Ian Potter Foundation

The Wnt pathway plays vital roles in bone and in cancer. In this issue of The Journal of Pathology, Cai and colleagues report results that suggest that the Wnt pathway is inactivated in bone cancers, a finding that could have significant implications for the development of Wnt agonists as bone anabolic agents. While these findings are at odds with the prevailing view that the Wnt pathway is oncogenic in all systems studied to date, they remind us how complex and still poorly understood this important signalling pathway remains. At the very least, these findings should provoke debate and stimulate further research into the role of Wnt signalling in osteosarcoma.

Osteosarcoma is a devastating but rare disease, whose study has illuminated both the basic biology and clinical management of cancer over the past 30 years. These contributions have included insight into the roles of key cancer genes such as the retinoblastoma tumor suppressor gene and TP53, the identification of familial cancer syndromes implicating DNA helicases, and dramatic improvements in survival by the use of adjuvant chemotherapy. This review provides a synoptic overview of our current understanding of the molecular causes of osteosarcoma, and suggests future directions for study.

Microarray technology has undergone a rapid evolution. With widespread interest in large-scale genomic research, an abundance of equipment and reagents have now become available and affordable to a large cross section of the scientific community. As protocols become more refined, careful investigators are able to obtain good quality microarray data quickly. In most recent times, however, perhaps one of the biggest obstacles researchers face is not the manufacture and use of microarrays at the bench, but storage and analysis of the array data. This review discusses the most recent equipment, reagents and protocols available to the researcher, as well as describing data analysis and storage options available from the evolving field of microarray informatics.

Alpha1-antitrypsin deficiency, which can lead to both emphysema and liver disease, is a result of the accumulation of alpha1-antitrypsin polymers within the hepatocyte. A wealth of biochemical and biophysical data suggests that alpha1-antitrypsin polymers form via insertion of residues from the reactive center loop of one molecule into the beta-sheet of another. However, this long-standing hypothesis has not been confirmed by direct structural evidence. Here, we describe the first crystallographic evidence of a beta-strand linked polymer form of alpha1-antitrypsin: the crystal structure of a cleaved alpha1-antitrypsin polymer.

Human theta-class glutathione S-transferases (GST's) appear to play a critical role in the metabolism of a variety of environmental pollutants but in some cases the products of the reaction are carcinogenic. Crystals of a human theta-class GST, namely hGSTT2-2, have been grown from polyethylene glycol by the hanging-drop vapour-diffusion method. The crystals belong to the trigonal space group P3121 with cell dimensions of a = b = 94.0 and c = 120.5 A. They contain two monomers in the asymmetric unit and diffract to 3.0 A resolution.

The possible role of the hydroxyl group of Tyr 108 in the catalytic mechanism of human glutathione transferase P1-1 has been investigated by means of site-directed mutagenesis, steady-state kinetic analysis, and crystallographic studies. Three representative cosubstrates have been used, i.e. ethacrynic acid, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and 1-chloro-2,4-dinitrobenzene. In the presence of ethacrynic acid, the enzyme follows a rapid equilibrium random bi-bi mechanism with a rate-limiting step which occurs after the addition of the substrates and before the release of products. The replacement of Tyr 108 with Phe yields a 14-fold decrease of k(cat), while it does not change appreciably the affinity of the H site for the substrate. In this case, it would appear that the role of the hydroxyl function is to stabilize the transition state for the chemical step, i.e. the Michael addition of GSH to the electrophilic substrate. Crystallographic data are compatible with this conclusion showing the hydroxyl group of Y108 in hydrogen bonding distance of the ketone moiety of ethacrynic acid [Oakley, A. J., Rossjohn, J., Lo Bello, M., Caccuri, A. M., Federici, G., & Parker, M. W. (1997) Biochemistry 36, 576-585]. Moreover, no structural differences are observed between the Y108F mutant and the wild type, suggesting that the removal of the hydroxyl group is solely responsible for the loss of activity. A different involvement of Tyr 108 appears in the catalyzed conjugation of 7-chloro-4-nitrobenz-2-oxa-1,3-diazole with GSH in which the rate-limiting step is of a physical nature, probably a structural transition of the ternary complex. The substitution of Tyr 108 yields an approximately 7-fold increase of k(cat) and a constant k(cat)/Km(NBD-Cl) value. Lack of a critical hydrogen bond between 7-chloro-4-nitrobenz-2-oxa-1,3-diazole and Tyr 108 appears to be the basis of the increased k(cat). In the 1-chloro-2,4-dinitrobenzene/GSH system, no appreciable changes of kinetics parameters are found in the Y108F mutant. We conclude that Y108 has a multifunctional role in glutathione transferase P1-1 catalysis, depending on the nature of the electrophilic cosubstrate.