Measurements of magnetic field fluctuations in TJ-II plasmas with new in-vessel helical arrays of magnetic coils.

This paper describes two new helical arrays of magnetic coils recently installed inside the TJ-II vacuum vessel. Their main objective is the precise measurement of the spatial periodicity of the magnetohydrodynamic perturbations usually found in the TJ-II plasmas. Given the high probability of coil failures due to the harsh plasma environment and in view of the extremely difficult access to the TJ-II vessel interior for maintenance, the coil system has been divided in two quasi-identical helical arrays. Both arrays consist of 32 triaxial sensors measuring orthogonal components of the local magnetic field along an ideal helical path whose trajectory runs close to the plasma edge. A description of the main characteristics of coils and arrays as well as their nominal positioning along an ideal helical path, inside the vessel, is given. A precise experimental determination of the real spatial orientation of the coils is performed by comparing the signals measured in current ramp-up and ramp-down experiments with calculations based on a filamentary model for the TJ-II magnetic coils. After this fine calibration procedure, it is possible to analyze the dependence of the amplitude of the measured magnetic field and its fluctuations as a function of the coil distance to the last closed flux surface. The study of the phase evolution of the parallel and perpendicular oscillatory components is also enabled. Finally, two examples of mode number determination are shown. One corresponds to a low frequency mode appearing in pure electron cyclotron resonance heating plasma, and the other one shows several modes observed during combined injection of both co and counter neutral beams and identified as shear Alfvén waves.

Function and regulation of RhoE.

The three Rnd proteins, Rnd1, Rnd2 and RhoE/Rnd3, are a subset of Rho family proteins that are unusual in that they bind but do not hydrolyse GTP, and are therefore not regulated by the classical GTP/GDP conformational switch of small GTPases. Increased expression of each Rnd protein induces loss of stress fibres in cultured fibroblasts and epithelial cells, acting antagonistically to RhoA, which stimulates stress fibre formation. RhoE is farnesylated and localizes partly on membranes, including the Golgi and plasma membrane, and in the cytosol. RhoE inhibits RhoA signalling in part by binding to the RhoA-activated serine/threonine kinase ROCK I (Rho-associated kinase I), thereby preventing it from phosphorylating its targets. RhoE activity is itself regulated by phosphorylation by ROCK I on multiple sites. RhoE phosphorylation enhances its stability, leading to an increase in RhoE levels. In addition, phosphorylation reduces its association with membranes and correlates with its ability to induce loss of stress fibres. RhoE also acts independently of ROCK to inhibit cell cycle progression, in part by preventing translation of cyclin D1, and to inhibit transformation of fibroblasts by oncogenic H-Ras. RhoE is therefore a multifunctional protein whose localization and actions are regulated by phosphorylation.

Calmodulin binds to K-Ras, but not to H- or N-Ras, and modulates its downstream signaling.

Activation of Ras induces a variety of cellular responses depending on the specific effector activated and the intensity and amplitude of this activation. We have previously shown that calmodulin is an essential molecule in the down-regulation of the Ras/Raf/MEK/extracellularly regulated kinase (ERK) pathway in cultured fibroblasts and that this is due at least in part to an inhibitory effect of calmodulin on Ras activation. Here we show that inhibition of calmodulin synergizes with diverse stimuli (epidermal growth factor, platelet-derived growth factor, bombesin, or fetal bovine serum) to induce ERK activation. Moreover, even in the absence of any added stimuli, activation of Ras by calmodulin inhibition was observed. To identify the calmodulin-binding protein involved in this process, calmodulin affinity chromatography was performed. We show that Ras and Raf from cellular lysates were able to bind to calmodulin. Furthermore, Ras binding to calmodulin was favored in lysates with large amounts of GTP-bound Ras, and it was Raf independent. Interestingly, only one of the Ras isoforms, K-RasB, was able to bind to calmodulin. Furthermore, calmodulin inhibition preferentially activated K-Ras. Interaction between calmodulin and K-RasB is direct and is inhibited by the calmodulin kinase II calmodulin-binding domain. Thus, GTP-bound K-RasB is a calmodulin-binding protein, and we suggest that this binding may be a key element in the modulation of Ras signaling.

[Lys61]N-Ras is able to induce full activation and nuclear accumulation of Cdk4 in NIH3T3 cells.

The elements of the cell cycle regulatory machinery activated by the oncogenic form of Ras, [Lys61]N-Ras, have been analysed in NIH3T3 cells. We demonstrate that [Lys61]N-Ras expression is able to induce full cdk4 activation. As already reported, oncogenic Ras expression was sufficient to induce cyclin D1 and p21cip1 expression and their association with cdk4. Furthermore, serum-starved [Lys61]N-Ras NIH3T3 cells showed nuclear accumulation of cyclin D1 and cdk4 not observed in serum-starved NIH3T3 cells. This accumulation of cdk4 into the cell nucleus observed in serum-starved [Lys61]N-Ras NIH3T3 cells was inhibited by a microinjection of neutralizing anti-Ras antibodies. Thus, active [Lys61]N-Ras was a sufficient signal to induce nuclear accumulation of cyclin D1/cdk4, leading to its full activation. Transfection of [Lys61]N-Ras NIH3T3 cells with an inactive form of MEK or their treatment with PD 98059, showed that nuclear translocation of cdk4 was MEK dependent. Interestingly, cells constitutively expressing [Lys61]N-Ras did not inactivate pRb and did not proliferate in the absence of serum. This may be due to the fact that although association of cdk2 with cyclin E and the translocation of those complexes to the nucleus were achieved, [Lys61]N-Ras expression was not sufficient to induce cdk2 activation. The high levels of p27(kip1) that were found in cyclin E/cdk2 complexes may be responsible for the inability of oncogenic Ras to activate this kinase. In consequence, oncogenic alterations that lead to a decrease in p27kip1 bound to cyclin E may cooperate with Ras to induce full cdk2 activation, pRb inactivation and thus cell proliferation.

Disruption of the antiproliferative TGF-beta signaling pathways in human pancreatic cancer cells.

Resistance to TGF-beta1 occurred in pancreatic cancer cells suggesting that inactivation of TGF-beta inhibitory signaling pathways may play an important role in human pancreatic cancer. The aim of our study was to determine the presence of alterations in the main putative components of the TGF-beta inhibitory signaling pathways (p15, Smad4, Smad2, TGFbeta-RII, CDC25A). A panel of human carcinomas of the exocrine pancreas orthotopically implanted and perpetuated in nude mice and pancreatic cancer cell lines were studied. p15 gene alterations, mainly homozygous deletions that involved exons 1 and/or 2, were found in the 62.5% (5 of 8) of pancreatic xenografts whereas Smad4 gene aberrations were found in one of eight xenografts and in two of seven cell lines. Additional aberrations in these genes were acquired during in vivo perpetuation and distal dissemination. Paradoxically, TGFbeta-RII overexpression and a decrease in CDC25A protein levels were found in all tumors and cell lines. In one cell line, resistance to TGF-beta1 occurred in the absence of alterations in the genes analysed so far. We conclude that all human pancreatic tumor cells analysed herein have non-functional TGF-beta pathways. The majority of cells harbor alterations in at least one of the putative components of TGF-beta pathways, mainly in p15 and Smad4 genes. These results suggest that inactivation of TGF-beta signaling pathways plays an important role in human pancreatic tumorigenesis.