Molecular and functional interactions between Escherichia coli nucleoside-diphosphate kinase and the uracil-DNA glycosylase Ung

Escherichia coli nucleoside-diphosphate kinase (Ndk) catalyzes nucleoside triphosphate synthesis and maintains intracellular triphosphate pools. Mutants of E. coli lacking Ndk exhibit normal growth rates but show a mutator phenotype that cannot be entirely attributed to the absence of Ndk catalytic activity or to an imbalance in cellular triphosphates. It has been suggested previously that Ndk, similar to its human counterparts, possesses nuclease and DNA repair activities, including the excision of uracil from DNA, an activity normally associated with the Ung and Mug uracil-DNA glycosylases (UDGs) in E. coli. Here we have demonstrated that recombinant Ndk purified from wild-type E. coli contains significant UDG activity that is not intrinsic, but rather, is a consequence of a direct physical and functional interaction between Ung and Ndk, although a residual amount of intrinsic UDG activity exists as well. Co-purification of Ung and Ndk through multicolumn low pressure and nickel-nitrilotriacetic acid affinity chromatography suggests that the interaction occurs in a cellular context, as was also suggested by co-immunoprecipitation of endogenous Ung and Ndk from cellular extracts. Glutathione S-transferase pulldown and far Western analyses demonstrate that the interaction also occurs at the level of purified protein, suggesting that it is specific and direct. Moreover, significant augmentation of Ung catalytic activity by Ndk was observed, suggesting that the interaction between the two enzymes is functionally relevant. These findings represent the first example of Ung interacting with another E. coli protein and also lend support to the recently discovered role of nucleoside-diphosphate kinases as regulatory components of multiprotein complexes.     

Extension of the Nef reaction to C-glycosylnitromethanes

Acid-catalysed methanolysis of 3,4,5,6-tetra-O-acetyl-1,2-dideoxy-l-arabino-hex-1-enitol proceeds via a cascade set of consecutive reactions resulting in its regiospecific conversion to a mixture of alpha- and beta-C-L-arabinofuranosylmethanal dimethyl acetals and a mixed internal methyl acetal. Structures of the final products of the overall process provide unique evidence that a kinetically controlled, five-membered-ring closure precedes a six-membered-ring closure in reversible systems capable of giving both five-membered and six-membered all-sp3-atom rings. Determination of the reaction intermediate enabled extension of the Nef reaction to C-glycosylnitromethanes. Protonated aci-nitro forms of C-glycosylnitromethanes that are resistant to the Nef reaction in aqueous acidic media undergo a modified Nef reaction in acidified methanol, and the corresponding C-glycosylmethanal dimethyl acetals with alpha-L-arabinopyranosyl, beta-D-glucopyranosyl, beta-D-galactopyranosyl, beta-D-mannopyranosyl and beta-L-rhamnopyranosyl configurations were obtained in moderate yields.     

Chronic thyroiditis--current issue in the cytological diagnostics of the thyroid gland

INTRODUCTION: Lowered and non-homogenous echogenicity in patients with chronic thyroiditis (CT) may cause problems in revealing focal lesions in ultrasound scans. Moreover, frequent anisocytosis of thyroid follicular cells and of oncocytes may falsely suggest neoplastic growth. The aim of the study was to evaluate the frequency of CT diagnosed in patients subjected to fine-needle aspiration biopsy (FNAB) of the thyroid in years 1994-2005, as well as to assess the cancer frequency in the operated patients with CT. MATERIAL AND METHODS: As much as 23 173 cytological reports and corresponding outcomes were analyzed and in patients treated surgically verified against postoperative histopathological examinations. Moreover, the analysis of cytological smears in CT with distinguishing its types (lymphocytic, epithelial-hyperplastic, colloidal, oxyphillic) was performed in 50 patients; the changes between subsequent FNABs in the picture of the smears were also assessed. RESULTS: The frequency of diagnosed CT had been increasing in the evaluated time span from 2.6% to 9.7% (p < 0.0001). There were 11 cases of papillary carcinoma found coexisting with CT. The analysis of cytological types of CT showed that the lymphocytic type prevailed over the others (lymphocytic - 43%, colloidal--21%, epithelial-hyperplastic--20%, oxyphilic--16%, p < 0.001). The evolution in time of the cytological picture was observed. CONCLUSION: The increase in the frequency of CT observed in smears obtained from the thyroid gland and the reports on the coexistence of CT with papillary carcinoma suggest the necessity of prudent diagnostics and follow-up of patients with CT.     

Role of pro-oxidant properties of flavonoids in the induction of detoxifying enzymes gene expression

Flavonoids are one of the most important components of human daily diet. In recent years flavonoids have became the subject of extensive investigations mostly due to their heath-promoting properties. Beneficial health effects of flavonoids are mainly ascribed to their antioxidant activity. However, there is increasing evidence of the positive role of pro-oxidant properties of flavonoids, considered previously as highly unfavorable, through the induction of detoxifying enzymes gene expression. The article discusses recent reports on the EpRE-mediated induction of NQO1 (NAD(P)H: quinone oxidoreductase) gene expression by the flavonoids (flavonols, flavones and flavan-3-oles) and the role of pro-oxidant properties of the flavonoids in the mechanism of this induction.     

Characterization of Pasteurellaceae‐like bacteria isolated from clinically affected psittacine birds

Aims: The aim of the present investigation was to identify and characterize Pasteurella‐like isolates obtained from clinically affected psittacine birds. Methods and Results: A total of 37 isolates from psittacine birds tentatively classified with the family Pasteurellaceae were characterized phenotypically. The genetic relationship was investigated by sequencing of partial rpoB and 16S rRNA genes for selected isolates. The results obtained were compared with the data from 16 reference strains. Nine isolates were identified as Gallibacterium spp., 16 as Volucribacter spp. or Volucribacter‐like, while 11 isolates were classified as taxon 44 of Bisgaard. A single isolate was identified as Pasteurella multocida. Conclusions: Characterization of Pasteurellaceae by traditional methods is often inconclusive because of inconsistent reactions and phenotypic diversity. For the same reason, genotyping is essential to allow proper classification as demonstrated in the present study. Significance and Impact of the Study: Limited information exists on the isolation and significance of Pasteurellaceae associated with clinically affected psittacine birds showing signs of digestive and/or respiratory disorders. The present investigations demonstrated that these organisms are widely distributed among clinically affected birds, but isolation of these taxa cannot be unambiguously correlated with the symptoms observed.     

Porcine pregnancy-associated glycoprotein family (pPAGs) - as in vitro-produced chorionic ligands for luteal and uterine gonadotropin receptors

This paper compares proteomic interaction-types and binding-effectiveness of secretory chorionic ligands (including pPAGs) with other proteins, i.e. gonadotropin membrane receptors (Rc) isolated from luteal-phase corpora lutea, uterine myometrium and endometrium of cyclic (cCLRc, cMYORc and cENDRc) or pregnant (pCLRc, pMYORc and pENDRc) pigs. Binding-effectiveness of miscellaneous in vitro-produced chorionic ligands (+pPAGs) was compared by radioreceptor assay (RRA) with endometrial (END) proteins of cyclic, pseudopregnant and pregnant gilts - as negative control ligands and porcine LH and hCG - as positive control ligands. The binding-comparison suggests that the pPAGs may play an important role as potential antiluteolytic or luteoprotective chorionic-origin signals during pregnancy, according to the binding-effectiveness of secretory chorionic ligands (+pPAGs) that was relatively comparable to LH/hCG - as classical ligands competing for luteal and uterine gonadotropin receptors of cyclic and pregnant pigs.     

First detection of Anaplasma phagocytophilum in quill mites (Acari: Syringophilidae) parasitizing passerine birds

Forest passerine birds and their ectoparasites: Ixodes ricinus ticks and Syringophilidae quill mites were surveyed for infection with Anaplasma phagocytophilum in west-central Poland. Of 126 birds captured from May to June of 2002, 71 (56.3%) comprising eight species, hosted immature I. ricinus ticks. A total of 383 ticks and 71 blood samples collected from tick-infested birds were investigated by PCR. The pathogen was not detected in either bird-derived ticks or in blood samples. Among the captured birds, a total of 14 individuals representing four species hosted quill mites from the family Syringophilidae. Three of the 14 mite pools recovered from the 14 mite-infested birds harbored A. phagocytophilum DNA by amplifying both the epank1 and p44 gene. The PCR-positive pools originated from one blackbird and two starlings. The specific biology of syringophilid mites, which parasitize exclusively inside the quill of feathers, feeding on host subcutaneous fluids, implies that they must have acquired the pathogen from a bacteremic bird. These results provide the first indirect evidence that at least some passerine hosts are prone to develop systemic infection with A. phagocytophilum under natural conditions. Consequently, the infected quill mites may serve as a "biological marker" of past or current infection with the agent within birds.     

A solid-phase glycosyltransferase assay for high-throughput screening in drug discovery research

Glycosyltransferases mediate changes in glycosylation patterns which, in turn, may affect the function of glycoproteins and/or glycolipids and, further downstream, processes of development, differentiation, transformation and cell-cell recognition. Such enzymes, therefore, represent valid targets for drug discovery. We have developed a solid-phase glycosyltransferase assay for use in a robotic high-throughput format. Carbohydrate acceptors coupled covalently to polyacrylamide are coated onto 96-well plastic plates. The glycosyltransferase reaction is performed with recombinant enzymes and radiolabeled sugar-nucleotide donor at 37°C, followed by washing, addition of scintillation counting fluid, and measurement of radioactivity using a 96-well -counter. Glycopolymer construction and coating of the plastic plates, enzyme and substrate concentrations, and linearity with time were optimized using recombinant Core 2 1-6-N-acetylglucosaminyltransferase (Core 2 GlcNAc-T). This enzyme catalyzes a rate-limiting reaction for expression of polylactosamine and the selectin ligand sialyl-Lewis^x in -glycans. A glycopolymer acceptor for 1-6-N-acetylglucosaminyltransferase V was also designed and shown to be effective in the solid-phase assay. In a high-throughput screen of a microbial extract library, the coefficient of variance for positive controls was 9.4%, and high concordance for hit validation was observed between the Core 2 GlcNAc-T solid-phase assay and a standard solution-phase assay. The solid-phase assay format, which can be adapted for a variety of glycosyltransferase enzymes, allowed a 5–6 fold increase in throughput compared to the corresponding solution-phase assay.     

Time course of proteolytic, cytokine, and myostatin gene expression after acute exercise in human skeletal muscle.

The aim of this study was to examine the time course induction of select proteolytic [muscle ring finger-1 (MuRF-1), atrogin-1, forkhead box 3A (FOXO3A), calpain-1, calpain-2], myostatin, and cytokine (IL -6, -8, -15, and TNF-alpha) mRNA after an acute bout of resistance (RE) or run (RUN) exercise. Six experienced RE (25 +/- 4 yr, 74 +/- 14 kg, 1.71 +/- 0.11 m) and RUN (25 +/- 4 yr, 72 +/- 5 kg, 1.81 +/- 0.07 m) subjects had muscle biopsies from the vastus lateralis (RE) or gastrocnemius (RUN) before, immediately after, and 1, 2, 4, 8, 12, and 24 h postexercise. RE increased (P < 0.05) mRNA expression of MuRF-1 early (3.5-fold, 1-4 h), followed by a decrease in atrogin-1 (3.3-fold) and FOXO3A (1.7-fold) 8-12 h postexercise. Myostatin mRNA decreased (6.3-fold; P < 0.05) from 1 to 24 h postexercise, whereas IL-6, IL-8, and TNF-alpha mRNA were elevated 2-12 h. RUN increased (P < 0.05) MuRF-1 (3.6-fold), atrogin-1 (1.6-fold), and FOXO3A (1.9-fold) 1-4 h postexercise. Myostatin was suppressed (3.6-fold; P < 0.05) 8-12 h post-RUN. The cytokines exhibited a biphasic response, with immediate elevation (P < 0.05) of IL-6, IL-8, and TNF-alpha, followed by a second elevation (P < 0.05) 2-24 h postexercise. In general, the timing of the gene induction indicated early elevation of proteolytic genes, followed by prolonged elevation of cytokines and suppression of myostatin. These data provide basic information for the timing of human muscle biopsy samples for gene expression studies involving exercise. Furthermore, this information suggests a greater induction of proteolytic genes following RUN compared with RE.     

Assessment of susceptibility to metronidazole and vancomycin of Clostridium difficile strains isolated between 1998-2002

The drugs of choice used to treat C. diffcile associated diarrhoea (CDAD) are metronidazole and vancomycin. C. difficile strains isolated in most laboratories are susceptible to metronidazole and vancomycin. Communication about emergence of antimicrobial resistance among C. difficile strains in some countries to metronidazole and intermediate resistance to vancomycin are alarming. This study was performed to determine the susceptibility to metronidazole and vancomycin of 140 C. difficile strains isolated from patients with CDAD hospitalised in academic hospital between 1999-2002. Resistance to metronidazole and vancomycin was not observed.