELC is a component of a high-molecular-weight complex. Yeast two-hybrid and immunoprecipitation assays showed that ELC interacts with Arabidopsis homologs of the ESCRT I complex. The elc mutant shows multiple nuclei in various cell types, indicating a role in cytokinesis. Double-mutant analysis with kaktus shows that increased ploidy levels do not influence the cytokinesis effect of elc mutants, suggesting that ELC is only important during the first endoreduplication cycle. Double mutants with tubulin folding cofactor a mutants show a synergistic phenotype, suggesting that ELC regulates cytokinesis through the microtubule cytoskeleton.     

A new β-galactosidase with a low temperature optimum isolated from the Antarctic <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. 20B: gene cloning,purification and characterization

A psychrotrophic bacterium producing a cold-adapted β-galactosidase upon growth at low temperatures was classified as Arthrobacter sp. 20B. A genomic DNA library of strain 20B introduced into Escherichia coli TOP10F′ and screening on X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside)-containing agar plates led to the isolation of β-galactosidase gene. The β-galactosidase gene (bgaS) encoding a protein of 1,053 amino acids, with a calculated molecular mass of 113,695 kDa. Analysis of the amino acid sequence of BgaS protein, deduced from the bgaS ORF, suggested that it is a member of the glycosyl hydrolase family 2. A native cold-adapted β-galactosidase was purified to homogeneity and characterized. It is a homotetrameric enzyme, each subunit being approximately 116 kDa polypeptide as deduced from native and SDS–PAGE, respectively. The β-galactosidase was optimally active at pH 6.0–8.0 and 25°C. P-nitrophenyl-β-d-galactopyranoside (PNPG) is its preferred substrate (three times higher activity than for ONPG—o-nitrophenyl-β-d-galactopyranoside). The Arthrobacter sp. 20B β-galactosidase is activated by thiol compounds (53% rise in activity in the presence of 10 mM 2-mercaptoethanol), some metal ions (activity increased by 50% for Na⁺, K⁺ and by 11% for Mn²⁺) and inactivated by pCMB (4-chloro-mercuribenzoic acid) and heavy metal ions (Pb²⁺, Zn²⁺, Cu²⁺).     

Tunicamycin desensitizes store-operated Ca entry to ATP and mitochondrial potential

Tunicamycin effect on thapsigargin-induced store-operated calcium entry was investigated. Ca²⁺ influx was stimulated by 50% upon exposure of Jurkat cells to tunicamycin. Moreover, tunicamycin efficiently prevented the inhibition of store-operated calcium entry caused by dissipation of mitochondrial membrane potential. Protective action of tunicamycin on store-operated Ca²⁺ entry was also partially preserved in Jurkat cells depleted of ATP, while Ca²⁺ entry into ATP-deprived cells grown in tunicamycin-free medium was almost completely inhibited. Tunicamycin-evoked changes in cellular Ca²⁺ fluxes coincided with decreased glycosylation of STIM1 protein. Although the latter observation is correlative and needs additional confirmation it may suggest that deglycosylation of STIM1 protein deprives store-operated calcium entry system of an important regulatory mechanism. This study suggests a novel mechanism of modulation of the activity of store-operated calcium channels in lymphoidal cells.     

pH Effect of the Sphingomyelin Membrane Interfacial Tension

The effect of pH on the interfacial tension of a sphingomyelin membrane in aqueous solution has been studied. Three models describing H⁺ and OH⁻ ion adsorption on the bilayer lipid surface are presented. In models I and II, the membrane surface is continuous, with uniformly distributed functional groups as centers of H⁺ and OH⁻ ion adsorption. In model III, the membrane surface is composed of lipid molecules, with and without adsorbed H⁺ and OH⁻ ions. The contribution of each individual lipid molecule to the overall interfacial tension of the bilayer was assumed to be additive in models I and II. In model III, the Gibbs isotherm was used to describe adsorption of H⁺ and OH⁻ ions at the bilayer surface. Theoretical equations are derived to describe the interfacial tension as a function of pH for all three models. Maximum interfacial tension was observed experimentally at the isoelectric point.     

<Emphasis Type="Italic">Dicrogaster perpusilla</Emphasis> Looss, 1902 <Emphasis Type="Italic">sensu</Emphasis> Sarabeev &; Balbuena (Digenea: Haploporidae): a note of caution

Sarabeev & Balbuena (2003) considered Dicrogaster perpusilla Looss, 1902 and D. contracta Looss, 1902 (Digenea: Haploporidae) synonymous. They designated a neotype for the type-species, D. perpusilla, from a sample of specimens ex Chelon labrosus off West Thurrock, UK. The morphology of the material (three specimen lots) studied by these authors was re-examined in detail and compared with their data. The material labelled ‘D. perpusilla’ from off West Thurrock, from which the neotype specimen was selected, consists of 14 specimens; of these one might belong to Haploporus Looss, 1902 and one to Haplosplanchnus Looss 1902. A well-developed genital atrium was observed in 11 of the 12 remaining specimens, and they all possess large saccular caeca and a vitellarium consisting of two groups of loosely coalesced follicles rather than two compact masses. These features suggest that the 12 specimens of this lot may belong to Saccocoelium Looss, 1902, but the state of the material does not permit its reliable identification. The specimen designated by Sarabeev & Balbuena (2003) as the neotype of D. perpusilla represents a neogravid dorso-laterally mounted specimen and is unrecognisable. Although five of the six voucher specimens of these authors may represent Dicrogaster spp., the poor state of this material does not allow its accurate identification. The metrical data obtained from this voucher material indicate that juvenile and laterally mounted specimens have been used in the comparisons upon which the synonymy of D. perpusilla and D. contracta was suggested. The overall conclusion of the study is that the synonymy of D. contracta and D. perpusilla proposed by Sarabeev & Balbuena (2003) is based on questionable material. Since the neotype of D. perpusilla is unrecognisable, and a number of qualifying conditions of the ICZN in its designation were not met, the usage of the original conception of the type-species of Dicrogaster given by Looss (1902) is recommended.     

New records of rare derogenids (Digenea: Hemiuroidea) from Mediterranean sparids,including the description of a new species of <Emphasis Type="Italic">Magnibursatus</Emphasis> Naidenova, 1969 and redescription of <Emphasis Type="Italic">Derogenes adriaticus</Emphasis> Nikolaeva, 1966

Records of derogenid digeneans in the Mediterranean and Black Sea region are scarce and tend to be restricted to a small number of host-groups, but especially to sparid fishes. This work reports on the presence of derogenine and halipegine derogenids from two sparids, Diplodus annularis (L.) and D. sargus (L.), from off the Mediterranean coast of Spain. Five derogenid forms were recovered. Derogenes adriaticus Nikolaeva, 1966 is redescribed from Diplodus annularis, and Derogenes sp. is described from the same host but differentiated from the former species. Magnibursatus barretti n. sp. is described from Diplodus sargus and distinguished from other species of the genus especially by its smaller body size and smaller eggs. M. bartolii Kostadinova, Power, Fernández, Balbuena, Raga & Gibson, 2003 is redescribed from D. sargus, a new host for this species. A single specimen from D. sargus, somewhat similar to M. minutus Kostadinova, Power, Fernández, Balbuena, Raga & Gibson, 2003, is also described, as it exhibits some morphometric differences from the latter species.