Oncogenes,protein tyrosine kinases,and signal transduction

Many oncogenes encode protein tyrosine kinases (PTKs). Oncogenic mutations of these genes invariably result in constitutive activation of these PTKs. Autophosphorylation of the PTKs and tyrosine phosphorylation of their cellular substrates are essential events for transmission of the mitogenic signal into cells. The recent discovery of the characteristic amino acid sequences, of thesrc homology domains 2 and 3 (SH2 and SH3), and extensive studies on proteins containing the SH2 and SH3 domains have revealed that protein tyrosine-phosphorylation of PTKs provides phosphotyrosine sites for SH2 binding and allows extracellular signals to be relayed into the nucleus through a chain of protein-protein interactions mediated by the SH2 and SH3 domains. Studies on oncogenes, PTKs and SH2/SH3-containing proteins have made a tremendous contribution to our understanding of the mechanisms for the control of cell growth, oncogenesis, and signal transduction. This review is intended to provide an outline of the most recent progress in the study of signal transduction by PTKs.     

Managing the pattern of forest harvest: lessons from wildfire

Managing forests for sustainable use requires that both the biological diversity of the forests and a viable forest industry be maintained. A current approach towards maintaining biological diversity is to pattern forest management practices after those of natural disturbance events. This paradigm hypothesizes that ecological processes will be maintained best where active management approximates natural disturbance events. The forest management model now used in most sub-boreal and boreal forests calls for regularly dispersed clearcuts no greater than 60–100 ha in size. However, the spatial characteristics of the landscape produced by this model are distinctly different from the historic pattern generated by wildfire, which was heretofore the dominant stand-replacing process in these forests. Wildfire creates a more complex landscape spatial pattern with greater range in patch size and more irregular disturbance boundaries. Individual wildfires are often over 500 ha but leave patches of unburned forest within them. The combination of these attributes is not present in recent clearcuts. Allowing a proportion of larger (i.e.>500ha) harvest units may provide distinct economic advantages that could outweight the opportunity costs of leaving some patches of forest behind. For the forest type examined, further evaluation of modelling forest harvest patterns more closely after the patterns created by wildfire is required as it may achieve a good balance and strike a suitable compromise between certain ecological and economic objectives of sustainable development.     

Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

Aggregation-dependent turnover of flagellar adhesion molecules in chlamydomonas gametes

Previous studies on flagellar adhesion in chlamydomonas (Snell, W. and S. Roseman. 1979. J. Biol. Chem. 254:10820-10829.) have shown that as gametes adhere to flagella isolated from gametes of the opposite mating type, the adhsiveness of the added flagella but not of the gametes is lost. The studies reported here show that the addition of protein synthesis inhibitors (cycloheximide [CH] or anisomycin) to the medium of such cell- flagella mixtures causes the cells to lose their adhesiveness. This loss, however, occurs only after the cells have interacted with 4-8 flagella/cell and does not occur if the cells are kept in CH (7 h) without aggregating. The availability of an impotent (imp) mating type plus (MT(+)) mutant (provided by U.W. Goodenough), which adheres but is unable to undergo the fusion that normally follows adhesion, made it possible to determine whether a similar loss of adhesiveness occurs in mixtures of matting type minus (mt(-)) and imp mt(+) gametes. In the absence of inhibitor, mt(-) and imp mt(+) gametes adhered to each other (without fusing) for several hours; however, in the presence of CH or anisomycin, the gametes began to de-adhere 35 min after mixing, and, by 90 min, 100 percent of the cells were single again. This effect was reversible, and the rapid turnover of cells were single again. This effect was reversible, and the rapid turnover of molecules involved in adhesion occurred only during adhesion inasmuch as gametes pretreated for 4 h with CH were able to aggregate in CH for the same length of time as nonpretreated cells aggregated in CH. By the addition of CH at various times after the mt(-) and imp mt(+) gametes were mixed, measurements were made of the “pool size” of the molecules involved in adhesion. The pool reached a minimum after 25 min of aggregation, rapidly increased for the next 25 min, and then leveled off at the premixing level. These results suggest that flagellar adhesion in chlamydomonas causes modification of surface molecules (receptors, ligands), which brings about their inactivation and stimulates their replacement.     

ArfGAP1 inhibits mTORC1 lysosomal localization and activation

The mammalian target of rapamycin complex 1 (mTORC1) integrates nutrients, growth factors, stress, and energy status to regulate cell growth and metabolism. Amino acids promote mTORC1 lysosomal localization and subsequent activation. However, the subcellular location or interacting proteins of mTORC1 under amino acid‐deficient conditions is not completely understood. Here, we identify ADP‐ribosylation factor GTPase‐activating protein 1 (ArfGAP1) as a crucial regulator of mTORC1. ArfGAP1 interacts with mTORC1 in the absence of amino acids and inhibits mTORC1 lysosomal localization and activation. Mechanistically, the membrane curvature‐sensing amphipathic lipid packing sensor (ALPS) motifs that bind to vesicle membranes are crucial for ArfGAP1 to interact with and regulate mTORC1 activity. Importantly, ArfGAP1 represses cell growth through mTORC1 and is an independent prognostic factor for the overall survival of pancreatic cancer patients. Our study identifies ArfGAP1 as a critical regulator of mTORC1 that functions by preventing the lysosomal transport and activation of mTORC1, with potential for cancer therapeutics.     

Metapopulation Dynamics Enable Persistence of Influenza A,Including A/H5N1, in Poultry

Highly pathogenic influenza A/H5N1 has persistently but sporadically caused human illness and death since 1997. Yet it is still unclear how this pathogen is able to persist globally. While wild birds seem to be a genetic reservoir for influenza A, they do not seem to be the main source of human illness. Here, we highlight the role that domestic poultry may play in maintaining A/H5N1 globally, using theoretical models of spatial population structure in poultry populations. We find that a metapopulation of moderately sized poultry flocks can sustain the pathogen in a finite poultry population for over two years. Our results suggest that it is possible that moderately intensive backyard farms could sustain the pathogen indefinitely in real systems. This fits a pattern that has been observed from many empirical systems. Rather than just employing standard culling procedures to control the disease, our model suggests ways that poultry production systems may be modified.     

The Clinical Significance and Risk Factors of Anti-Platelet Factor 4/heparin Antibody on Maintenance Hemodialysis Patients: A Two-Year Prospective Follow-up

Background

Heparin-induced thrombocytopenia is an immune response mediated by anti-PF4/heparin antibody, which is clinically characterized by thrombocytopenia and thromboembolic events. In this study, a prospective and multi-center clinical investigation determined the positive rate of anti-PF4/heparin antibody in maintenance hemodialysis patients in China, identified the related risk factors, and further explored the effect of the anti-PF4/heparin antibody on bleeding, thromboembolic events, and risk of death in the patients.

Methods

The serum anti-PF4/heparin antibody was measured in 661 patients from nine hemodialysis centers, detected by IgG-specific ELISA and followed by confirmation with excess heparin. Risk factors of these patients were analyzed. Based on a two-year follow-up, the association between the anti-PF4/heparin antibody and bleeding, thromboembolic events, and risk of death in the patients was investigated.

Results

The positivity rate of the anti-PF4/heparin antibody in maintenance hemodialysis patients was 5.6%. With diabetes as an independent risk factor, the positivity rate of the anti-PF4/heparin antibody decreased in the patients undergoing weekly dialyses ≥3 times. The positivity rate of the anti-PF4/heparin antibody was not related to the occurrence of clinical thromboembolic events and was not a risk factor for death within two years in maintenance hemodialysis patients. Negativity for the anti-PF4/heparin antibody combined with a reduction of the platelet count or combined with the administration of antiplatelet drugs yielded a significant increase in bleeding events. However, the composite determination of the anti-PF4/heparin antibody and thrombocytopenia, as well as the administration of antiplatelet drugs, was not predictive for the risk of thromboembolic events in the maintenance hemodialysis patients.

Conclusions

A single detection of the anti-PF4/heparin antibody did not predict the occurrence of clinical bleeding, thromboembolic events, or risk of death in the maintenance hemodialysis patients.     

The RCN1-encoded A subunit of protein phosphatase 2A increases phosphatase activity in vivo

Protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine-specific protein phosphatase, comprises a catalytic C subunit and two distinct regulatory subunits, A and B. The RCN1 gene encodes one of three A regulatory subunits in Arabidopsis thaliana. A T-DNA insertion mutation at this locus impairs root curling, seedling organ elongation and apical hypocotyl hook formation. We have used in vivo and in vitro assays to gauge the impact of the rcn1 mutation on PP2A activity in seedlings. PP2A activity is decreased in extracts from rcn1 mutant seedlings, and this decrease is not due to a reduction in catalytic subunit expression. Roots of mutant seedlings exhibit increased sensitivity to the phosphatase inhibitors okadaic acid and cantharidin in organ elongation assays. Shoots of dark-grown, but not light-grown seedlings also show increased inhibitor sensitivity. Furthermore, cantharidin treatment of wild-type seedlings mimics the rcn1 defect in root curling, root waving and hypocotyl hook formation assays. In roots of wild-type seedlings, RCN1 mRNA is expressed at high levels in root tips, and accumulates to lower levels in the pericycle and lateral root primordia. In shoots, RCN1 is expressed in the apical hook and the basal, rapidly elongating cells in etiolated hypocotyls, and in the shoot meristem and leaf primordia of light-grown seedlings. Our results show that the wild-type RCN1-encoded A subunit functions as a positive regulator of the PP2A holoenzyme, increasing activity towards substrates involved in organ elongation and differential cell elongation responses such as root curling.