Sequence-Specific and Strand-Specific Cleavage In Oligodeoxyribonucleotides and DNA Containing 3'-Thiothymidine

Oligonucleotides containing a 3'-thiothymidine residue (T3's) at the cleavage site for the EcoRV restriction endonuclease (between the central T and A residues of the sequence GATATC) have been prepared on an automated DNA synthesizer using 5'-O-monomethoxytritylthymidine 3'-S-(2-cyanoethyl N,N-di-isopropylphosphorothioamidite). The self-complementary sequence GACGAT3'sATCGTC was completely resistant to cleavage by EcoRV, while the heteroduplex composed of 5'-TCTGAT3'sATCCTC and 5'-GAGGATATCAGA (duplex 4) was cleaved only in the unmodified strand (5'-GAGGATATCAGA). In contrast, strands containing a 3'-S-phosphorothiolate linkage could be chemically cleaved specifically at this site with Ag+. A T3's residue has also been incorporated in the (-) strand of double-stranded closed circular (RF IV) M13mp18 DNA at the cleavage site of a unique EcoRV recognition sequence by using 5'-pCGAGCTCGAT3'sATCGTAAT as a primer for polymerization on the template (+) strand of M13mp18 DNA. On treatment of this substrate with EcoRV, only one strand was cleaved to produce the RF II or nicked DNA. Taken in conjunction with the cleavage studies on the oligonucleotides, this result demonstrates that the 3'-S-phosphorothiolate linkage is resistant to scission by EcoRV. Additionally, the phosphorothiolate-containing strand of the M13mp18 DNA could be cleaved specifically at the point of modification using iodine in aqueous pyridine. The combination of enzymatic and chemical techniques provides, for the first time, a demonstrated method for the sequence-specific cleavage of either the (+) or (-) strand.

Divergence of chemical function in the alkaline phosphatase superfamily: Structure and mechanism of the P-C bond cleaving enzyme phosphonoacetate hydrolase

Phosphonates constitute a class of natural products that mimic the properties of the more common organophosphate ester metabolite yet are not readily degraded owing to the direct linkage of the phosphorus atom to the carbon atom. Phosphonate hydrolases have evolved to allow bacteria to utilize environmental phosphonates as a source of carbon and phosphorus. The work reported in this paper examines one such enzyme, phosphonoacetate hydrolase. By using a bioinformatic approach, we circumscribed the biological range of phosphonoacetate hydrolase to a select group of bacterial species from different classes of Proteobacteria. In addition, using gene context, we identified a novel 2-aminoethylphosphonate degradation pathway in which phosphonoacetate hydrolase is a participant. The X-ray structure of phosphonoformate-bound phosphonoacetate hydrolase was determined to reveal that this enzyme is most closely related to nucleotide pyrophosphatase/diesterase, a promiscuous two-zinc ion metalloenzyme of the alkaline phosphatase enzyme superfamily. The X-ray structure and metal ion specificity tests showed that phosphonoacetate hydrolase is also a two-zinc ion metalloenzyme. By using site-directed mutagenesis and P-32-labeling strategies, the catalytic nucleophile was shown to be Thr64. A structure-guided, site-directed mutation-based inquiry of the catalytic contributions of active site residues identified Lys126 and Lys128 as the most likely candidates for stabilization of the aci-carboxylate dianion leaving group. A catalytic mechanism is proposed which combines Lys12/Lys128 leaving group stabilization with zinc ion activation of the Thr64 nucleophile and the substrate phosphoryl group.

Unfunded pension liabilities and sponsoring firm credit risk: an international analysis of corporate bond spreads

This paper tests empirically whether pension information derived by corporate pension accounting disclosures is priced in corporate bond spreads. The model represents a hybrid of more traditional accounting ratio-based models of credit risk and structural models of bond spreads initiated by Merton (1974). The model is fitted to 5 years of data from 2002 to 2006 featuring companies from the US and Europe. The paper finds that while unfunded pension liabilities are priced in the overall sample, they are not priced as aggressively as traditional leverage. Furthermore, an extended model shows that the pension–credit risk relation is most evident in the US and Germany, where unfunded pension liabilities are priced more aggressively than traditional forms of leverage. No pension–credit risk relation is found in the other countries sampled, notably the UK, Netherlands and France.

Characterization of the highly conserved VFMGD motif in a bacterial polyisoprenyl-phosphate N-acetylaminosugar-1-phosphate transferase

Polyisoprenyl-phosphate N-acetylaminosugar-1-phosphate transferases (PNPTs) constitute a family of eukaryotic and prokaryotic membrane proteins that catalyze the transfer of a sugar-1-phosphate to a phosphoisoprenyl lipid carrier. All PNPT members share a highly conserved 213-Valine-Phenylalanine-Methionine-Glycine-Aspartic acid-217 (VFMGD) motif. Previous studies using the MraY protein suggested that the aspartic acid residue in this motif, D267, is a nucleophile for a proposed double-displacement mechanism involving the cleavage of the phosphoanhydride bond of the nucleoside. Here, we demonstrate that the corresponding residue in the E. coli WecA, D217, is not directly involved in catalysis, as its replacement by asparagine results in a more active enzyme. Kinetic data indicate that the D217N replacement leads to more than twofold increase in V(max) without significant change in the K(m) for the nucleoside sugar substrate. Furthermore, no differences in the binding of the reaction intermediate analog tunicamycin were found in D217N as well as in other replacement mutants at the same position. We also found that alanine substitutions in various residues of the VFMGD motif affect to various degrees the enzymatic activity of WecA in vivo and in vitro. Together, our data suggest that the highly conserved VFMGD motif defines a common region in PNPT proteins that contributes to the active site and is likely involved in the release of the reaction product.

Phosphoenolpyruvate phosphomutase activity in an L-phosphonoalanine-mineralizing strain of Burkholderia cepacia

A strain of Burkholderia cepacia isolated by enrichment culture utilized L-2-amino-3-phosphonopropionic acid (phosphonoalanine) at concentrations up to 20 mM as a carbon, nitrogen, and phosphorus source in a phosphate-insensitive manner. Cells contained phosphoenolpyruvate phosphomutase activity, presumed to be responsible for cleavage of the C-P bond of phosphonopyruvate, the transamination product of L-phosphonoalanine; this was inducible in the presence of phosphonoalanine.

A comparison of three bacterial phosphonoacetate hydrolases from different environmental sources

Cleavage of the carbon-phosphorus bond of the xenobiotic phosphonoacetate by phosphonoacetate hydrolase: represents a novel route for the microbial metabolism of organophosphonates, and is unique in that it: is substrate-inducible and its expression is independent of the phosphate status of the cell. The enzyme has previously only been demonstrated in cell extracts of Pseudomonas fluorescens 23F. Phosphonoacetate hydrolase activity is now reported in extracts of environmental Curtobacterium sp. and Pseudomonas sp. isolates capable of the phosphate-insensitive mineralization of phosphonoacetate as the sole source of carbon, energy and phosphorus at concentrations up to 40 mmol l(-1) and 100 mmol l(-1), respectively. The enzymes in both strains were similarly inducible by phosphonoacetate and had a unique specificity ibr this substrate. However, they differed significantly from each other, and from the previously described Ps. fluorescens 23F enzyme, in respect of their apparent molecular masses, temperature optima, thermostability, sensitivity to inhibition by chelating agents and by structural analogues of phosphonoacetate, and in their affinities for the substrate.

Functional study of elafin cleaved by Pseudomonas aeruginosa metalloproteinases

Elafin is a 6-kDa innate immune protein present at several epithelial surfaces including the pulmonary epithelium. It is a canonical protease inhibitor of two neutrophil serine proteases [neutrophil elastase (NE) and proteinase 3] with the capacity to covalently bind extracellular matrix proteins by transglutamination. In addition to these properties, elafin also possesses antimicrobial and immunomodulatory activities. The aim of the present study was to investigate the effect of Pseudomonas aeruginosa proteases on elafin function. We found that P aeruginosa PAO1-conditioned medium and two purified Pseudomonas metalloproteases, pseudolysin (elastase) and aeruginolysin (alkaline protease), are able to cleave recombinant elafin. Pseudolysin was shown to inactivate the anti-NE activity of elafin by cleaving its protease-binding loop. Interestingly, antibacterial properties of elafin against PAO1 were found to be unaffected after pseudolysin treatment. In contrast to pseudolysin, aeruginolysin failed to inactivate the inhibitory properties of elafin against NE. Aeruginolysin cleaves elafin at the amino-terminal Lys6-Gly7 peptide bond, resulting in a decreased ability to covalently bind purified fibronectin following transglutaminase activity. In conclusion, this study provides evidence that elafin is susceptible to proteolytic cleavage at alternative sites by P aeruginosa metalloproteinases, which can affect different biological functions of elafin.

Proteolytic cleavage of elafin by 20S proteasome may contribute to inflammation in acute lung injury

RATIONALE:
We hypothesise that elafin levels in acute lung injury (ALI) decrease over time due, in part, to proteolytic degradation as observed in other lung diseases.
OBJECTIVES:
The aim of this study was to characterise temporal changes in elafin concentration in patients with ALI and to evaluate whether a decrease in elafin levels is due to elevated protease activity.
METHODS:
Bronchoalveolar lavage fluid (BALF) was obtained from patients with ALI within 48 h of onset of ALI (day 0), at day 3 and at day 7. Elafin levels were quantified by ELISA. Elafin susceptibility to proteolytic cleavage by ALI BALF was assessed by Western blot and by high-performance liquid chromatography-mass spectrometry.
MEASUREMENTS AND MAIN RESULTS:
Elafin levels were found to be significantly increased at the onset of ALI compared with healthy volunteers and fell significantly by day 7 compared with day 0. In contrast, levels of secretory leukocyte protease inhibitor did not decrease over time. This decrease in elafin was due to cleavage by the 20S proteasome which was significantly increased in ALI BALF. Incubation of ALI BALF with the proteasome inhibitor epoxomicin confirmed that 20S proteasome protease activity was responsible for proteolytic cleavage of elafin, resulting in diminished anti-elastase activity. In addition, free neutrophil elastase activity significantly increased in ALI BALF from day 0 to day 7.
CONCLUSIONS:
Elafin concentrations fall within the pulmonary compartment over the course of ALI as a result of proteolytic degradation. This loss of elafin may predispose people, in part, to excessive inflammation in ALI.

Beyond Sex, Bond and Star Wars : Exhibition Data from the Southampton Odeon 1972-1980

This article uses the personal ledgers of a cinema manager to explore programming and film exhibition at the Southampton Odeon in the 1970s. The detailed accounts provide a rare insight into cinema exhibition and challenge the notion that 1970s cinema was all about sex, violence, horror and exploitation, suggesting instead that audiences at this cinema, favoured very different fare.