BL-02: a versatile X-ray scattering and diffraction beamline for engineering applications at Indus-2 synchrotron source.

A hard X-ray engineering applications beamline (BL-02) was commissioned recently and started operation in March 2019 at the Indian synchrotron source, Indus-2. This bending-magnet-based beamline is capable of operating in various beam modes, viz. white, pink and monochromatic beam. The beamline utilizes the X-ray diffraction technique in energy-dispersive and angle-dispersive modes to carry out experiments mainly focused on engineering problems, viz. stress measurement, texture measurement and determination of elastic constants in a variety of bulk as well as thin-film samples. An open-cradle six-circle diffractometer with ∼12 kg load capacity allows accommodation of a wide variety of engineering samples and qualifies the beamline as a unique facility at Indus-2. The high-resolution mode of this beamline is suitably designed so as to carry out line profile analysis for characterization of micro- and nano-structures. In the present article the beamline is described starting from the beamline design, layout, optics involved, various operational modes and experimental stations. Experiments executed to validate the beamline design parameters and to demonstrate the capabilities of the beamline are also described. The future facilities to be incorporated to enhance the capabilities of the beamline are also discussed.

The p75 neurotrophin receptor activates Akt (protein kinase B) through a phosphatidylinositol 3-kinase-dependent pathway.

The Akt kinase plays a crucial role in supporting Trk-dependent cell survival, whereas the p75 neurotrophin receptor (p75NTR) facilitates cellular apoptosis. The precise mechanism that p75NTR uses to promote cell death is not certain, but one possibility is that p75NTR-dependent ceramide accumulation inhibits phosphatidylinositol 3-kinase-mediated Akt activation. To test this hypothesis, we developed a system for examining p75NTR-dependent apoptosis and determined the effect of p75NTR on Akt activation. Surprisingly, p75NTR increased, rather than decreased, Akt phosphorylation in a variety of cell types, including human Niemann-Pick fibroblasts, which lack acidic sphingomyelinase activity. The p75NTR expression level required to elicit Akt phosphorylation was much lower than that required to activate the JNK pathway or to mediate apoptosis. We show that p75NTR-dependent Akt phosphorylation was independent of TrkA signaling, required active phosphatidylinositol 3-kinase, and was associated with increased tyrosine phosphorylation of p85 and Shc and with reduced cytosolic tyrosine phosphatase activity. Finally, we show that p75NTR expression increased survival in cells exposed to staurosporine or subjected to serum withdrawal. These findings indicate that p75NTR facilitates cell survival through novel signaling cascades that result in Akt activation.

NRAGE, a novel MAGE protein, interacts with the p75 neurotrophin receptor and facilitates nerve growth factor-dependent apoptosis.

The mechanisms employed by the p75 neurotrophin receptor (p75NTR) to mediate neurotrophin-dependent apoptosis are poorly defined. Two-hybrid analyses were used to identify proteins involved in p75NTR apoptotic signaling, and a p75NTR binding partner termed NRAGE (for neurotrophin receptor-interacting MAGE homolog) was identified. NRAGE binds p75NTR in vitro and in vivo, and NRAGE associates with the plasma membrane when NGF is bound to p75NTR. NRAGE blocks the physical association of p75NTR with TrkA, and, conversely, TrkA overexpression eliminates NRAGE-mediated NGF-dependent death, indicating that interactions of NRAGE or TrkA with p75NTR are functionally and physically exclusive. NRAGE overexpression facilitates cell cycle arrest and permits NGF-dependent apoptosis within sympathetic neuron precursors cells. Our results show that NRAGE contributes to p75NTR-dependent cell death and suggest novel functions for MAGE family proteins.

The p75 neurotrophin receptor (p75NTR) alters tumor necrosis factor-mediated NF-kappaB activity under physiological conditions, but direct p75NTR-mediated NF-kappaB activation requires cell stress.

The p75 neurotrophin receptor (p75NTR) has been linked to activation of the NF-kappaB transcriptional complex in oligodendrocytes, Schwann cells, and PCNA cells. In this report, tumor necrosis factor (TNF)- and neurotrophin-mediated NF (nuclear factor)-kappaB activation were compared in several cell lines. All cell types showed TNF-mediated activation of NF-kappaB, but direct neurotrophin-dependent activation of NF-kappaB was never observed under normal growth conditions. In PCNA cells, a modest nerve growth factor (NGF)-dependent induction of NF-kappaB was detected but only after cells were subjected to severe stress. Although NGF binding did not directly activate NF-kappaB under normal conditions, NGF consistently altered TNF-dependent NF-kappaB activation in each cell type examined, and extended exposure to NGF and TNF always increased NF-kappaB activation over that achieved with TNF alone. The increase in NF-kappaB activity mediated by NGF correlated with reduced levels of IkappaBalpha; NGF added alone had no effect on IkappaBalpha levels, but when added with TNF, NGF treatment significantly reduced IkappaBalpha levels. We propose that modulation of cytokine receptor signaling is a significant physiological function of the p75 neurotrophin receptor and that previous reports of direct NF-kappaB activation through p75NTR reflect this modulatory activity.

p75 neurotrophin receptor expression on adult human oligodendrocytes: signaling without cell death in response to NGF.

Oligodendrocytes (OLs) are the primary targets in the autoimmune disease multiple sclerosis (MS). Cell receptors belonging to the tumor necrosis factor receptor (TNF-R) superfamily, such as TNF receptors and fas, are implicated in signaling the injury response of OLs. The p75 neurotrophin receptor (p75(NTR)), another member of the TNF-R superfamily, has been reported to mediate nerve growth factor (NGF)-induced apoptosis in some neural systems. To address the potential relationship between p75(NTR) signaling and OL injury, we assayed adult human OLs cultured under several different conditions for p75(NTR) and tyrosine kinase receptor trkA expression, for NGF-mediated apoptosis, and for NGF-mediated jun kinase (JNK) or nuclear factor (NF) kappaB activation. In the current study, we have found expression of p75(NTR) on cultured adult CNS-derived human OLs but not on other glial cells. TrkA was not detected on these OLs in any of the culture conditions tested. Treatment of the OLs with varying concentrations of NGF over a range of time periods resulted in no significant increase in numbers of terminal transferase (TdT)-mediated d-uridine triphosphate (UTP)-biotin nick-end labeling positive OLs, whereas significant cell death was observed using TNF-alpha. Furthermore, unlike TNF-alpha treatment, NGF treatment did not significantly activate JNK, although both TNF-alpha and NGF induced nuclear translocation of NF-kappaB. These findings contrast with the recent report of NGF-mediated apoptosis in the OLs of neonatal rats matured in vitro, which express p75(NTR) but not trkA (), and suggest that, at least in humans, p75(NTR) signaling may mediate responses other than apoptosis of OLs.

Transgenic mice expressing the intracellular domain of the p75 neurotrophin receptor undergo neuronal apoptosis.

We have asked whether p75(NTR) may play a role in neuronal apoptosis by producing transgenic mice that express the p75(NTR) intracellular domain within peripheral and central neurons. These animals showed profound reductions in numbers of sympathetic and peripheral sensory neurons as well as cell loss in the neocortex, where there is normally little or no p75(NTR) expression. Developmental loss of facial motor neurons was not observed, but induced expression of the p75(NTR) intracellular domain within adult animals led to increased motor neuron death after axotomy. Biochemical analyses suggest that these effects were not attributable to a p75(NTR)-dependent reduction in trk activation but instead indicate that the p75(NTR) intracellular domain may act as a constitutive activator of signaling cascades that regulate apoptosis in both peripheral and central neurons.

Metabolism and biologic effects of 5-oxoeicosanoids on human neutrophils.

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a recently discovered metabolite of arachidonic acid that activates human neutrophils by a mechanism independent of the receptor for leukotriene B4 (LTB4). The objectives of this study were to identify the major metabolites of 5-oxo-ETE in neutrophils and to compare the biologic activities of 5-oxo-ETE with those of its metabolites and other 5-oxoeicosanoids. Neutrophils rapidly converted 5-oxo-ETE to its omega-oxidation product, 5-oxo-20-hydroxy-6E,8Z,11Z,14Z- eicosatetraenoic acid. This compound was nearly 100 times less potent than 5-oxo-ETE in elevating cytosolic calcium levels in neutrophils. Methylation of the carboxyl group of 5-oxo-ETE resulted in a 20-fold loss of potency, whereas replacement of the 8,9-cis double bond by a trans double bond reduced potency by about sixfold. Similar results were obtained for the effects of the above compounds on neutrophil migration. 5-Oxo-20-hydroxy-6E,8Z,11Z,14Z- eicosatetraenoic acid, 5-oxo-8-trans-ETE, and 5-oxo-ETE methyl ester desensitized neutrophils to 5-oxo-ETE. 5-Oxo-ETE-induced calcium mobilization was inhibited by pretreatment of the cells with pertussis toxin. 5-Oxo metabolites of 6-trans-LTB4 and 12-epi-6-trans-LTB4 had weak stimulatory effects on calcium levels and migration that appeared to be mediated primarily by stimulation of LTB4 receptors. These studies indicate that the 5-oxo group, the omega-end of the molecule, and the carboxyl group are all important for the biologic activity of 5-oxo-ETE, which may be mediated by a G protein-linked receptor. The biologic activity of 5-oxo-ETE can be terminated by omega-oxidation.