Role of pinch in Argon impurity transport in ohmic discharges of Aditya-U Tokamak.

We present experimental results of the trace argon impurity puffing in the ohmic plasmas of Aditya-U tokamak performed to study the argon transport behaviour. Argon line emissions in visible and Vacuum Ultra Violet (VUV) spectral ranges arising from the plasma edge and core respectively are measured simultaneously. During the experiments, space resolved brightness profile of Ar1+ line emissions at 472.69 nm (3p44s 2P3/2-3p44p 2D3/2), 473.59 nm (3p44s 4P5/2-3p44p 4P3/2), 476.49 nm (3p44s 2P1/2-3p44p 2P3/2), 480.60 nm (3p44s 4P5/2-3p44p 4P5/2) are recorded using a high resolution visible spectrometer. Also, a VUV spectrometer has been used to simultaneously observe Ar13+ line emission at 18.79 nm (2s22p 2P3/2-2s2p2 2P3/2) and Ar14+ line emission at 22.11 nm (2s2 1S0-2s2p 1P1). The diffusivity and convective velocity of Ar are obtained by comparing the measured radial emissivity profile of Ar1+ emission and the line intensity ratio of Ar13+ and Ar14+ ions, with those simulated using the impurity transport code, STRAHL. Argon diffusivities ~ 12 m2/s and ~ 0.3 m2/s have been observed in the edge (ρ > 0.85) and core region of the Aditya-U, respectively. The diffusivity values both in the edge and core region are found to be higher than the neo-classical values suggesting that the argon impurity transport is mainly anomalous in the Aditya-U tokamak. Also, an inward pinch of ~ 10 m/s mainly driven by Ware pinch is required to match the measured and simulated data. The measured peaked profile of Ar density suggests impurity accumulation in these discharges.

Initial results from near-infrared spectroscopy on ADITYA-U tokamak.

Spectroscopy in vacuum ultraviolet (VUV) and visible ranges plays an important role in the investigation and diagnosis of tokamak plasmas. However, under harsh environmental conditions of fusion grade devices, such as ITER, VUV-visible systems encounter many issues due to the degradation of optical components used in such systems. Here, near-infrared (NIR) spectroscopy has become an effective tool in understanding the edge plasma dynamics. Considering its importance, a NIR spectroscopic diagnostic has been developed and installed on the ADITYA-U tokamak. The system consists of a 0.5 m spectrometer having three gratings with different groove densities, and it is coupled with a linear InGaAs photodiode array. Radiation from the ADITYA-U edge plasma has been collected using a collimating lens and optical fiber combination and transported to the spectrometer. The spectrum in the NIR range from the ADITYA-U plasma has been recorded using this system, in which Paß and Paγ along with many spectral lines from neutral and singly ionized impurities have been observed. The influxes of H and C have been estimated from measurements. The H influx value is found to be 2.8 × 1016 and 1.9 × 1016 particles cm-2 s-1 from neutral hydrogen lines Hα and Paß, respectively, and the C influx value is found to be 3.5 × 1015 and 2.9 × 1015 particles cm-2 s-1 from the neutral carbon and singly ionized carbon, respectively. A good agreement is seen between these results and the results obtained by using a routine photomultiplier tube based diagnostic.

Observations of visible argon line emissions and its spatial profile from Aditya-U tokamak plasma.

The spectroscopic studies of medium and high Z impurities have been the subject of interest in fusion research due to their role in mitigating plasma disruption and reducing heat load on the plasma facing components. Line emissions from these impurities provide the rotation velocity and ion temperature measurements along with the understanding of the overall impurity behavior in plasma. In the Aditya-U tokamak, the spatially resolved Ar II line emissions have been observed using a high resolution multi-track spectroscopic diagnostic consisting of a 1 m Czerny-Turner spectrometer coupled with a charge coupled device (CCD) detector using seven lines of sight viewing plasma tangentially along the toroidal direction. The spatially resolved Ar II lines at 458.96 nm have been observed. The singly ionized Ar emission peaks at the radial location of ρ = 0.8 of the plasma having a minor radius of 25 cm. Moreover, a 0.5 m UV-visible spectrometer coupled with a CCD detector and having a line of sight passing through the plasma midplane from the radial port was used to record visible Ar survey spectra within the 670-810 nm wavelength range, and all these lines have been identified for further analysis.

Oxidative phosphorylation-dependent regulation of cancer cell apoptosis in response to anticancer agents.

Cancer cells tend to develop resistance to various types of anticancer agents, whether they adopt similar or distinct mechanisms to evade cell death in response to a broad spectrum of cancer therapeutics is not fully defined. Current study concludes that DNA-damaging agents (etoposide and doxorubicin), ER stressor (thapsigargin), and histone deacetylase inhibitor (apicidin) target oxidative phosphorylation (OXPHOS) for apoptosis induction, whereas other anticancer agents including staurosporine, taxol, and sorafenib induce apoptosis in an OXPHOS-independent manner. DNA-damaging agents promoted mitochondrial biogenesis accompanied by increased accumulation of cellular and mitochondrial ROS, mitochondrial protein-folding machinery, and mitochondrial unfolded protein response. Induction of mitochondrial biogenesis occurred in a caspase activation-independent mechanism but was reduced by autophagy inhibition and p53-deficiency. Abrogation of complex-I blocked DNA-damage-induced caspase activation and apoptosis, whereas inhibition of complex-II or a combined deficiency of OXPHOS complexes I, III, IV, and V due to impaired mitochondrial protein synthesis did not modulate caspase activity. Mechanistic analysis revealed that inhibition of caspase activation in response to anticancer agents associates with decreased release of mitochondrial cytochrome c in complex-I-deficient cells compared with wild type (WT) cells. Gross OXPHOS deficiencies promoted increased release of apoptosis-inducing factor from mitochondria compared with WT or complex-I-deficient cells, suggesting that cells harboring defective OXPHOS trigger caspase-dependent as well as caspase-independent apoptosis in response to anticancer agents. Interestingly, DNA-damaging agent doxorubicin showed strong binding to mitochondria, which was disrupted by complex-I-deficiency but not by complex-II-deficiency. Thapsigargin-induced caspase activation was reduced upon abrogation of complex-I or gross OXPHOS deficiency whereas a reverse trend was observed with apicidin. Together, these finding provide a new strategy for differential mitochondrial targeting in cancer therapy.

Investigations of the potential effects of phosphorylation of the MWFE and ESSS subunits on complex I activity and assembly.

There have been several reports on the phosphorylation of various subunits of NADH-ubiquinone oxidoreductase (complex I) in mammalian mitochondria. The effects of phosphorylation on assembly or activity of these subunits have not been investigated directly. The cAMP-dependent phosphorylation of the MWFE and ESSS subunits in isolated bovine heart mitochondria has been recently reported. We have investigated the significance of potential phosphorylation of these two subunits in complex I assembly and function by mutational analysis of the phosphorylation sites. Chinese hamster mutant cell lines missing either the MWFE or the ESSS subunits were transfected and complemented with the corresponding wild type and mutant cDNAs made by site-directed mutagenesis. In MWFE the serine 55 was substituted by alanine, glutamate, glutamine, and aspartate (S55A, S55E, S55Q, and S55D, respectively). The glutamate substitutions might be expected to mimic the phosphorylated state of the protein. With the exception of the MWFE(S55A) mutant protein the assembly of complex I was completely blocked, and no activity could be detected. Various substitutions in the ESSS protein (S2A, S2E, S8A, S8E, T21A, T21E, S30A, S30E) appeared to cause lower levels of mature protein and a significantly reduced complex I activity measured polarographically. The ESSS (S2/8A) double mutant protein caused a complete failure to assemble. These mutational analyses suggest that if phosphorylation occurs in vivo, the effects on complex I activity are significant.

Molecular genetics of the mammalian NADH-ubiquinone oxidoreductase.

A serendipitous observation led to the first characterization of a respiration-deficient Chinese hamster mutant cell line. It has guided the design of an enrichment scheme for the isolation of additional mutant cell lines. Several complementation groups were identified with mutations affecting complex I. The X-linked NDUFA1 gene encoding the MWFE protein represents one group. Several mutant alleles isolated independently are described that yield very low activities and demonstrate that the MWFE protein is essential for activity. A phylogenetic sequence analysis of this highly conserved protein has directed attention to species-specific differences that make the primate MWFE protein inactive in hamster cells. Based on such comparisons, mutant alleles made by site-directed mutagenesis were expressed in a null mutant and reduced complex I activities were observed, with the mutant protein assembled into the complex. These and other mutants promise to be valuable for structure-function analyses, especially in conjunction with a high-resolution structure to be expected in the future. The possibility for transgenic and knock-in mice as models for mitochondrial diseases is being explored.

Regulated protein degradation controls PKA function and cell-type differentiation in Dictyostelium.

Cullins function as scaffolds that, along with F-box/WD40-repeat-containing proteins, mediate the ubiquitination of proteins to target them for degradation by the proteasome. We have identified a cullin CulA that is required at several stages during Dictyostelium development. culA null cells are defective in inducing cell-type-specific gene expression and exhibit defects during aggregation, including reduced chemotaxis. PKA is an important regulator of Dictyostelium development. The levels of intracellular cAMP and PKA activity are controlled by the rate of synthesis of cAMP and its degradation by the cAMP-specific phosphodiesterase RegA. We show that overexpression of the PKA catalytic subunit (PKAcat) rescues many of the culA null defects and those of cells lacking FbxA/ChtA, a previously described F-box/WD40-repeat-containing protein, suggesting CulA and FbxA proteins are involved in regulating PKA function. Whereas RegA protein levels drop as the multicellular organism forms in the wild-type strain, they remain high in culA null and fbxA null cells. Although PKA can suppress the culA and fbxA null developmental phenotypes, it does not suppress the altered RegA degradation, suggesting that PKA lies downstream of RegA, CulA, and FbxA. Finally, we show that CulA, FbxA, and RegA are found in a complex in vivo, and formation of this complex is dependent on the MAP kinase ERK2, which is also required for PKA function. We propose that CulA and FbxA regulate multicellular development by targeting RegA for degradation via a pathway that requires ERK2 function, leading to an increase in cAMP and PKA activity.

An F-Box/WD40 repeat-containing protein important for Dictyostelium cell-type proportioning, slug behaviour, and culmination.

FbxA is a novel member of a family of proteins that contain an F-box and WD40 repeats and that target specific proteins for degradation via proteasomes. In fruiting bodies formed from cells where the fbxA gene is disrupted (fbxA(-) cells), the spore mass fails to fully ascend the stalk. In addition, fbxA(-) slugs continue to migrate under environmental conditions where the parental strain immediately forms fruiting bodies. Consistent with this latter behaviour, the development of fbxA(-) cells is hypersensitive to ammonia, the signaling molecule that regulates the transition from the slug stage to terminal differentiation. The slug comprises an anterior prestalk region and a posterior prespore region and the fbxA mRNA is highly enriched in the prestalk cells. The prestalk zone of the slug is further subdivided into an anterior pstA region and a posterior pstO region. In fbxA(-) slugs the pstO region is reduced in size and the prespore region is proportionately expanded. Our results indicate that FbxA is part of a regulatory pathway that controls cell fate decisions and spatial patterning via regulated protein degradation.

Episomal expression of specific sense and antisense mRNAs in Leishmania amazonensis: modulation of gp63 level in promastigotes and their infection of macrophages in vitro.

The major surface glycoprotein (gp63) of Leishmania amazonensis is a metalloprotease implicated in the infection of mammalian macrophages. The expression of gp63 and its participation in this infection were further examined by modulating the level of this molecule in a virulent gp63-abundant wild-type clone. Promastigotes were transfected with gp63 genes cloned into a Leishmania-specific vector in two different orientations, leading to the expression of gp63 sense and antisense RNAs. With increasing selective pressure, cell surface gp63 was increasingly augmented in the transfectants with sense transcripts and suppressed to a very low level in those with antisense transcripts. Thus, the expression of gp63 from chromosomal, repetitive genes is not stringently regulated at the protein level and can be substantially reduced by episomal antisense transcription of a single copy. The transfectants differed significantly only in the level of gp63, thereby allowing specific evaluation of this molecule in leishmanial infection of macrophages in vitro. Kinetic studies of infection in vitro indicate that gp63 plays a role not only in the binding of this parasite to these macrophages but also in its intramacrophage survival and replication.

Characterization of EhCaBP, a calcium-binding protein of Entamoeba histolytica and its binding proteins.

A novel calcium-binding protein (EhCaBP) has been recently identified and characterized from the protozoan parasite Entamoeba histolytica. In order to decipher the function of this protein, a few basic properties were investigated and compared with the ubiquitous Ca(2+)-signal transducing protein calmodulin (CaM). Indirect immunofluorescence and immunoprecipitation analyses using specific antibodies against EhCaBP suggest that it is a soluble cytoplasmic protein with no major post-translational modification. EhCaBP did not stimulate cAMP-phosphodiesterase activity, differentiating it from all known CaMs. Affinity chromatography of [35S]methionine-labelled proteins of E. histolytica trophozoites using EhCaBP-sepharose column showed Ca(2+)-dependent binding of a group of proteins. Radiolabelled proteins from the same extract also bound to CaM-sepharose. However, the proteins bound to the two columns were different as revealed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. At least one of the EhCaBP-binding proteins became phosphorylated as revealed by in vivo phosphorylation analysis. The binding-proteins could not be detected in E. invadens (a species that is pathogenic in reptiles) and E. moshkovskii (which is found in the human gut but is not pathogenic), two species in which EhCaBP-like protein has not been found. Two distinct Ca(2+)-dependent protein kinases, which get activated by EhCaBP and CaM respectively, were detected in E. histolytica. These kinases require different levels of Ca2+ for their maximal activities. Affinity chromatography also showed the binding of protein kinase(s) to EhCaBP in a Ca(2+)-dependent manner. Our data suggest that there may be novel Ca(2+)-signal transduction pathway in E. histolytica mediated by EhCaBP.