Systematic mapping of potential binding sites for Shc and Grb2 SH2 domains on insulin receptor substrate-1 and the receptors for insulin, epidermal growth factor, platelet-derived growth factor, and fibroblast growth factor.

Multipin peptide synthesis has been employed to produce biotinylated 11-mer phosphopeptides that account for every tyrosine residue in insulin receptor substrate-1 (IRS-1) and the cytoplasmic domains of the insulin-, epidermal growth factor-, platelet-derived growth factor- and basic fibroblast growth factor receptors. These phosphopeptides have been screened for their capacity to bind to the SH2 domains of Shc and Grb in a solution phase enzyme-linked immunosorbent assay. The data revealed new potential Grb2 binding sites at Tyr-1114 (epidermal growth factor receptor (EGFR) C-tail); Tyr-743 (platelet-derived growth factor receptor (PDGFR) insert region), Tyr-1110 from the E-helix of the catalytic domain of insulin receptor (IR), and Tyr-47, Tyr-939, and Tyr-727 in IRS-1. None of the phosphopeptides from the juxtamembrane or C-tail regions of IR bound Grb2 significantly, and only one phosphopeptide from the basic fibroblast growth factor receptor (Tyr-556) bound Grb2 but with medium strength. Tyr-1068 and -1086 from the C-tail of EGFR, Tyr-684 from the kinase insert region of PDGFR, and Tyr-895 from IRS-1 were confirmed as major binding sites for the Grb2 SH2 domain. With regard to Shc binding, the data revealed new potential binding sites at Tyr-703 and Tyr-789 from the catalytic domain of EGFR and at Tyr-557 in the juxtamembrane region of PDGFR. It also identified new potential Shc binding sites at Tyr-764, in the C-tail of basic fibroblast growth factor receptor, and Tyr-960, in the juxtamembrane of IR, a residue previously known to be required for Shc phosphorylation in response to insulin. The study confirmed the previous identification of Tyr-992 and Tyr-1173 in the C-tail of EGFR and several phosphopeptides from the PDGFR as medium strength binding sites for the SH2 domain of Shc. None of the 34 phosphopeptides from IRS-1 bound Shc strongly, although Tyr-690 showed medium strength binding. The specificity characteristics of the SH2 domains of Grb2 and Shc are discussed. This systematic peptide mapping strategy provides a way of rapidly scanning candidate proteins for potential SH2 binding sites as a first step to establishing their involvement in kinase-mediated signaling pathways.

Comparative analysis of the restriction endonuclease profiles of the Dumas and Singapore strains of varicella-zoster virus.

The incidence of varicella in Singapore has been increasing since 1984. In 1991, 17,930 cases were reported in a population of about 3 million. A serological survey completed in 1990 demonstrated that only 43% of the cohort had antibodies to varicella-zoster virus (VZV), indicating inadequate herd immunity. To exclude novel VZV strains, representative VZV isolates from 9 chicken pox and 4 zoster patients were characterised by restriction endonuclease analysis. DNAs were extracted from viral isolates propagated in MRC5 human embryo lung cells and were digested separately with BglII, EcoRI, PstI, SalI, and XbaI enzymes. The cleavage profiles of these VZV strains derived from both chicken pox and zoster lesions revealed no distinct differences. This observation implies that the current upsurge of chicken pox most likely stems from closely related VZV genotypes infecting a susceptible population with insufficient herd immunity. Comparison of the restriction fragments of the Singapore and the Dumas strains revealed polymorphisms of the SalI-D, SalI-E, and XbaI-I fragment lengths, which correlated with variable regions I, II, and III of the VZV genome, thereby representing geographically distinct genotypic variants of VZV.

Metabolic clearance rate of cortisol in pigs: relationship to adrenal responsiveness.

Experiments were conducted on 12 prepuberal (18- to 20-week-old) Landrace cross Large White gilts to establish if differences in adrenal responsiveness between individuals could be explained by differences in the metabolic clearance rate (MCR) of cortisol. Pigs with the highest (n = 6) and lowest (n = 6) cortisol concentration 60 minutes after challenge with ACTH were selected from a pool of 36 commercial pigs. Tritium-labelled cortisol was infused (17 to 27 ml h-1) continuously for 120 minutes to establish 'steady state' conditions. Blood samples (10 ml) were collected at 90, 100, 110 and 120 minutes. Replicate experiments were performed on some pigs. Classification of individual pigs as high or low adrenal responders to ACTH challenge was confirmed at the end of the clearance rate experiments. The MCR of cortisol in the group classed as low adrenal responders was 59.7 +/- 7.8 litres h-1 or 1.01 litres h-1 kg-1 (n = 7) which was not significantly different from the average MCR in the group classed as high adrenal responders 60.2 +/- 5.9 litres h-1 or 1.19 litres h-1 kg-1 (n = 10). These results suggest that the repeatable differences in adrenal responsiveness to ACTH that exist between individuals within a particular strain of pig depend on differences in the rate of synthesis of cortisol in response to ACTH stimulation, rather than on differences in its rate of metabolism.