Tryptophan 86 of the alpha subunit in the Torpedo nicotinic acetylcholine receptor is important for channel activation by the bisquaternary ligand suberyldicholine

Suberyldicholine, a bisquaternary compound, is a potent nicotinic acetylcholine receptor agonist. Previously, we suggested that at least some of the unusual binding properties of this ligand may be a consequence of its ability to cross-link two binding "subsites" within each of the high-affinity agonist binding domains [Dunn, S. M. J., and Raftery, M. A. (1997) Biochemistry 36, 3846-3853]. Tryptophan 86 of the alpha subunit has previously been implicated in the binding of agonist to this receptor. However, on the basis of the crystal structure of a homologous acetylcholine binding protein, this residue is predicted to lie 15-20 A from the high-affinity site, i.e., a distance that approximates the interonium distance of suberyldicholine. Tryptophan 86 was mutated to either an alanine or a phenylalanine, and the mutated subunit was coexpressed with wild-type beta, gamma, and delta subunits in Xenopus oocytes. Although the alanine mutation resulted in a loss of receptor expression, the alphaW86F mutant receptor was expressed on the oocyte surface, albeit with a much reduced efficiency. Acetylcholine-evoked currents of the alphaW86F receptor were not significantly different from those of the wild type with respect to the concentration dependence of channel activation, receptor desensitization, or d-tubocurarine inhibition. In contrast, the EC(50) for suberyldicholine-mediated activation of the alphaW86F receptor was increased by approximately 500-fold. Furthermore, suberyldicholine-evoked currents in the mutant receptor did not desensitize and were insensitive to block by d-tubocurarine. Thus, tryptophan 86 of the Torpedo receptor alpha subunit may be part of a subsite for recognition of suberyldicholine and other bisquaternary ligands.     

Rapid purification method for the 26S proteasome from the filamentous fungus Trichoderma reesei

We have developed a fast and simple two column chromatographic method for the purification of the 26S proteasome from the filamentous fungus Trichoderma reesei that simplifies the overall procedure and reduces the purification time from 5 to 2.5 days. The combination of only the anionic exchange POROS^® HQ column (Applied Biosystems) together with a size exclusion column has not been used previously for proteasome purification. The purified complex was analysed further by two-dimensional electrophoresis (2DE) and examined by transmission electron microscopy (TEM). A total of 102 spots separated by 2DE were identified by mass spectrometry using cross-species identification (CSI) or an in-house custom-made protein database derived from the T. reesei sequencing project. Fifty-one spots out of 102 represented unique proteins. Among them, 30 were from the 20S particle and eight were from the 19S particle. In addition, seven proteasome-interacting proteins as well as several non-proteasome related proteins were identified. Co-purification of the 19S regulatory particle was confirmed by TEM and Western blotting. The rapidity of the purification procedure and largely intact nature of the complex suggest that similar procedure may be applicable to the isolation and purification of the other protein complexes.     

The neurobiology of schizophrenia: new leads and avenues for treatment

Recent large-scale genetic studies have provided robust evidence implicating several novel susceptibility genes for schizophrenia. These include ZNF804A, TCF4 and NRGN, which contain common variants that weakly increase schizophrenia susceptibility, and NRXN1, in which rare copy number variants have a greater impact on schizophrenia risk. Investigation of these and other substantiated susceptibility genes are providing valuable insight into the primary neurobiological mechanisms underlying schizophrenia, which may lead to novel therapeutic interventions for the disorder. In the meantime, several novel pharmacological strategies, including activation of mGluRs, elevation of synaptic glycine and inhibition of phosphodiesterase 10A, have recently shown promise for the treatment of schizophrenia in clinical trials.     

Direct activation of the ventral striatum in anticipation of aversive stimuli

The brain "reward" system, centered on the limbic ventral striatum, plays a critical role in the response to pleasure and pain. The ventral striatum is activated in animal and human studies during anticipation of appetitive/pleasurable events, but its role in aversive/painful events is less clear. Here we present data from three human fMRI studies based on aversive conditioning using unpleasant cutaneous electrical stimulation and show that the ventral striatum is reliably activated. This activation is observed during anticipation and is not a consequence of relief after the aversive event. Further, the ventral striatum is activated in anticipation regardless of whether there is an opportunity to avoid the aversive stimulus or not. Our data suggest that the ventral striatum, a crucial element of the brain "reward" system, is directly activated in anticipation of aversive stimuli.     

Functional and biochemical consequences of abrogating the activation of multiple diverse early signaling pathways in Kit. Role for Src kinase pathway in Kit-induced cooperation with erythropoietin receptor

Kit receptor tyrosine kinase and erythropoietin receptor (Epo-R) cooperate in regulating blood cell development. Mice that lack the expression of Kit or Epo-R die in utero of severe anemia. Stimulation of Kit by its ligand, stem cell factor activates several distinct early signaling pathways, including phospholipase C gamma, phosphatidylinositol 3-kinase, Src kinase, Grb2, and Grb7. The role of these pathways in Kit-induced growth, proliferation, or cooperation with Epo-R is not known. We demonstrate that inactivation of any one of these early signaling pathways in Kit significantly impairs growth and proliferation. However, inactivation of the Src pathway demonstrated the most profound defect. Combined stimulation with Epo also resulted in impaired cooperation between Src-defective Kit mutant and Epo-R and, to a lesser extent, with Kit mutants defective in the activation of phosphatidylinositol 3-kinase or Grb2. The impaired cooperation between the Src-defective Kit mutant and Epo-R was associated with reduced transphosphorylation of Epo-R and expression of c-Myc. Remarkably, restoration of only the Src pathway in a Kit receptor defective in the activation of all early signaling pathways demonstrated a 50% correction in proliferation in response to Kit stimulation and completely restored the cooperation with Epo-R. These data demonstrate an essential role for Src pathway in regulating growth, proliferation, and cooperation with Epo-R downstream from Kit.     

Evaluation of parameters for high efficiency gene transfer via particle bombardment in Indian mulberry

Particle bombardment is a popular method of direct gene delivery into cell, tissue and organs since it requires minimum pre- and post-bombardment manipulation. In addition, this technique is much easier and fast to perform with intact tissue/organ and reduces the period of in vitro culture. Genetic transformation of mulberry, Morus indica cv. K2 was attempted by particle bombardment using hypocotyl, cotyledon, leaf and leaf callus explants. The effect of various physical and biological parameters during bombardment were studied by the histochemical localization of GUS reporter gene following two days of bombardment and by assessing the number of blue spots per explant. p35SGUSINT was used for optimization of different parameters. The percentage of GUS positive explants was very low with tungsten (20%) as compared to gold particles (36%) indicating tungsten toxicity to the tissue. Maximum GUS activity was observed at 1100 psi helium pressure and 9 cm target distance for hypocotyl, cotyledon and leaf. Double bombardment of explants with 10 microg of DNA loaded on macrocarriers clearly yielded a better (up to 56%) result as compared to a single bombardment (30%). Amongst the various plasmids tested, pBI221 gave the highest (100%) GUS positive explants in the leaf callus.     

Genomics and genetics of Cryptosporidium parvum: the key to understanding cryptosporidiosis

This paper focuses on recent advances in the genetics and genomics of Cryptosporidium parvum. The approach to and the relevance of sequencing the genomes of C. parvum type 1 and type 2 are discussed, as well as new insights into the genetic heterogeneity of this species.