SCCRO (DCUN1D1) promotes nuclear translocation and assembly of the neddylation E3 complex

SCCRO/DCUN1D1/DCN1 (squamous cell carcinoma-related oncogene/defective in cullin neddylation 1 domain containing 1/defective in cullin neddylation) serves as an accessory E3 in neddylation by binding to cullin and Ubc12 to allow efficient transfer of Nedd8. In this work we show that SCCRO has broader, pleiotropic effects that are essential for cullin neddylation in vivo. Reduced primary nuclear localization of Cul1 accompanying decreased neddylation and proliferation in SCCRO(-/-) mouse embryonic fibroblasts led us to investigate whether compartmentalization plays a regulatory role. Decreased nuclear localization, neddylation, and defective proliferation in SCCRO(-/-) mouse embryonic fibroblasts were rescued by transgenic expression of SCCRO. Expression of reciprocal SCCRO and Cul1-binding mutants confirmed the requirement for SCCRO in nuclear translocation and neddylation of cullins in vivo. Nuclear translocation of Cul1 by tagging with a nuclear localization sequence allowed neddylation independent of SCCRO, but at a lower level. We found that in the nucleus, SCCRO enhances recruitment of Ubc12 to Cul1 to promote neddylation. These findings suggest that SCCRO has an essential role in neddylation in vivo involving nuclear localization of neddylation components and recruitment and proper positioning of Ubc12.     

Solution structure and dynamics of the single-chain hepatitis C virus NS3 protease NS4A cofactor complex

The backbone assignments, secondary structure, topology, and dynamics of the single-chain hepatitis C virus NS3 protease NS4A cofactor complex have been determined by NMR spectroscopy. Residues I34 to S181 of NS3 and the central three residues of the NS4A cofactor were assigned and the secondary structure was verified for these residues. In several X-ray structures of NS4A-bound NS3 protease, residues 1 to 28 are stabilized by crystal packing, which allows for the formation of the A0 strand and alpha0 helix. In solution, these N-terminal residues are largely unassigned and no evidence of a well-structured A0 strand or alpha0 helix was detected. NOEs between residues in the E1-F1 loop (containing D81) and the alpha1 helix (containing H57) together with the detection of a D81-H57 hydrogen bond indicate that in solution the catalytic triad (D81, H57, S139) of the protease is better ordered in the presence of the NS4A cofactor. This is consistent with the earlier crystallographic results and may explain the observed increase in catalytic activity of the enzyme due to NS4A binding. A model-free analysis of our relaxation data indicates substantial exchange rates for residues V51-D81, which comprise the upper part of the N-terminal beta-barrel. A comparison of chemical-shift differences between NS3 protease and the NS3 protease-NS4A complex shows extensive chemical-shift changes for residues V51-D81 indicating that non-local structural changes occur upon NS4A binding to the NS3 protease that are propagated well beyond the protease-cofactor interaction site. This is consistent with crystallographic data that reveal large structural rearrangements of the strand and loop regions formed by residues V51-D81 as a result of NS4A binding. The coincidence of large exchange rates for the NS3 protease-NS4A complex with chemical-shift differences due to NS4A binding suggests that residues V51-D81 of the NS3 protease NS4A complex are in slow exchange with a NS4A-free conformation of NS3 protease.     

Experimental methods for evaluation of psychotropic agents in rodents: II-Antidepressants

Rodent models of clinical depression are extensively used for the evaluation of putative antidepressants. In the present review, the available experimental methods which can be utilized by most laboratories involved in preclinical screening of antidepressants, have been discussed. The methods have been categorized on the basis of induction of the depressive state or on the assumption that monoamine deficiency leads to depression. These methods have been critically validated in terms of efficacy of standard antidepressants in these tests and, in some cases, by the neurochemical basis of depression, namely, the deficient monoaminergic theory of clinical depression.     

Time course studies on the functional evaluation of experimental chronic myocardial infarction in rats

In vivo models of myocardial infarction induced by coronary artery ligation (CAL) in rats usually suffer from high early mortality and a low rate of induction. This study investigated the time course initiation of chronic myocardial infarction (CMI) in albino rats and the possibility of reducing early mortality rate due to myocardial infarction by modification of the surgical technique. CAL was carried out by passing the suture through the epicardial layer around the midway of the left anterior descending coronary artery including a small area of the myocardium to avoid mechanical damage to the heart geometry. In addition, the role of endothelin-1 (ET-1) in rat heart with congestive heart failure was critically assessed. Time course initiation experiments were designed by sacrificing the animals at different time intervals and by carrying out physiological, biochemical, histopathological, electron microscopical and immunohistochemical studies. Specific markers of myocardial injury, viz. cardiac troponin-T (cTnT), high sensitivity C-reactive protein, lactate dehydrogenase and fibrinogen were measured at different time points. Serum marker enzymes and activities of lysosomal hydrolases were found to be elevated on the eighth day post-ligation. Histopathological studies demonstrated focal areas showing fibrovascular tissue containing fibroblasts, collagenous ground substance and numerous small capillaries replacing cardiac muscle fibers. Transmission electron micrographs exhibited mitochondrial changes of well-developed irreversible cardiac injury, viz. swelling, disorganization of cristae, appearance of mitochondrial amorphous matrix densities, significant distortion of muscle fibers and distinct disruption of the intercalated discs. Immunoblotting studies confirmed the presence of alpha 2-macroglobulin which supported the inflammatory response. The severity of the CMI was inferred by the measurement of the level of ET-1 in plasma and left ventricle which was significantly higher in the CMI rats than in the sham-operated rats. Immunohistochemical studies at different time intervals showed that there was a significant immunoexpression of ET-1 on the eighth day post-ligation. This study conclusively showed that ligation of left anterior descending artery minimized mortality and ET-1 was expressed during CMI.     

In vitro study of the electromagnetic interaction between wireless phones and an implantable neural stimulator

Several clinical and laboratory studies have demonstrated electromagnetic interaction between implantable medical devices like pacemakers and cell phones being operated in close proximity. Those devices are largely now immune to phone interaction or procedures have been established to limit their interaction. The use of cell phones near people with implanted neural stimulators has not been studied. This research was initiated to investigate electromagnetic interaction between current cell phone technology and specific models of Cyberonics neural stimulators. Out of 1080 test runs conducted for this study, no interactions were observed, and it was concluded that the phone technologies examined in this study did not adversely affect the Cyberonics NeuroStar (Model 102) NeuroCybernetic Prosthesis (NCP) System. This article provides details on the experimental procedure that was used, which can also be used to test other neural stimulators and test technologies, and the results obtained.     

Immobilization of Candida bombicola cells on free-standing organic-gold nanoparticle membranes and their use as enzyme sources in biotransformations

Preparation of chemically functionalized biocompatible surfaces is of current interest, with application in the immobilization of various bioactive species such as DNA, enzymes, whole cells, etc. We report herein the one-step synthesis of a self-supporting gold nanoparticle membrane, its surface modification, and application in the immobilization of Candida bombicola (yeast) cells. The gold nanoparticle membrane is prepared by the spontaneous reduction of aqueous chloroaurate ions by a diamine at a liquid-liquid interface. The gold nanoparticles in the polymeric membrane may be capped with octadecylamine (ODA) molecules, thereby rendering the nanoparticle membrane hydrophobic. Exposure of the hydrophobized organic-gold nanoparticle membrane to C. bombicola yeast cells results in their binding to the membrane, possibly through nonspecific interactions such as hydrophobic interactions between the yeast cell walls and the ODA molecules. The enzyme cytochrome P450 present in the yeast cells immobilized on the organic-gold nanoparticle membrane was then used in the transformation of the arachidonic acid (AA) to sophorolipids followed by acid hydrolysis to form 20-hydroxyeicosatetraneoic acid (20-HETE). The organic-gold nanoparticle membrane-C. bombicola bioconjugate could be easily separated from the reaction medium and reused a number of times.     

Genomic effects of IFN-beta in multiple sclerosis patients

The purpose of this report was to characterize the dynamics of the gene expression cascades induced by an IFN-beta-1a treatment regimen in multiple sclerosis patients and to examine the molecular mechanisms potentially capable of causing heterogeneity in response to therapy. In this open-label pharmacodynamic study design, peripheral blood was obtained from eight relapsing-remitting multiple sclerosis patients just before and at 1, 2, 4, 8, 24, 48, 120, and 168 h after i.m. injection of 30 micro g of IFN-beta-1a. The total RNA was isolated from monocyte-depleted PBL and analyzed using cDNA microarrays containing probes for >4000 known genes. IFN-beta-1a treatment resulted in selective, time-dependent effects on multiple genes. The mRNAs for genes implicated in the anti-viral response, e.g., double-stranded RNA-dependent protein kinase, myxovirus resistance proteins 1 and 2, and guanylate binding proteins 1 and 2 were rapidly induced within 1-4 h of IFN-beta treatment. The mRNAs for several genes involved in IFN-beta signaling, such as IFN-alpha/beta receptor-2 and Stat1, were also increased. The mRNAs for lymphocyte activation markers, such as IFN-induced transmembrane protein 1 (9-27), IFN-induced transmembrane protein 2 (1-8D), beta(2)-microglobulin, and CD69, were also increased in a time-dependent manner. The findings demonstrate that IFN-beta treatment induces specific and time-dependent changes in multiple mRNAs in lymphocytes of multiple sclerosis patients that could provide a framework for rapid monitoring of the response to therapy.     

Evaluation of cognitive function of fluoxetine, sertraline and tianeptine in isolation and chronic unpredictable mild stress-induced depressive Wistar rats

Depressive illness is generally associated with cognitive impairment. Serotonergic selective antidepressant drugs, fluoxetine (FLX), sertraline (SER) and tianeptine (TIA), are claimed to have less or no effect on cholinergic system, the key system involved in memory. In the present study, these drugs were evaluated for their influence on cognitive behavior in both depressive and non-depressive animals. Depression was induced by two models, (i) 60 days social isolation of litter; and ii) by applying chronic unpredictable mild stress for 21 days. Depression in the rats was confirmed by behavioral despair test. Transfer latency on elevated plus maze and inflexion ratio in passive avoidance step through behavior were employed to assess learning and memory. The results indicated that administration of fluoxetine; sertraline and tianeptine attenuated the cognitive deficits observed in depressive rats. In non-depressive rats these drugs produced retention deficit, which was found to be parameter and model dependent. Data suggested that, FLX and SER (SSRI's) effectively attenuated the isolation-induced depression and cognitive deficit, whereas TIA (SSRE) produced better effect in stress-induced depressive conditions. It was concluded that behavioral profiles of fluoxetine, sertraline and tianeptine on cognition were model and parameter dependent.     

Disabling receptor ensembles with rationally designed interface peptidomimetics

Members of the erbB family receptor tyrosine kinases (erbB1, erbB2, erbB3, and erbB4) are overexpressed in a variety of human cancers and represent important targets for the structure-based drug design. Homo- and heterodimerization (oligomerization) of the erbB receptors are known to be critical events for receptor signaling. To block receptor self-associations, we have designed a series of peptides derived from potential dimerization surfaces in the extracellular subdomain IV of the erbB receptors (erbB peptides). In surface plasmon resonance (BIAcore) studies, the designed peptides have been shown to selectively bind to the erbB receptor ectodomains and isolated subdomain IV of erbB2 with submicromolar affinities and to inhibit heregulin-induced interactions of erbB3 with different erbB receptors. A dose-dependent inhibition of native erbB receptor dimerization by the erbB peptides has been observed in 32D cell lines transfected with different combinations of erbB receptors. The peptides effectively inhibited growth of two types of transformed cells overexpressing different erbB receptors, T6-17 and 32D, in standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and cell viability assays. The study identifies distinct loops within the membrane-proximal part of the subdomain IV as potential receptor-receptor interaction sites for the erbB receptors and demonstrates the possibility of disabling receptor activity by structure-based targeting of the dimerization interfaces. Molecular models for possible arrangement of the erbB1.EGF complex, consistent with the involvement of subdomain IV in inter-receptor interactions, are proposed. Small dimerization inhibitors described herein can be useful as probes to elucidate different erbB signaling pathways and may be developed as therapeutic agents.