Recovered src genes are polymorphic and contain host markers

Analysis of recovered sarcoma viruses (rASV) and their parental sarcoma virus SR-D by oligonucleotide fingerprinting revealed multiple differences in the src region of the viral genomes. This heterogeneity was further investigated by tryptic peptide mapping of the in vitro translated products of rASV and SR-D RNA. No differences were found in the pr76^gag proteins encoded by the various rASVs or SR-D, but the p60^src proteins showed considerable variation. The p60^src proteins of rASV could be distinguished from that of SR-D on the basis of their mobility in SDS-polyacrylamide gels. Furthermore, two peptides which were absent from SR-D but consistently found in rASV p60^src proteins were also demonstrated in a tryptic peptide map of the cellular src-related protein, p60^sarc. These results provide strong support for the hypothesis that rASV arose by recombination of residual viral src sequences with cellular src-related sequences.     

Structural,Functional, and Clinical Characterization of a Novel PTPN11 Mutation Cluster Underlying Noonan Syndrome

Germline mutations in PTPN11, the gene encoding the Src‐homology 2 (SH2) domain‐containing protein tyrosine phosphatase (SHP2), cause Noonan syndrome (NS), a relatively common, clinically variable, multisystem disorder. Here, we report on the identification of five different PTPN11 missense changes affecting residues Leu²⁶¹, Leu²⁶², and Arg²⁶⁵ in 16 unrelated individuals with clinical diagnosis of NS or with features suggestive for this disorder, specifying a novel disease‐causing mutation cluster. Expression of the mutant proteins in HEK293T cells documented their activating role on MAPK signaling. Structural data predicted a gain‐of‐function role of substitutions at residues Leu²⁶² and Arg²⁶⁵ exerted by disruption of the N‐SH2/PTP autoinhibitory interaction. Molecular dynamics simulations suggested a more complex behavior for changes affecting Leu²⁶¹, with possible impact on SHP2's catalytic activity/selectivity and proper interaction of the PTP domain with the regulatory SH2 domains. Consistent with that, biochemical data indicated that substitutions at codons 262 and 265 increased the catalytic activity of the phosphatase, while those affecting codon 261 were only moderately activating but impacted substrate specificity. Remarkably, these mutations underlie a relatively mild form of NS characterized by low prevalence of cardiac defects, short stature, and cognitive and behavioral issues, as well as less evident typical facial features.     

Mechanism of the cardiotoxic action of palytoxin

Palytoxin (PTX) is a non-12-O-tetradecanoylphorbol-13-acetate-type tumor promoter that has potent cardiotoxic properties. In embryonic chick ventricular cells, PTX increased [Ca2+]i (K0.5 = 5 nM) in a manner that was dependent on the presence of extracellular Ca2+. The action of PTX was not consequent to its depolarizing action, to the opening of voltage-dependent Ca2+ channels, to an intracellular Na+ load, or to intracellular acidification. Flow cytometric analysis of the [Ca2+]i distribution in PTX-treated cells showed that only the largest ventricular cells responded to the toxin. All ventricular cells responded to PTX by intracellar acidification. PTX also increased 22Na+ uptake by cardiac cells (K0.5 = 100 nM) via a pathway that was sensitive to 3,4-dichlorobenzamil (K0.5 = 8 microM), suggesting a possible involvement of the Na+/Ca2+ antiporter. We conclude that the action of PTX in chick cardiac cells is distinct from that in erythrocytes or in fibroblasts and that it likely involves several distinct mechanisms. A primary action of PTX could be to open a Ca2+ uptake pathway in the plasma membrane, which would then trigger 22Na+ uptake by the Na+/Ca2+ antiporter.     

Identification and pharmacological properties of binding sites for the atypical thiazide diuretic, indapamide

[3H]Indapamide bound to a single class of binding sites in pig renal cortex membranes with a dissociation constant Kd = 35 +/- 13 nM and a binding site density Bmax = 40 +/- 9 pmol/mg of protein. [3H]Indapamide binding was inhibited by the carbonic anhydrase inhibitor, acetazolamide, and by thiazide diuretics with the following rank order of potency: chlorothiazide greater than hydrochlorothiazide approximately metolazone greater than hydroflumethiazide. The effect of the latter drugs to inhibit [3H]indapamide binding was not related to their activity as thiazide diuretics, but was significantly correlated with their inhibitory effect on carbonic anhydrase II. These results suggest that the major renal binding site of [3H]indapamide is a membrane form of carbonic anhydrase. Inhibition of carbonic anhydrase may play a role in the antihypertensive effect of indapamide.