Novel nicotinamide adenine dinucleotide analogues as selective inhibitors of NAD-dependent enzymes

Three novel dinucleotide analogues of nicotinamide adenine dinucleotide (NAD+) have been synthesised from -ribonolactone. These compounds incorporate a thiophene moiety in place of nicotinamide and are hydrolytically stable. They have been evaluated as inhibitors of adenosine diphosphate ribosyl cyclase, glutamate dehydrogenase and Sir2 acyltransferase activities. Enzyme specificity and a high level of inhibition was observed for the dehydrogenase.

The DUB/USP17 deubiquitinating enzymes, a multigene family within a tandemly repeated sequence

Here we report the identification of 10 human, 1 murine, and 2 rat ORFs, all of which represent additional members of the DUB/USP17 family of deubiquitinating enzymes. In addition, we demonstrate that this family constitutes part of a tandemly repeated sequence conserved throughout humans, mice, and rats. Furthermore, upon examination of the known family members we have found that the multiple genes observed, in contrast to other gene families, have arisen due to the independent expansion of an ancestral sequence within each species. This premise is further strengthened by the observation that the murine and rat genes span two exons while their human counterparts have one. These observations, in conjunction with previous work demonstrating that the DUB/USP17's are cytokine inducible and that they regulate both cell growth and survival, suggest that the DUB/USP17's are a large highly conserved family of genes that may play an important role in controlling cell fate.

Detection of glucocorticoid bioactivity in bovine urine samples using a reporter gene assay.

The illegal use of anabolic substances in the meat producing industry is an ongoing problem due to the continual production of new synthetic compounds and/or the practice of low-level cocktail administration to avoid detection by the surveillance schemes of EU member states National Plan surveillance systems.

We present a highly sensitive reporter gene assay and sample extraction procedure based on a two step solid phase extraction and high performance liquid chromatography, developed for the detection of glucocorticoid abuse in bovine urine. The assay is capable of detecting compounds with glucocorticoid bioactivity and is extremely sensitive with an EC50 of 0.79 ng mL-1 for dexamethasone. New or unknown compounds with glucocorticoid bioactivity and low-level cocktail mixtures are detectable by this assay.

Cross-reactivity data for a range of 11ß-hydroxyglucocorticoids has been provided. This assay shows low interference from the 11-keto prohormones and other steroidal hormones. The assay may be suitable for application in other matrices such as hair. In conclusion this screening assay offers advantages over existing analytical techniques.

Association of airway cathepsin B and S with inflammation in cystic fibrosis.

Irreversible tissue damage within the cystic fibrosis (CF) lung is mediated by proteolytic enzymes during an inflammatory response. Serine proteinases, in particular neutrophil elastase (NE), have been implicated however, members of the cysteine proteinase family may also be involved. The aim of this study was to determine cathepsin B and S levels in cystic fibrosis (CF) sputum and to assess any relationship to recognized markers of inflammation such as sputum NE, interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-a), urine TNF receptor 1 (TNFr1), plasma IL-6, and serum C-reactive protein (CRP). Proteinase activities were measured in the sputum of 36 clinically stable CF patients using spectrophotometric and fluorogenic assays. Immunoblots were also used to confirm enzyme activity data. All other parameters were measured by ELISA. Patients had a mean age of 27.2 (8.2) years, FEV. of 1.6 (0.79) L and BMI of 20.7 (2.8). Both cathepsin B and S activities were detected in all samples, with mean concentrations of 18.0 (13.5)?µg/ml and 1.6 (0.88)?µg/ml, respectively and were found to correlate not only with each other but with NE, TNF-a and IL-8 (in all cases .?<?0.05). Airway cathepsin B further correlated with circulatory IL-6 and CRP however, no relationship for either cathepsin was observed with urine TNFr1. This data indicates that cathepsin B and S may have important roles in the pathophysiology of CF lung disease and could have potential as markers of inflammation in future studies. Pediatr. Pulmonol. 2010; 45:860–868.

Emergence of CTX-M Group 1-ESBL producing Klebsiella pneumonia from a tertiary care centre in Karachi, Pakistan.

http://www.jidc.org/index.php/journal/article/view/20818098/422 Background: Extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae have been reported previously from Pakistan but the genotypic characteristics of these enzymes is not known. Hence the aim of the study was first to characterise the genotypic content of these beta-lactamases and secondly to assess the clonal relationship of these isolates. Methodology: We analysed 65 non-duplicate ESBL positive, K. pneumoniae isolates prospectively collected based on phenotype as detected using the two-disc method. Isolates were collected from different sources: blood cultures (46.15%; n = 30); tracheal aspirates (24.6%; n = 16); urine (10.7%; n = 7); wound swabs, pus and tissue (18.4%; n = 12). ESBL production was confirmed by the ESBL E-test method and the presence of the blaCTX-M encoding genes was confirmed by polymerase chain reaction. The clonal relationship of clinical isolates was studied by Pulsed Field Gel Electrophoresis. Results: The results showed that 93.84% (n = 61) isolates of K. pneumoniae were positive for the blaCTX-M-1 group. One isolate showed PCR signals for blaCTX-M-25 group. None of our isolates were positive for CTX-M groups 2, 8 and 9. The majority of blaCTX-M positive isolates were genetically unrelated and no epidemic clones were identified. Conclusion: This study reports the emergence of CTX-M groups 1 and 25 producing isolates of K. pneumoniae with genetic diversity in Karachi, Pakistan.