A cytosolic activity distinct from crm1 mediates nuclear export of protein kinase inhibitor in permeabilized cells

The leucine-rich nuclear export signal (NES) is used by a variety of proteins to facilitate their delivery from the nucleus to the cytoplasm. One of the best-studied examples, protein kinase inhibitor (PKI), binds to the catalytic subunit of protein kinase A in the nucleus and mediates its rapid export to the cytoplasm. We developed a permeabilized cell assay that reconstitutes nuclear export mediated by PKI, and we used it to characterize the cytosolic factors required for this process. The two-step assay involves an import phase and an export phase, and quantitation is achieved by digital fluorescence microscopy. During the import phase, a fluorescent derivative of streptavidin is imported into the nuclei of digitonin-permeabilized HeLa cells. During the export phase, biotinylated PKI diffuses into the nucleus, binds to fluorescent streptavidin, and mediates export of the complex to the cytoplasm. Nuclear export of the PKI complex is cytosol dependent and can be stimulated by addition of the purified NES receptor, Crm1. HeLa cell cytosol treated with N-ethylmaleimide (NEM) or phenyl-Sepharose to inactivate or deplete Crm1, respectively, is still fully active in the PKI export assay. Significantly, the export activity can be depleted from cytosol by preadsorption with a protein conjugate that contains a functional NES. These data indicate that cytosol contains an export activity that is distinct from Crm1 and is likely to correspond to an NES receptor.     

Assessment of conformational parameters as predictors of limited proteolytic sites in native protein structures

Despite the importance of limited proteolysis in biological systems it isoften difficult to rationalize why a proteinase hydrolyses a particularbond, given a simple sequence specificity alone. Understanding of thestructural properties limiting the proteolysis represents a first step onthe pathway to control and manipulation of this phenomena. An expanded setof nick-sites in proteins of known tertiary structure, cut by both narrowand broad specificity proteinases, has been generated yielding a robustdata set of strictly limited sites. A critical evaluation of an expandedset of conformational parameters revealed a strong correlation with limitedproteolytic sites, although they are only modest predictors in isolation.The overall predictive power is significantly improved when theconformational parameters are combined in a weighted predictive scheme thatpermits their relative importance to be compared via a Metropolis searchprotocol. A subset of the parameters performs equally well demonstratingthe key determinants of susceptibility. The derived predictive algorithmhas been made available via the internet. Its utility for predicting othersurface-correlated features is also discussed.     

The effect of montmorillonite and compatibilizer quantities on stiffness and toughness of polyamide nanoblends

Although polymer blend nanocomposites are widely studied, the balance between stiffness and toughness has not yet been investigated in detail. Some materials producers as well as some sectors in the automotive industry try to improve the toughness of materials without an important loss in stiffness. With this in mind, the aim of the study reported here was to obtain a good balance between toughness and stiffness of polymer blends with different amounts of clay and compatibilizer. In this context, the microstructure of polyamide 6/ethylene–propylene–diene metallocene terpolymer/(ethylene–propylene–diene copolymer)‐graft‐(maleic anhydride) blends with various amounts of clay (2, 3, 4 and 5 wt%) and compatibilizer (10 and 20 wt%) was studied to analyse the achieved morphology to understand the macroscopic properties. The morphology of the rubber phase and the dispersion of the montmorillonite (MMT) are the main factors that influence the mechanical properties. In this sense, the highest Young's modulus is achieved for nanoblends with 5 wt% of MMT, although this nanoblend has the lowest value of notched Izod impact strength. The results obtained suggest that there is a clear trade‐off between stiffness, toughness and temperature behaviour when the ratio of (ethylene–propylene–diene copolymer)‐graft‐(maleic anhydride) to MMT is 5:1. Copyright © 2009 Society of Chemical Industry     

Effect of menopause on femoral and vertebral bone loss

The aim of this study was to investigate the effect of menopause on bone loss in the proximal femur and the lumbar spine. The rates of change in bone mineral density (BMD) were measured longitudinally by dual X-ray absorptiometry (DXA) at the femoral neck (FN), Ward's triangle (WT), and trochanter (TR) together with the lumbar spine in 81 healthy postmenopausal women (45-65 years of age) who had passed a natural menopause, 6 months to 12 years before. A significant correlation between the rate of change and interval since menopause was evidenced. The best fit of the data was a binomial function of interval since menopause at the spine, FN, and WT and a simple linear regression at TR level. At each skeletal site, the rate of bone loss (mean +/- SD) was significantly different (p<0.05) and twice as high in women who were between 6 months and 2 years postmenopausal at enrollment (FN, -1.82 +/- 1.1%; WT, -2.43 +/- 1.7%; TR, -1.12 +/- 1.7%) than in those who were beyond 5 years of menopause (FN, -0.48 +/- 0.8%; WT, -0.68 +/- 2.1% TR, 0.41 +/- 1.2%). A poor correlation (r = 0.39 - 0.42, p<0.001) was found between the rate of vertebral and that of femoral postmenopausal bone loss. This study demonstrates that menopause is associated with a rapid and transient bone loss in BMD of the proximal femur, which declines with time after 3 years. These data suggest that therapy should be initiated as early as possible after menopause to prevent bone loss.     

Towards a rapid, non-contact shaping method for fibre metal laminates using a laser source

Since their initial development, fibre metal laminates (FMLs) have slowly started to be used by industry, particularly the aerospace sector. One of the reasons for the relatively slow adoption of FMLs is due to the difficulties faced in shaping them to the desired geometry. Whilst traditional processes such as roll forming are effective in shaping monolithic materials, these processes could potentially destroy the mechanical properties of the composite layer. The approached investigated here uses thermal or laser forming (LF) to shape flat panels of thermosetting glass fibre based FMLs into 2D geometries. This initial empirical investigation covers the effectiveness of the various LF processes and the effects of various parameters have on the forming process. These include laser parameters such as power and velocity and material parameters such as FML lay-up strategy, fibre orientation and comparison with monolithic materials.     

Investigations on New Scanning Pattern for Stereolithography

The basic layer-based manufacturing mechanism of stereolithography is built upon a scanning pattern for the entire cross section for each layer. In this investigation, a new schema of scanning is proposed and its effects on dimensional accuracy and surface profile are benchmarked against an industry standard scanning pattern. Experimental results show that the new scanning pattern offers further improvements in terms of dimensional accuracy and geometrical profile as supported by a higher value of process capability index C_pk. The use of Finite Element method to simulate the new scanning pattern is also described in order to provide an analogous insight on process effects and residual stress distribution.     

A heroin-, but not a cocaine-expecting, self-administration state preferentially alters endogenous brain peptides

The effects of L-type voltage-dependent Ca2+ channel blockers on apomorphine, bromocriptine and morphine-induced changes in locomotor activity were examined in mice. Apomorphine (4 mg/kg) and morphine (20 mg/kg) produced locomotor stimulation. Bromocriptine (8 mg/kg) produced a biphasic effect on motor behaviour, an early depressant phase, followed by locomotor stimulation. Amlodipine (2.5 mg/kg), nicardipine (10 mg/kg), diltiazem (10 mg/kg) and verapamil (10 mg/kg), which by itself did not affect locomotor activity, inhibited the stimulant phase of bromocriptine without altering the depressant phase, while they did not affect apomorphine- and morphine-induced locomotor stimulation. Apomorphine, bromocriptine and morphine-induced locomotor stimulation was decreased by SCH 23390 (R-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7- ol) (0.05 mg/kg) or haloperidol (0.1 mg/kg). These results indicate that L-type voltage-dependent Ca2+ channels are involved in the motor stimulant effect of bromocriptine, but not in apomorphine- and morphine-induced locomotor stimulation. The effects of Ca2+ channel blockers on the dopaminergic system appears not to be directly related to dopamine receptor blockade.     

Posterior column ataxia with retinitis pigmentosa (AXPC1) maps to chromosome 1q31-q32

OBJECTIVE: To establish a genetic linkage between highly polymorphic microsatellite loci and the disease locus responsible for an autosomal recessive neurodegenerative syndrome that causes posterior column ataxia and retinitis pigmentosa. BACKGROUND: The authors reported previously a genetic syndrome that causes visual impairment, proprioceptive loss, sensory ataxia, and areflexia in affected individuals from a large, inbred family belonging to a sectarian population that has been genetically semi-isolated from mainstream society for several centuries. METHODS: To find the disease locus responsible for this condition, the authors performed a genome-wide search using genetic loci spaced at 10 to 20-cM intervals spanning human chromosomes (chr) 1-22. Pairwise linkage analysis, multipoint linkage analysis, and haplotype reconstruction were used to delineate the candidate region containing the disease gene. RESULTS: After testing 226 loci that covered the entire genome, the authors identified a maximum lod score of 8.94 at a recombination fraction of 0.00 for locus D1S2692. Additional analyses placed the disease gene, AXPC1, in an 8.3-cM interval flanked by markers D1S2692 and D1S414 on chr 1q31-q32. CONCLUSIONS: This study suggests that a single genetic mutation can cause selective degeneration of the posterior columns of the spinal cord and retina. Finding the gene responsible for this syndrome may increase our understanding of the molecular basis of diseases that affect sensory neurons.