N-Phenyl-N -(2- benzothiazolylazo)hydroxylamine as an Analytical Reagent for Pd(II), Cu(II) & Ni(II)

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Nef-mediated enhancement of cellular activation and human immunodeficiency virus type 1 replication in primary T cells is dependent on association with p21-activated kinase 2

<p>Abstract</p> <p>Background</p> <p>The HIV-1 accessory protein Nef is an important determinant of lentiviral pathogenicity that contributes to disease progression by enhancing viral replication and other poorly understood mechanisms. Nef mediates diverse functions including downmodulation of cell surface CD4 and MHC Class I, enhancement of viral infectivity, and enhancement of T cell activation. Nef interacts with a multiprotein signaling complex that includes Src family kinases, Vav1, CDC42, and activated PAK2 (p21-activated kinase 2). Although previous studies have attempted to identify a biological role for the Nef-PAK2 signaling complex, the importance of this complex and its constituent proteins in Nef function remains unclear.</p> <p>Results</p> <p>Here, we show that Nef mutants defective for PAK2-association, but functional for CD4 and MHC Class I downmodulation and infectivity enhancement, are also defective for the ability to enhance viral replication in primary T cells that are infected and subsequently activated by sub-maximal stimuli (1 μg/ml PHA-P). In contrast, these Nef mutants had little or no effect on HIV-1 replication in T cells activated by stronger stimuli (2 μg/ml PHA-P or anti-CD3/CD28-coated beads). Viruses bearing wild-type Nefs, but not Nef mutants defective for PAK2 association, enhanced NFAT and IL2 receptor promoter activity in Jurkat cells. Moreover, expression of wild-type Nefs, but not mutant Nefs defective for PAK2 association, was sufficient to enhance responsiveness of primary CD4 and CD8 T cells to activating stimuli in Nef-expressing and bystander cells. siRNA knockdown of PAK2 in Jurkat cells reduced NFAT activation induced by anti-CD3/CD28 stimulation both in the presence and absence of Nef, and expression of a PAK2 dominant mutant inhibited Nef-mediated enhancement of CD25 expression.</p> <p>Conclusion</p> <p>Nef-mediated enhancement of cellular activation and viral replication in primary T cells is dependent on PAK2 and on the strength of the activating stimuli, and correlates with the ability of Nef to associate with PAK2. PAK2 is likely to play a role in Nef-mediated enhancement of viral replication and immune activation <it>in vivo</it>.</p

Pain during ice water test distinguishes clinical bladder hypersensitivity from overactivity disorders.

BACKGROUND: The Bladder cooling reflex (BCR) i.e. uninhibited detrusor contractions evoked by intravesical instillation of cold saline, is a segmental reflex believed to be triggered by menthol sensitive cold receptors in the bladder wall, with the afferent signals transmitted by C fibres. The BCR is a neonatal reflex that becomes suppressed by descending signals from higher centres at approximately the time when the child gains full voluntary control of voiding. It re-emerges in adults with neurogenic detrusor overactivity as a consequence of loss of central descending inhibition, resulting from conditions such as spinal cord injury or multiple sclerosis. We have recently shown an increase of nerve fibres expressing the cool and menthol receptor TRPM8 in both overactive (IDO) and painful bladder syndrome (PBS), but its functional significance is unknown. We have therefore studied the bladder cooling reflex and associated sensory symptoms in patients with PBS and overactivity disorders. METHODS: The BCR, elicited by ice water test (IWT) was performed in patients with painful bladder syndrome (PBS, n = 17), idiopathic detrusor overactivity (IDO, n = 22), neurogenic detrusor overactivity (NDO, n = 4) and stress urinary incontinence (as controls, n = 21). The IWT was performed by intravesical instillation of cold saline (0 - 4 degrees C). A positive IWT was defined as presence of uninhibited detrusor contraction evoked by cold saline, associated with urgency or with fluid expulsion. Patients were asked to report and rate any pain and cold sensation during the test. RESULTS: A positive IWT was observed in IDO (6/22, 27.3%) and NDO (4/4, 100%) patients, but was negative in all control and PBS patients. Thirteen (76.5%) PBS patients reported pain during the IWT, with significantly higher pain scores during ice water instillation compared to the baseline (P = 0.0002), or equivalent amount of bladder filling (100 mls) with saline at room temperature (P = 0.015). None of the control or overactive (NDO/IDO) patients reported any pain during the IWT. CONCLUSION: The BCR in DO may reflect loss of central inhibition, which appears necessary to elicit this reflex; the pain elicited in PBS suggests afferent sensitisation, hence sensory symptoms are evoked but not reflex detrusor contractions. The ice water test may be a useful and simple marker for clinical trials in PBS, particularly for novel selective TRPM8 antagonists

Proteomic analysis of HIV-1 Nef cellular binding partners reveals a role for exocyst complex proteins in mediating enhancement of intercellular nanotube formation

<p>Abstract</p> <p>Background</p> <p>HIV-1 Nef protein contributes to pathogenesis via multiple functions that include enhancement of viral replication and infectivity, alteration of intracellular trafficking, and modulation of cellular signaling pathways. Nef stimulates formation of tunneling nanotubes and virological synapses, and is transferred to bystander cells via these intercellular contacts and secreted microvesicles. Nef associates with and activates Pak2, a kinase that regulates T-cell signaling and actin cytoskeleton dynamics, but how Nef promotes nanotube formation is unknown.</p> <p>Results</p> <p>To identify Nef binding partners involved in Pak2-association dependent Nef functions, we employed tandem mass spectrometry analysis of Nef immunocomplexes from Jurkat cells expressing wild-type Nef or Nef mutants defective for the ability to associate with Pak2 (F85L, F89H, H191F and A72P, A75P in NL4-3). We report that wild-type, but not mutant Nef, was associated with 5 components of the exocyst complex (EXOC1, EXOC2, EXOC3, EXOC4, and EXOC6), an octameric complex that tethers vesicles at the plasma membrane, regulates polarized exocytosis, and recruits membranes and proteins required for nanotube formation. Additionally, Pak2 kinase was associated exclusively with wild-type Nef. Association of EXOC1, EXOC2, EXOC3, and EXOC4 with wild-type, but not mutant Nef, was verified by co-immunoprecipitation assays in Jurkat cells. Furthermore, shRNA-mediated depletion of EXOC2 in Jurkat cells abrogated Nef-mediated enhancement of nanotube formation. Using bioinformatic tools, we visualized protein interaction networks that reveal functional linkages between Nef, the exocyst complex, and the cellular endocytic and exocytic trafficking machinery.</p> <p>Conclusions</p> <p>Exocyst complex proteins are likely a key effector of Nef-mediated enhancement of nanotube formation, and possibly microvesicle secretion. Linkages revealed between Nef and the exocyst complex suggest a new paradigm of exocyst involvement in polarized targeting for intercellular transfer of viral proteins and viruses.</p

A novel C-terminal HSP90 inhibitor KU135 induces apoptosis and cell cycle arrest in melanoma cells

Heat shock protein 90 (Hsp90) is differentially expressed in tumor cells including melanoma and involved in proper folding, stabilization and regulation of cellular proteins. We investigated a novobiocin-derived Hsp90 C-terminal inhibitor, KU135, for anti-proliferative effects in melanoma cells. The results indicate that KU135 reduced cell viability and cell proliferation in melanoma cells and IC50 values for A735(DRO), M14(NPA), B16F10 and SKMEL28 cells were 0.82, 0.92, 1.33 and 1.30 M respectively. KU135 induced a more potent anti-proliferative effect in most melanoma cells versus N-terminal Hsp90 inhibitor 17AAG. KU135 induced apoptosis in melanoma cells, as indicated by annexin V/PI staining, reduction in the mitochondrial membrane potential, mitochondrial cytochrome C release and caspase 3 activation. KU135 reduced levels of Hsp90 client proteins Akt, BRAF, RAF-1, cyclin B and cdc25 proteins. Additionally, it reduced Hsp70, Hsp90 paralog, GRP94 and HSF1 levels. KU135 induced strong G2/M cell cycle arrest, associated with decreased expression of cdc25c, cyclin B and increased phosphorylation of cdc25c. These finding show that KU135 reduced cell survival, proliferation, and induces apoptosis in melanoma cells. We suggest that KU135 may be a potential candidate for cancer therapy against melanoma

Molecular analysis of autosomal dominant hereditary ataxias in the Indian population: high frequency of SCA2 and evidence for a common founder mutation

Expansion of CTG/CAG trinucleotide repeats has been shown to cause a number of autosomal dominant cerebellar ataxias (ADCA) such as SCA1, SCA2, SCA3/MJD, SCA6, SCA7, SCA8 and DRPLA. There is a wide variation in the clinical phenotype and prevalence of these ataxias in different populations. An analysis of ataxias in 42 Indian families indicates that SCA2 is the most frequent amongst all the ADCAs we have studied. In the SCA2 families, together with an intergenerational increase in repeat size, a horizontal increase with the birth order of the offspring was also observed, indicating an important role for parental age in repeat instability. This was strengthened by the detection of a pair of dizygotic twins with expanded alleles showing the same repeat number. Haplotype analysis indicates the presence of a common founder chromosome for the expanded allele in the Indian population. Polymorphism of CAG repeats in 135 normal individuals at the SCA loci studied showed similarity to the Caucasian population but was significantly different from the Japanese population

Hierarchical model for the scale-dependent velocity of seismic waves

Elastic waves of short wavelength propagating through the upper layer of the Earth appear to move faster at large separations of source and receiver than at short separations. This scale dependent velocity is a manifestation of Fermat's principle of least time in a medium with random velocity fluctuations. Existing perturbation theories predict a linear increase of the velocity shift with increasing separation, and cannot describe the saturation of the velocity shift at large separations that is seen in computer simulations. Here we show that this long-standing problem in seismology can be solved using a model developed originally in the context of polymer physics. We find that the saturation velocity scales with the four-third power of the root-mean-square amplitude of the velocity fluctuations, in good agreement with the computer simulations.Comment: 7 pages including 3 figure