Glutathione-S-transferase (GST)-pp60 cÀSrc (GSTSrc) expressed in Escherichia coli is as catalytically active as puri¢ed, activated pp60 cÀSrc protein derived from human platelets. We utilized the bacterially expressed enzyme, together with information about the structures of Src family kinases in complex with their inhibitors PP1 and PP2, to modify PP1 in a quest for improved inhibitors. Despite the detailed structural information on Hck-PP1 and Lck-PP2 complexes, which shows that PP1 and PP2 bind to the adenosine triphosphate (ATP) pocket, we were unable to improve the a⁄nity between modi¢ed PP1 and Src. Puzzled, we examined in detail the mechanism by which PP1 inhibits the kinase activity of Src. Here we report that PP1 is non-competitive with ATP for the inhibition of Src, at variance with what is currently accepted, and is a 'mixed competitive inhibitor' vis-a '-vis the substrate. These ¢ndings shed new light on the mechanism whereby PP1-like molecules inhibit Src. Examination of the homology between the kinase domain of Src and those of Hck and Lck reveals signi¢cant di¡erences outside the ATP binding pocket, whereas they are identical within the ATP binding domain. These results suggest that PP1 may be a leading compound for ATP non-competitive inhibitors of Src family kinases. Since Src in its active form is the hallmark of numerous cancers, understanding how PP1 inhibits activated Src will aid in the discovery of potent and selective Src kinase inhibitors. ß

Objective: Data from prospective studies indicate a positive impact of gastric cancer screening programs on mortality associated with the disease. Unfortunately, endoscopic procedures, widely regarded as uncomfortable, face low patient compliance, thus underscoring the need for reliable biological markers capable of detection of tumor growth in bodily fluids. Furthermore, in light of the emerging patient-friendly, still devoid of histopathological capabilities, capsule endoscopy, gastric fluid may prove valuable for biomarker-assisted cancer diagnosis. We set out to determine whether CA72-4 measurement in gastric fluid may be of benefit for detection of gastric cancer.Design: Open prospective study.Setting: Sample collection was performed at a tertiary referral center for patients with gastroenterological diseases; immunological analysis was performed at the R&D facility of a commercial biotechnology company. Studies were part of an EU-FP6 project (NEMO).Patients: 176 patients referred for endoscopy due to gastrointestinal complaints.Interventions: Gastric juice was aspirated endoscopically according to standard operating procedures, volume and pH were measured immediately and samples stored at -80°C.Outcome measures: Concentration of CA72-4 tumor marker was evaluated by enzyme-linked immunosorbent assay (ELISA).Results: Median CA72-4 levels were about 4-fold higher in cancer patients compared with patients with normal gastric findings, gastric inflammation, intestinal metaplasia or other diseases (p=0.001). Multivariate linear regression analysis revealed that elevated CA72-4 was significantly predicted by gastric carcinoma adjusted for H. pylori status, age, smoking status, PPI dose, and pH of aspirate (R2=0.27, p<0.0001). In this model, diagnosis of gastric carcinoma had by far the greatest influence. At a cut-off level of 100 U/ml, CA72-4 had 75% sensitivity and 89% specificity for detection of gastric cancer.Conclusions: Based on our findings, CA72-4 level assessment in gastric fluid, featuring yet unmatched accuracy of malignant neoplasia detection may prove beneficial for gastric cancer screening.

Human I-mfa domain containing protein (HIC) differentially regulates transcription from viral promoters. HIC affects the Wnt pathway, the JNK/SAPK pathway and the activity of positive transcription elongation factor-b (P-TEFb). Studies exploring HIC function in mammalian cells used ectopically expressed HIC due to undetected endogenous HIC protein. HIC mRNA contains exceptionally long 5′ and 3′ untranslated regions (UTRs) compared to the average length of mRNA UTRs. Here we show that HIC protein is subject to strict repression at multiple levels. The HIC mRNA UTRs reduce the expression of HIC or of a reporter protein: The HIC 3′-UTR decreases both HIC and reporter mRNA levels, whereas upstream open reading frames located in the 5′-UTR repress the translation of HIC or of the reporter protein. In addition, ectopically expressed HIC protein is degraded by the proteasome, with a half-life of approximately 1 h, suggesting that upon activation, HIC expression in cells may be transient. The strict regulation of HIC expression at the levels of mRNA stability, translation efficiency and protein stability suggests that expression of the HIC protein and its involvement in the various pathways is required only under specific cellular conditions.

Data from prospective studies indicate a positive impact of Objective