The SH2- and SH3-containing Nck protein transforms mammalian fibroblasts in the absence of elevated phosphotyrosine levels.

We have established the human nck sequence as a new oncogene. Nck encodes one SH2 and three SH3 domains, the Src homology motifs found in nonreceptor tyrosine kinases, Ras GTPase-activating protein, phosphatidylinositol 3-kinase, and phospholipase C-gamma. Overexpression of human nck in 3Y1 rat fibroblasts results in transformation as judged by alteration of cell morphology, colony formation in soft agar, and tumor formation in nude BALB/c mice. However, overexpression of nck does not induce detectable elevation of the phosphotyrosine content of specific proteins, as is observed for v-crk, another SH2/SH3-containing oncogene. Despite this fact, we demonstrate that Nck retains the ability to bind tyrosine phosphorylated proteins in vitro, using a fusion protein of Nck with glutathione-S-transferase (GST). Moreover, when incubated with lysates prepared from v-src-transformed 3Y1 cells or the nck-overexpressing cell lines, GST-Nck binds to both p60v-src and serine/threonine kinases, respectively. Although phosphotyrosine levels are not elevated in the nck-expressing fibroblasts, vanadate treatment of these cells results in a phosphotyrosine pattern that is altered from the parental 3Y1 pattern, suggestive of a perturbation of indigenous tyrosine kinase pathways. These results suggest the possibility that human nck induces transformation in 3Y1 fibroblasts by virtue of its altered affinity or specificity for the normal substrates of its rat homolog and that Nck may play a role in linking tyrosine and serine/threonine kinase pathways within the cell.     

Effect of chemical sympathectomy on renal hydroelectrolytic handling in dogs with chronic caval constriction

The effect of renal selective chemical sympathectomy by intrarenal infusion of 6-hydroxydopamine (6-OHDA, 5 mg/kg body weight) on the renal excretion of water and electrolytes was studied in 7 dogs in whom a syndrome of sodium and water retention and ascites formation was induced by partial constriction of thoracic inferior vena cava. Propranolol (1 mg) and phentolamine (3 mg) were also injected to obviate acute systemic hemodynamic changes. Sympathectomy was performed once in 4 dogs and three times in 3 dogs. Sympathectomy induced an abrupt and transient increase in urinary flow (from 170 +/- 30 to 890 +/- 60 ml/24 h) and sodium excretion (from 4.5 +/- 1.5 to 178 +/- 21 mEq/24 h). This was accompanied by an important fall in plasma renin activity (from 2.2 +/- 0.2 to 0.5 +/- 0.1 ng angiotensin I/ml/h) and aldosterone, and disappearance of ascites. It is concluded that chemical sympathectomy, by increasing renal sodium and water excretion, mobilizes the ascites induced by chronic caval constriction, a fact that highlights the role of the renal sympathetic system in the pathogenesis of sodium and water retention by the kidney.     

Expression of the v-crk oncogene product in PC12 cells results in rapid differentiation by both nerve growth factor- and epidermal growth factor-dependent pathways.

The transforming gene of the avian sarcoma virus CT10 encodes a fusion protein (p47gag-crk or v-Crk) containing viral Gag sequences fused to cellular sequences consisting primarily of Src homology regions 2 and 3 (SH2 and SH3 sequences). Here we report a novel function of v-Crk in the mammalian pheochromocytoma cell line, PC12, whereby stable expression of v-Crk induces accelerated differentiation, as assessed by induction of neurites following nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) treatment compared with the effect in native PC12 cells. Surprisingly, however, these cells also develop extensive neurite processes after epidermal growth factor (EGF) stimulation, an event which is not observed in native PC12 cells. Following EGF or NGF stimulation of the v-CrkPC12 cells, the v-Crk protein itself became tyrosine phosphorylated within 1 min. Moreover, in A431 cells or TrkA-PC12 cells, which overexpress EGF receptors and TrkA, respectively, a GST-CrkSH2 fusion protein was indeed capable of binding these receptors in a phosphotyrosine-dependent manner, suggesting that v-Crk can directly couple to receptor tyrosine kinase pathways in PC12 cells. In transformed fibroblasts, v-Crk binds to specific tyrosine-phosphorylated proteins of p130 and paxillin. Both of these proteins are also complexed to v-Crk in PC12 cells, as evidenced by their coprecipitation with v-Crk in detergent lysates, suggesting that common effector pathways may occur in both cell types. However, whereas PC12 cellular differentiation can occur solely by overexpression of the v-Src or oncogenic Ras proteins, that induced by v-Crk requires a growth factor stimulatory signal, possibility in a two-step process.     

A 31-amino-acid N-terminal extension regulates c-Crk binding to tyrosine-phosphorylated proteins.

Overproduction of v-Crk, but not of c-Crk, in chicken embryo fibroblasts results in cell transformation. The transforming activity of v-Crk mutants correlates with their ability to cause increased tyrosine phosphorylation of specific cellular proteins, a property that depends on the binding of v-Crk to phosphotyrosine residues via its SH2 domain. In this study, proteins translated in rabbit reticulocyte lysates were used to analyze interactions between Crk derivatives and tyrosine-phosphorylated proteins, particularly the epidermal growth factor (EGF) receptor. The results demonstrate that the binding affinity of c-Crk is much lower than that of v-Crk, despite the fact that both proteins contain identical SH2 domains. Moreover, a 31-amino-acid N-terminal extension of c-Crk, resulting from upstream translational initiation at a CUG codon, significantly increases the ability of the resulting protein to bind to phosphotyrosine-containing proteins. Of those 31 amino acids, 24 can be found in the 27-amino-acid region between Gag and Crk sequences in v-Crk, and removal of this region results in a protein with lower affinity toward the EGF receptor. In addition, fusion of Gag to the amino terminus of c-Crk yields a protein with a binding activity that is lower than that of v-Crk but significantly higher than that of c-Crk without the fusion. These data suggest that sequences N terminal to the Crk SH2 regulate binding activity to tyrosine-phosphorylated proteins and that the amino acids encoded immediately 5' to the c-Crk initiator AUG specifically increase binding affinity. In contrast, deletion of one or two SH3 domains of c-Crk proteins did not change their affinity for the EGF receptor. These results were confirmed in vivo by using A431-derived cell lines overproducing either the chicken c-Crk protein or c-Crk with the 31-amino-acid N-terminal extension. Furthermore, the in vivo experiments suggest that binding of Crk proteins to the stimulated EGF receptor results in Crk phosphorylation and subsequent loss of binding affinity.     

Post-treatment of fish canning effluents by sequential nitrification and autotrophic denitrification processes

In this research study a nitrifying/autotrophic denitrifying system was used for the post-treatment of an effluent coming from an anaerobic digester treating the wastewater produced in a fish canning industry. The nitrifying reactor achieved 100% of ammonia oxidation into nitrate. The effluent from this unit was fed to the autotrophic denitrifying reactor which treated a maximum sulphide loading rate (SLR) of 200 mg S^2?/L d with removal percentages of 100% and 30% for sulphide and nitrate, respectively. The low nitrate removal efficiency is attributed to sulphide limitations.The operational costs of this system were estimated as 0.92 €/kg N_removed, lower than those for conventional nitrification/denitrification processes. For nitrogen removal the SHARON/anammox processes is the cheapest option. However the combination of nitrification and autotrophic denitrification (using elemental sulphur) processes would present a better operational stability compared to the SHARON/anammox system.     

Simulated warming does not impair seedling survival and growth of Nothofagus pumilio in the southern Andes

It has been predicted that subalpine forests will be negatively affected by global warming; however, direct responses to experimental warming have been scarcely examined in these systems. In this study we evaluated the effects of higher temperatures with and without water addition on the survival and growth of recently emerged (small) and large seedlings of the widely distributed species Nothofagus pumilio in subalpine forests of the southern Chilean Andes. We also examined the variations in seedling traits related to carbon balance in order to infer the causal mechanisms of survival and growth responses. Treatments of open top chambers (OTCs) were combined with watering in two locations with differing climates: Antillanca (40°S, humid) and Cerro Castillo (46°S, drier). OTCs increased mean and maximum air temperatures by 0.6 °C and 2–3 °C, respectively, and decreased soil humidity by 56% in Antillanca and 30% in Cerro Castillo, fulfilling methodological expectations and climate model predictions. After two complete growing seasons, the survival, relative growth rate (RGR), biomass, and a suite of seedling traits were measured and analyzed using mixed-effects models. Warming and warming in combination with watering significantly increased large seedling survival in Cerro Castillo. In Antillanca, warmer conditions increased the height, biomass, and leaf area of small seedlings, and the RGR of large seedlings. In this location, warming also caused lower leaf carbon isotopic composition in both age classes and higher specific leaf area in small seedlings, suggesting whole-plant carbon gain improvements; warming did not produce any drought effects. Our results indicate that warming produces positive effects on the seedling establishment of N. pumilio in the southern Andes, highlighting the importance of site-specific effects in response to climate change in widespread species. Site-specific effects can most likely explain the discrepancies between the results of this study and the predictions outlined by previous studies for these forests.     

Increased Production of IL-4 and IL-12p40 from Bronchoalveolar Lavage Cells Are Biomarkers of Mycobacterium tuberculosis in the Sputum

Background

Tuberculosis (TB) causes 1.45 million deaths annually world wide, the majority of which occur in the developing world. Active TB disease represents immune failure to control latent infection from airborne spread. Acid-fast bacillus (AFB) seen on sputum smear is a biomarker for contagiousness.

Methods

We enrolled 73 tuberculosis patients with extensive infiltrates into a research study using bronchoalveolar lavage (BAL) to sample lung immune cells and assay BAL cell cytokine production. All patients had sputum culture demonstrating Mycobacterium tuberculosis and 59/73 (81%) had AFB identified by microscopy of the sputum. Compared with smear negative patients, smear positive patients at presentation had a higher proportion with smoking history, a higher proportion with temperature >38.5⁰ C, higher BAL cells/ml, lower percent lymphocytes in BAL, higher IL-4 and IL-12p40 in BAL cell supernatants. There was no correlation between AFB smear and other BAL or serum cytokines. Increasing IL-4 was associated with BAL PMN and negatively associated with BAL lymphocytes. Each 10-fold increase in BAL IL-4 and IL-12p40 increased the odds of AFB smear positivity by 7.4 and 2.2-fold, respectively, in a multi-variable logistic model.

Conclusion

Increasing IL-4 and IL-12p40 production by BAL cells are biomarkers for AFB in sputum of patients who present with radiographically advanced TB. They likely reflect less effective immune control of pathways for controlling TB, leading to patients with increased infectiousness.