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Biomedical subjects

D M Worrall

Publications and source records attributed to D M Worrall.

15 recordsLinked to original sources
PubMed2001

The serine protease inhibitor antithrombin III inhibits LPS-mediated NF-kappaB activation by TLR-4.

In Drosophila, the Toll family of proteins mediates the innate immune response. Toll is activated by Spaetzle, which is generated in response to pathogens via a serine protease cascade. We wished to investigate if lipopolysaccharides (LPS) might activate Toll-like receptor (TLR) 4 via a serine protease in humans. The serpin antithrombin III (ATIII) and the thrombin inhibitor hirudin both inhibited nuclear factor (NF)-kappaB activation by LPS and Lipid A. ATIII and hirudin were also able to inhibit LPS-induced NF-kappaB activation in cells stably transfected with TLR4. These results suggest that LPS may activate a mammalian serine protease, which generates a product required for TLR4 signalling.

Antithrombin III↗
PubMed2001

SCCA2 inhibits TNF-mediated apoptosis in transfected HeLa cells. The reactive centre loop sequence is essential for this function and TNF-induced cathepsin G is a candidate target.

The squamous cell carcinoma antigens, SCCA1 and SCCA2, are members of the serine protease inhibitors (serpin) superfamily and are transcribed by two tandomly arrayed genes. A number of serpins are known to inhibit apoptosis in mammalian cells. In this study we demonstrate the ability of SCCA2 to inhibit tumor necrosis factor-alpha (TNF alpha)-induced apoptosis. HeLa cells stably transfected with SCCA2 cDNA had increased percentage cell survival and reduced DNA fragmentation. We investigated if the reactive centre loop (RCL) was necessary to allow SCCA2 to inhibit TNF alpha-mediated apoptosis. The RCL amino acids (E353Q, L354G, S355A), flanking the predicted cleavage site, were mutated and the resulting SCCA2 lost both the ability to inhibit cathepsin G and to protect stably transfected cells from TNF alpha-induced apoptosis. The presence of SCCA2 caused a decrease in the activation of caspase-3 upon induction with TNF alpha but no direct inhibition of caspases by SCCA2 has been found. Expression of cathepsin G was found to be induced in HeLa cells following treatment with TNF alpha. This protease has recently been shown to have a role in apoptosis through cleavage of substrates, so maybe the relevant target for SCCA2 in this system.

Antigens, Neoplasm↗
PubMed2000

Immunoreactivity of recombinant squamous cell carcinoma antigen and leupin/SCCA-2: implications for tumor marker detection.

OBJECTIVES: Squamous cell carcinoma antigen (SCCA) is a member of the serpin superfamily, and has been used as a serological tumor marker for cervical squamous cell carcinomas. We have identified a closely related serpin gene, leupin (SCCA-2), which may be the fraction previously thought to be the acidic isoform of SCCA. The purpose of this study is to isolate the individual recombinant proteins, to examine their reactivity with current immunological detection methods, and to use a gene-specific method to examine their expression in the uterine cervix. METHODS: We have expressed and purified recombinant forms of SCCA and leupin individually. The proteins were characterized with respect to their isoelecric points and their reactivity with the monoclonal antibody from the current tumor marker diagnostic immunoassay (IMx SCC). Reverse transcription polymerase chain reaction (RT-PCR) with gene-specific primers was used to examine expression of both genes. RESULTS: Isoelectric focusing shows that leupin is the more acidic antigen with a determined pI for recombinant leupin (rLeupin) of 6.01, with rSCCA having a pI of 6.17. The IMx SCC monoclonal antibody recognized both rSCCA and rLeupin in immunoassays and immunoblots and both genes are expressed in normal cervix and in cervical carcinoma tissue. CONCLUSIONS: The findings from this study suggest that all previous clinical studies examining SCCA expression have used methodology that detects two gene products. The confirmation that leupin or SCCA-2 is the more acidic protein and that its expression is significantly elevated in cervical cancer suggests that this gene product may be the more important tumor marker.

Antibodies, Monoclonal↗
PubMed1999

The expanding superfamily of serpins: searching for the real targets.

The serpin superfamily of proteins has expanded rapidly in recent years as represented by the ovalbumin-type serpin subfamily. PCR methods have been used to identify new ovalbumin-type serpins, including leupin or SCCA-2, a close relative of SCCA. Although in vitro inhibition of proteases by these serpins has been shown, and we have evidence that leupin can protect cells against apoptosis, the exact role of these serpins is not well understood. Finding relevant targets is a major challenge in serpin biology, and we have investigated the yeast-2-hybrid trap for identification of new interactions. Preliminary studies suggest that serpins are at the upper limit for bait size, but this technique may be useful for identifying interactions where full-length serpin protein is not required.

Animals↗
PubMed1997

Analysis of serpin inhibitory function by mutagenesis of ovalbumin and generation of chimeric ovalbumin/PAI-2 fusion proteins.

Ovalbumin is a non-inhibitory serpin which lacks the ability to undergo the S --> R transition or conformational change. Amino acid residues in the hinge region (P11 to P14) of ovalbumin and other non-inhibitory serpins differ from the concensus sequence of this region of inhibitory serpins, and have been proposed to be responsible for lack of inhibitory properties, particularly the P14 charged residue. Site directed mutagenesis using PCR overlap extension was performed on these residues in ovalbumin to create a mutant with three amino acid changes, R340T, V342A and V343A. However analysis of the mutant recombinant ovalbumin with the consensus residues failed to show inhibitory activity or decreased stability, indicating that the hinge region alone is not responsible for lack of inhibition. A series of three fusion proteins were then constructed by replacing varying C-terminal regions of ovalbumin with the corresponding region of the inhibitory ov-serpin PAI-2 in order to further analyse serpin inhibitory function. Fusion proteins F1 and F2 contained approximately 16% and 35% PAI-2, respectively. This resulted in the replacing of structural features such as the reactive site loop, hinge region and beta sheet strands 5A and 6A. However both fusion proteins showed no inhibitory activity with the PAI-2 target protease urokinase (uPA) and no decrease in stability as analysed by transverse urea gradient (TUG) gels. The third chimeric fusion protein constructed (F3) contained 64% PAI-2 and did demonstrate inhibition of uPA, SDS-PAGE stable complex formation with uPA and increased instability on TUG gels. Structural differences between the inactive F2 and active F3 include the replacement of helix F and beta sheet strand 3A of ovalbumin with those of PAI-2, suggesting that these features may have a key role in serpin beta-sheet opening and inhibitory function.

Amino Acid Sequence↗
PubMed1995

Identification of a novel human serpin gene; cloning sequencing and expression of leupin.

A novel serpin gene has been isolated, cloned and sequenced. A PCR amplified fragment of the gene was originally identified from human genomic DNA, and the full-length cDNA was subsequently isolated from HeLa cells and sequenced. The novel serpin is very high in protein sequence similarity (91.8%) to the squamous cell carcinoma antigen (SCCA), but contains substantial differences in the reactive site loop sequence, including a different amino acid (leucine) in the P1 position. The gene product, named leupin, is expressed in HeLa cells, SKGIIIa cells and human placenta. The protein has a predicted M(r) of 44,857 and an isoelectric point of 6.04 which is consistent with the more acidic form of SCCA associated with squamous cell carcinomas.

Amino Acid Sequence↗
PubMed1989

The formation of biologically active beta-galactosidase inclusion bodies in Escherichia coli.

Culture conditions affecting the formation of beta-galactosidase inclusion bodies in E. coli were examined. High temperature, early induction, high salt concentration and low aeration were all found to favour an increase of insoluble beta-galactosidase and the formation of visible inclusion bodies. The ratio of soluble to insoluble beta-galactosidase decreased during the course of cell growth. When assayed for beta-galactosidase activity, the inclusion bodies were enzymatically active with a specific activity of one third that of soluble beta-galactosidase. The activity remained associated with the inclusion bodies on washing with detergent and high ionic strength buffers. These results suggest that inclusion bodies can contain correctly folded protein.

Bacteriological Techniques↗
PubMed1986

Kinetic studies on the major form of aldehyde reductase in ox kidney: a general kinetic mechanism to explain substrate-dependent mechanisms and the inhibition by anticonvulsants.

The inhibition of the major form of ox kidney aldehyde reductase (AR 1) by sodium barbitone revealed linear mixed kinetics. This behaviour is distinct from the non-linear intercept effect we reported for valproate [Daly and Mantle (1982) Biochem. J. 205, 381]. 4-Carboxybenzaldehyde exhibits partial uncompetitive substrate inhibition. These results are discussed in terms of a model that involves nucleotide-induced isomerization and an additional flux (with some substrates and inhibitors) through an enzyme.nucleotide.substrate/inhibitor ternary complex.

Aldehyde Dehydrogenase↗
PubMed1985

Limited proteolysis of pig liver CoA synthase: evidence for subunit identity.

The bifunctional enzyme CoA synthase can be nicked by trypsin without loss of its activities. The original dimer of subunit Mr approx. 61 000 yields fragments of Mr 41 000 and 22 000 as seen on gel electrophoresis in the presence of SDS, but the nicked enzyme retains the native Mr of 118 000. Further proteolysis occurs rapidly in the absence of protecting substrates. The N-terminal of native CoA synthase is proline, and proteolysis exposes glycine as a second N-terminal. This evidence strongly suggests that the subunits are identical.

Animals↗
PubMed1983

A bifunctional enzyme complex in coenzyme A biosynthesis: purification of pantetheine phosphate adenylyltransferase and dephospho-CoA kinase.

Pantetheine phosphate adenylyltransferase (EC 2.7.7.3) and dephospho-CoA kinase (EC 2.7.1.24) were purified to near homogeneity from pig liver. The purification steps included the use of Sepharose-linked triazine dyes and affinity elution by CoA. Both activities co-purified at every stage of the 18 000-fold purification. An Mr of 115 000 was obtained by gel filtration on Sephadex G-150, and the final preparation yielded one major band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, with a subunit Mr of 57 000. It is concluded that pantetheine phosphate adenylyltransferase and dephospho-CoA kinase exist as a bifunctional dimeric protein, which could be designated CoA synthetase.

Animals↗