Assisted RNP assembly: SMN and PRMT5 complexes cooperate in the formation of spliceosomal UsnRNPs

Although spliceosomal Sm proteins can assemble spontaneously onto UsnRNA in vitro, this process requires assisting factors in vivo. SMN, the protein involved in spinal muscular atrophy, is part of a complex that contains the Sm proteins and serves as a critical factor for this reaction. Here, we have reconstituted the SMN-dependent assembly of UsnRNPs in vitro. We demonstrate that the SMN complex is necessary and sufficient for the assembly reaction. The PRMT5 complex, previously implicated in methylation and storage of Sm proteins, interacts with the SMN complex and enhances its activity in an ATP-dependent manner. These data uncover the SMN-PRMT5 complex as a functional entity that promotes the assisted assembly of spliceosomal UsnRNPs, and potentially other, RNA-protein complexes.     

Effect of recombination in the parent populations on the means and combining ability variances in hybrid populations of maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

Recombination of selected genotypes plays a key role in plant breeding for generating new base populations. We investigated the influence of recombination in two parent populations on the means and combining ability variances of their hybrid population by (1) quantitative genetic theory and (2) experiments with maize. The two parent populations were founded by four early flint and four early dent inbred lines, respectively. Each population was studied in three generations: Syn-0, the four inbred lines themselves; Syn*-1, the six intrapool single crosses (SC); and Syn*-2, the three intrapool double crosses (DC). Four interpool hybrid populations were created: (1) all 16 SC and (2) all 36 DC were produced from generations Syn-0 and Syn*-1, respectively, (3) 168 biparental progenies (BIP) of type flint x dent (female x male), and (4) 168 BIP of type dent x flint were produced according to NC-design I with randomly sampled plants of generation Syn*-2. The half-sib and full-sib families obtained in this manner were evaluated for grain yield, dry matter concentration and plant height. According to theoretical results, differences in the population means of these hybrid populations indicate the presence of various types of epistasis. Changes in combining ability variances from SC to DC reflect different levels of parental inbreeding (F = 1 vs F = 0), whereas changes from DC to BIP only reflect the effects of recombination and are attributable to covariances between additive and dominance effects caused by linkage disequilibrium in the Syn-0 generations. The experimental results showed a significant decline in yield from DC to BIP due to a loss of gene combinations with favourable epistatic effects. Estimates of sigma(2)(GCA) attributable to flint or dent lines decreased or remained unchanged from SC to DC, but generally increased in the BIP populations. The consequences of these trends for developing improved interpool hybrids are discussed.     

Potential role of glycosidase inhibitors in industrial biotechnological applications

The nutrient content of food and animal feed may be improved through new knowledge about enzymatic changes in complex carbohydrates. Enzymatic hydrolysis of complex carbohydrates containing alpha or beta glycosidic bonds is very important in nutrition and in several technological processes. These enzymes are called glycosidases (Enzyme Class 3.2.1) and include amylases, pectinases and xylanases. They are present in many foods such as cereals, but their microbial analogues are often produced and added in many food processes, for instance to improve the shelf-life of bakery products, clear beer, produce glucose, fructose or dextrins, hydrolyse lactose, modify food pectins, or improve processes. However, many plant foods also contain endogenous inhibitors, which reduce the activity of glycosidases, in particular, proteins, peptides, complexing agents and phenolic compounds. The plant proteinaceous inhibitors of glycosidases are in focus in this review whose objective is to report the effect and implications of these inhibitors in industrial processes and applications. These studies will contribute to the optimisation of industrial processes by using modified enzymes not influenced by the natural inhibitors. They will also allow careful selection of raw material and reaction conditions, and future development of new genetic varieties low in inhibitors. These are all new and very promising concepts for the food and feed sector.     

Flow pattern and shear stress distribution of distal end-to-side anastomoses. A comparison of the instantaneous velocity fields obtained by particle image velocimetry

OBJECTIVE: To describe the local hemodynamics and pressure losses of crural bypass anastomoses using instantaneous velocity fields acquired by particle image velocimetry (PIV). METHODS: Silastic models of a Taylor patch, a Miller cuff and a femoro-crural patch prosthesis (FCPP) were attached to a circuit driven by a Berlin Heart, providing a pulsatile flow with an amplitude of 450 to 25 ml/min (mean 200 ml/min). An outflow resistance of 0.5 mmHg/ml/min (peripheral resistance units, PRU) was modeled using small silastic tubes providing a phase shift of -12 degrees between flow and pressure curves. The working fluid consisted of a glycerine/water mixture with a viscosity of 4 mPas. Hollow glass spheres with a mean size of 9-13 microm were used as tracer particles. Instantaneous velocity fields were obtained by means of PIV and shear rates as well as shear stresses were calculated. Triggered by the flowmeter signal, 10 measurements at 100 ms intervals per cardiac cycle were obtained. The pressures were measured on the inflow and at both distal outflows. The resulting mean pressure losses due to flow separation and distal fluid acceleration were calculated. RESULTS: Inside the Taylor patch anastomosis a large flow separation at the hood containing a clockwise rotating vortex was found. Additionally a smaller flow separation at the heel and a flow stagnation zone on the floor of the recipient artery were observed. Conversely, inside the Miller cuff a counterclockwise rotating vortex was seen inside a large heel flow separation. The FCPP also showed typical separation areas at the hood and heel of the anastomosis, although these were smaller compared to the other anastomoses. Inside the FCPP anastomosis no vortex creation was observed throughout the cardiac cycle. The mainstream velocities at the inlet levels were comparable for the three anastomoses. A significant fluid acceleration was present at the antegrade as well as the retrograde outlets of the Taylor and Miller cuff, while the fluid acceleration at the antegrade outflow of the FCPP was small, which was attributed to the end-to-end configuration of the antegrade FCPP leg. The calculated normalized antegrade and retrograde pressure losses for the Taylor form were 0.90 and 0.88, for the Miller cuff 0.89 and 0.86 and for the FCPP 0.94 and 0.86, respectively. The shear stresses inside the flow separations of the three anastomoses were significantly lower than normal wall shear stresses. High shear stress levels were found inside the transition zones between flow separation and high velocity mainstream. CONCLUSIONS: The flow pattern inside cuffed or funnel shaped anastomoses consists of large flow separation zones, which are thought to be associated with intimal hyperplasia development. In addition, fluid accelerations at the distal outlets result in pressure losses, which may contribute to impaired crural perfusion.     

Proteolytic cleavage of the catalytic subunit of DNA-dependent protein kinase during poliovirus infection

DNA-dependent protein kinase (DNA-PK) is a serine/threonine kinase that has critical roles in DNA double-strand break repair, as well as B- and T-cell antigen receptor rearrangement. The DNA-PK enzyme consists of the Ku regulatory subunit and a 450-kDa catalytic subunit termed DNA-PK(CS). Both of these subunits are autoantigens associated with connective tissue diseases such as systemic lupus erythematosus (SLE) and scleroderma. In this report, we show that DNA-PK(CS) is cleaved during poliovirus infection of HeLa cells. Cleavage was visible as early as 1.5 h postinfection (hpi) and resulted in an approximately 40% reduction in the levels of native protein by 5.5 hpi. Consistent with this observation, the activity of the DNA-PK(CS) enzyme was also reduced during viral infection, as determined by immunoprecipitation kinase assays. Although it has previously been shown that DNA-PK(CS) is a substrate of caspase-3 in vitro, the protein was still cleaved during poliovirus infection of the caspase-3-deficient MCF-7 cell line. Cleavage was not prevented by infection in the presence of a soluble caspase inhibitor, suggesting that cleavage in vivo was independent of host caspase activation. DNA-PK(CS) is directly cleaved by a picornaviral 2A protease in vitro, producing a fragment similar in size to the cleavage product observed in vivo. Taken together, our results indicate that DNA-PK(CS) is cleaved by the 2A protease during poliovirus infection. Proteolytic cleavage of DNA-PK(CS) during poliovirus infection may contribute to inhibition of host immune responses. Furthermore, cleavage of autoantigens by viral proteases may target these proteins for the autoimmune response by generating novel, or "immunocryptic," protein fragments.     

Redox-dependent effects of nitric oxide on microvascular integrity in oxygen-induced retinopathy

Opposing effects have been ascribed to nitric oxide (NO) on retinal microvascular survival. We investigated whether changes in the redox state may contribute to explain apparent conflicting actions of NO in a model of oxygen-induced retinal vasoobliteration. Retinal microvascular obliteration was induced by exposing 7-day-old rat pups (P7) for 2 or 5 days to 80% O(2). The redox state of the retina was assessed by measuring reduced glutathione and oxidative and nitrosative products malondialdehyde and nitrotyrosine. The role of NO on vasoobliteration was evaluated by treating animals with nitric oxide synthase (NOS) inhibitors (N-nitro-l-arginine; L-NA) and by determining NOS isoform expression and activity; the contribution of nitrosative stress was also determined in animals treated with the degradation catalyst of peroxynitrite FeTPPS or with the superoxide dismutase mimetic CuDIPS. eNOS, but not nNOS or iNOS, expression and activity were increased throughout the exposure to hyperoxia. These changes were associated with an early (2 days hyperoxia) decrease in reduced glutathione and increases in malondialdehyde and nitrotyrosine. CuDIPS, FeTPPS, and L-NA treatments for these 2 days of hyperoxia nearly abolished the vasoobliteration. In contrast, during 5 days exposure to hyperoxia when the redox state rebalanced, L-NA treatment aggravated the vasoobliteration. Interestingly, VEGFR-2 expression was respectively increased by NOS inhibition after short-term (2 days) exposure to hyperoxia and decreased during the longer hyperoxia exposure. Data disclose that the dual effects of NO on newborn retinal microvascular integrity in response to hyperoxia in vivo depend on the redox state and seem mediated at least in part by VEGFR-2.     

Death, autoantigen modifications, and tolerance

Autoantibodies present in the serum of patients with a variety of inflammatory diseases have proven useful as diagnostic markers and as probes with which to elucidate biochemical and signaling pathways. The mechanisms governing the generation of autoantibodies remain elusive, constituting a critical missing link in our understanding of rheumatologic illnesses. Several lines of experimentation in recent years have strongly implicated events surrounding cell death in this process. This review will address the potential role played by death-specific modifications of autoantigens in bypassing tolerance to highly conserved autoantigens, including nucleic acids, lipids, and proteins.