TENB2, a proteoglycan identified in prostate cancer that is associated with disease progression and androgen independence.

TENB2 encodes a putative transmembrane proteoglycan, related to the EGF/heregulin family of growth factors and follistatin, which has been identified through the application of a differential display technique to a xenograft model of prostate cancer. Northern analysis and competitive PCR were used to demonstrate significantly increased TENB2 expression (p = 0.0003) on the acquisition of androgen independence in the model system. TENB2 is also overexpressed in clinical prostate carcinoma vs. its benign counterpart (p < 0.0001), with particular prominence in high-grade tumours, and shows a high degree of tissue specificity, being detected on a multitissue Northern array exclusively in brain and prostate material. Studies of recombinant protein expression demonstrate that TENB2 is a chondroitin sulphate proteoglycan. The presence of an EGF and 2 follistatin domains suggests a role in the regulation of growth factor signalling either as a ligand precursor, a membrane-bound receptor or as a binding protein for growth factors. These data are indicative of a significant role for TENB2 in the progression of poorly differentiated tumour types, with implications for prostate cancer detection, prognosis and therapy.

BRCA1 expression levels predict distant metastasis of sporadic breast cancers.

The role of BRCA1 in progression of sporadic breast cancers has to date been equivocal, although preliminary studies on small numbers of samples have suggested an association between expression levels of this gene and acquisition of an invasive phenotype. We have further reasoned that loss of oestrogen receptor positivity may have a detrimental effect on BRCA1 expression. In order to test this hypothesis and extend earlier investigations we have applied a sensitive RT-PCR procedure to determine the associations between BRCA1 expression and a variety of clinical parameters in a sample cohort derived from sporadic breast tumour specimens. We have established that BRCA1 and ER mRNA expression are closely associated (p=0.013), indicating a possible functional relationship between these 2 genes. We have further identified an association between low levels of BRCA1 expression and acquisition of distant metastasis in sporadic disease (p=0.019). In light of our findings, we suggest that suppression of BRCA1 has a role to play in progression of a significant fraction of sporadic breast cancers and may additionally prove to be a useful, novel, prognostic marker for this disease type.

c-erbB3 and c-erbB4 expression is a feature of the endocrine responsive phenotype in clinical breast cancer.

We examined c-erbB3 and c-erbB4 mRNA expression in 47 primary breast cancer samples by simultaneous RT-PCR and have investigated correlations between these parameters and the expression of both ER and EGFR mRNA and protein as measured by RT-PCR and ICA and with Ki67 immunostaining. A direct association was found between c-erbB3 and c-erbB4 mRNA and ER marker status measured by either RT-PCR (c-erbB3 P = 0.0003; c-erbB4 P = 0.02) or ICA (c-erbB-3 P = 0.002; c-erbB4 P = 0.01). Inverse associations were seen between c-erbB3 and c-erbB4 mRNA marker status and EGFR membrane protein (c-erbB3: P = 0.003; cerbB4: P = 0.003) and mRNA (c-erbB4: P = 0.009) status. These associations were reinforced by Spearman Rank Correlation Tests. A significant relationship was seen between Ki67 and c-erbB4 mRNA status and level. Measurements of c-erbB3 protein levels in tumour samples removed from a further 89 patients of known response to endocrine therapy: (i) confirmed the relationship between c-erbB3 and ER and (ii) identified that patients whose ER positive tumours expressed high levels of c-erbB3 were most likely to benefit from endocrine measures. A non-significant trend was recorded between c-erbB3 levels and Ki67 immunostaining. These results clearly demonstrate that increased c-erbB3 and c-erbB4 expression appears to be associated with the prognostically-favourable ER phenotype.

Molecular evolution of the aldo-keto reductase gene superfamily.

The aldo-keto reductase enzymes comprise a functionally diverse gene family which catalyze the NADPH-dependant reduction of a variety of carbonyl compounds. The protein sequences of 45 members of this family were aligned and phylogenetic trees were deduced from this alignment using the neighbor-joining and Fitch algorithms. The branching order of these trees indicates that the vertebrate enzymes cluster in three groups, which have a monophyletic origin distinct from the bacterial, plant, and invertebrate enzymes. A high level of conservation was observed between the vertebrate hydroxysteroid dehydrogenase enzymes, prostaglandin F synthase, and rho-crystallin of Xenopus laevis. We infer from the phylogenetic analysis that prostaglandin F synthase may represent a recent recruit to the eicosanoid biosynthetic pathway from the hydroxysteroid dehydrogenase pathway and furthermore that, in the context of gene recruitment, Xenopus laevis rho-crystallin may represent a shared gene.

The Xenopus laevis homologue of the 64-kDa subunit of cleavage stimulation factor.

Cleavage stimulation factor (CstF) is composed of three subunits of 50, 64 and 77 kDa, respectively. We report here the identification of a cDNA clone from Xenopus laevis encoding a homologue of the 64-kDa subunit of human CstF. Comparative sequence analysis reveals that these two proteins are highly conserved with the exception of a unique repeat structure found in the human, but not in the X. laevis, protein. Analysis of expression of this mRNA during X. laevis tadpole development indicates a requirement for this protein throughout all stages of development.

S-adenosyl-L-homocysteine hydrolase from Xenopus laevis--identification, developmental expression and evolution.

S-Adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) is an important enzyme in the trans-sulphuration pathway, mediating the conversion of S-adenosyl-L-homocysteine to adenosine and L-homocysteine. We have identified a cDNA clone from Xenopus laevis, encoding a protein of 433 aa, which is highly conserved with S-Adenosyl-L-homocysteine hydrolases (Adohcyases) from other species. Expression of Adohcyase mRNA in X.laevis tadpoles is detectable from developmental Stage 27 onwards. Phylogenetic analysis of available Adohcyase sequences indicates that species cluster essentially as predicted from morphological data. Furthermore, we estimate that S-adenosyl-L-homocysteine hydrolase is evolving very slowly, almost 10 times slower than the average rate.

Identification and characterization of a cDNA encoding ribosomal protein S12 from Xenopus laevis.

The complete nucleotide (nt) sequence of the cDNA clone XL-S12, encoding a Xenopus laevis (Xl) homologue of the mammalian ribosomal protein S12, has been determined. The sequence predicts a Xl S12 protein of 132 amino acids (aa) with a molecular mass of 14.7 kDa. Xl S12 shares 95 and 97% aa sequence identity with the human and murine S12 proteins, respectively. Analysis of nt substitution patterns and rates indicates that S12 is a very highly constrained protein, evolving at an estimated rate of only 0.03 x 10(-9) non-synonymous (protein-altering) substitutions per site per year.

Characterization, expression and evolution of mouse beta 2-glycoprotein I (apolipoprotein H).

beta 2-glycoprotein I (beta 2I) is a 50kDa serum glycoprotein of ill defined function. Based upon its capacity to bind negatively charged phospholipids a number of possible inhibitory roles for beta 2I have been proposed. We have cloned and sequenced a full length mouse beta 2I cDNA clone and demonstrated that mouse beta 2I does not behave as an acute phase reactant following an experimentally induced inflammation. Phylogenetic analysis of the known mammalian beta 2I homologues has provided evidence that mouse beta 2I is the most divergent and is evolving at a faster rate than beta 2I in other species.

Comparative analysis of the pC194 group of rolling circle plasmids.

pC194-type plasmids have been isolated from widely divergent species of bacteria: Gram positive, Proteobacteria, Spirochaetes and Cyanobacteria. We have examined the three essential replication elements of these plasmids, i.e., the Rep protein, and the origins of double and single stranded synthesis. Comparative analysis of Rep protein sequences from these plasmids indicates that they are highly divergent. Those isolated from Gram positive species fall into five groups: a Bacillus group, a Lactobacillus group, a Streptococcus group and two Staphylococcus aureus groups. The two S. aureus clusters are quite separate, suggesting that there has been at least one plasmid transfer between divergent Gram positive species. The double stranded origin of replication and the active site of the Rep protein display similarities across species indicating that these motifs can function in very divergent hosts. In contrast the single stranded origin of replication is typical of the host from which the plasmid is isolated. This is exemplified by (i) pKYM where the single stranded origins are similar to the minus origins found on the single-stranded coliphages, and (ii) pTD1 (isolated from a Spirochaete), pNostoc, pMA1 and pRF1 (all isolated from Cyanobacteria) which have no sequence homology to the minus origins identified in Gram positive or Gram negative species. This points to the single stranded origin as a feature critical to the determination of the host range of the plasmid.

Identification of a novel member of the pentraxin family in Xenopus laevis.

Pentraxins are a family of acute phase reactants. Two family members, C-reactive protein (CRP) and serum amyloid P component (SAP), are known in a range of mammalian species. CRP and SAP are both about 200 residues long, and arose from a gene duplication event, apparently before the divergence of the mammalian orders. To elucidate the origins of mammalian pentraxins, we have searched for pentraxin-coding genes in the amphibian Xenopus laevis. We have identified a gene determining a protein (XL-PXN1) which is about twice the size expected: the XL-PXN1 gene appears to be a fusion between regions encoding an amino-terminal peptide of unknown function and a carboxy-terminal pentraxin. The pentraxin domain is more divergent from CRP and SAP than they are from each other: it provides an outgroup for analysis of the evolution of mammalian pentraxins and confirms that putative CRP and SAP proteins partly characterized in non-vertebrate species cannot be true homologues of the mammalian proteins.