PRODECOMPv3: decompositions of NMR projections for protein backbone and side-chain assignments and structural studies

PRODECOMP (projection decomposition) is an implementation of a multi-way decomposition algorithm for the analysis of two-dimensional projections of high-dimensional nuclear magnetic resonance spectra. The newest version, PRODECOMPv3, features a dramatic speedup, more reliable decompositions, a substantial reduction in memory demands, a new graphical user interface and integration into third-party software. These improvements extend the applicability of decompositions to novel types of NMR data on proteins, yielding backbone and side-chain assignments as well as structural information, and therewith enabling complete characterizations of proteins. AVAILABILITY: Program, short manual and an example calculation are freely available at www2.chem.gu.se/bcbp/nmr/prodecomp.html.     

Macrophages and nitric oxide as the possible cellular and molecular basis for strain and gender differences in susceptibility to autoimmune central nervous system inflammation

PVG rats are resistant to actively induced experimental autoimmune encephalomyelitis (EAE) and this appears to be directly related to high and sustained systemic levels of reactive nitrogen intermediates(RNI) following sensitization for EAE when compared to the highly susceptible Lewis rat. An apparent cellular basis for the different EAE susceptibility between the two rat strains is described. Spleens of PVG rats have increased monocyte/macrophage numbers(NO producing cells) and lower erythrocyte (NO scavengers) to nucleated spleen cell ratios compared with Lewis rats. Splenectomy demonstrated the pivotal role of the spleen in resistance to EAE as splenectomized PVG rats were rendered completely susceptible to disease induction.It was further demonstrated that EAE resistance in PVG rats is limited only to females and that only female PVG rats have increased splenic macrophage and an enhanced NO production following immunization. The males are fully susceptible to EAE and their spleen cell populations are similar to those of Lewis rats of either gender. Despite being resistant to active disease induction, immunized female PVG rats can generate EAE effector cells that are capable of passively transferring disease.Furthermore, female PVG rats are fully susceptible to passively transferred EAE. Thus, there appears to be no defect in the female PVG target tissue or in the processing or presentation of antigen,but a block at the level of effector cell expansion and/or recirculation and transmigration into the target tissue in actively induced EAE.     

Synthesis,in vitro biological activity and docking of new analogs of BIM-23052 containing unnatural amino acids

Amino Acids - Somatostatin (SST) is an endogenous cyclic tetradecapeptide hormone that exerts multiple biological activities via a family of five receptors. BIM-23052 (DC-23-99)...     

Assignment of protein NMR spectra based on projections,multi-way decomposition and a fast correlation approach

We present an approach for the assignment of protein NMR resonances that combines established and new concepts: (a) Based on published reduced dimensionality methods, two 5-dimensional experiments are proposed. (b) Multi-way decomposition (PRODECOMP) applied simultaneously to all acquired NMR spectra provides the assignment of resonance frequencies under conditions of very low signal-to-noise. (c) Each resulting component characterizes all spin (1/2) nuclei in a (doubly-labeled) CbetaH(n)-CalphaH-C'-NH-CalphaH-CbetaH(n) fragment in an unambiguous manner, such that sequentially neighboring components have about four atoms in common. (d) A new routine (SHABBA) determines correlations for all component pairs based on the common nuclei; high correlation values yield sequential chains of a dozen or more components. (e) The potentially error-prone peak picking is delayed to the last step, where it helps to place the component chains within the protein sequence, and thus to achieve the final backbone assignment. The approach was validated by achieving complete backbone resonance assignments for ubiquitin.     

Nutritional value of raw and micronised field beans (Vicia faba L. var. minor) with and without enzyme supplementation containing tannase for growing chickens

An experiment examined the effects of two field bean cultivar samples with different tannin contents, the effect of heat treatment (micronising) and the effect of dietary supplementation of a proprietary enzyme preparation containing tannase, pectinase, and xylanase activities on metabolisable energy (ME), total tract dry matter digestibility (DMD) and ether extract digestibility (EED), nitrogen retention (NR), tannin degradability, gastrointestinal tract (GIT) development, and endogenous mucin losses excretion in broiler chickens. The Control diet contained per kg 221 g crude protein and 12.83 MJ ME. Four additional diets contained 300 g/kg of each of the two untreated or micronised experimental field bean cultivar samples. Each diet was then split into two batches and one of them was supplemented with 3400 units tannase per kg diet resulting in 10 diets in total. Each diet was fed to seven pens with two randomly selected male broilers each. Birds fed the high tannin bean sample had a lower weight gain (p < 0.001), and a lower determined apparent ME (p < 0.05), and DMD (p < 0.001) but a higher tannin degradability (p < 0.001). Compared to the Control diet, feeding field beans increased (p < 0.001) the weights of the proventriculus and gizzard of the birds, and also increased endogenous mucin losses (p < 0.05). Supplementing diets with the tannase-containing enzyme preparation improved dietary ME (p < 0.001), DMD (p < 0.001), NR (p < 0.001) and DEE (p < 0.05), but did not change tannin degradability. Heat treatment of the beans reduced the degradability of condensed tannins and increased endogenous mucin losses (p < 0.05). The differences in the feeding value of the different field bean samples were not improved by heat treatment, but enzyme supplementation improved the feeding value of all diets regardless of the bean samples or heat treatment. Further research is warranted to study the effectiveness of tannase supplementation in poultry diet formulations by dose response trials with purified tannase preparations.     

Localization of cellulases and β-D-glucosidases inAspergillus terreus upon substrate-induced biosynthesis

In conditions which stimulated the production of cellulases (introduction of lint into a medium) and β-D-glucosidases (addition of cellobiose to a medium) byAspergillus terreus, the localization of these enzymes was studied. Irrespective of the nature of the inducer, cellulases were shown to be predominantly excreted into the medium, and β-D-glucosidases were found to be mostly localized inside the cells and on the cell surface. It was demonstrated cytochemically that cellulases were transported from the periplasm to the medium in vesicles, whereas β-D-glucosidases were diffusely distributed in the periplasm and the cell-wall.     

Cell surface expression of the 300 kDa mannose-6-phosphate receptor by activated T lymphocytes

Phosphosugars, such as mannose-6-phosphate (M6P), have been shown previously to display anti-inflammatory properties, notably inhibition of experimental autoimmune encephalomyelitis (EAE) and adjuvant-induced arthritis in rats. It has been proposed that M6P exerts its anti-inflammatory effect by displacing lysosomal enzymes, which are involved in T-cell extravasation into inflammatory sites, from the 300 kDa mannose-6- phosphate receptor (MPR-300) on the surface of T cells. If this model is correct MPR-300 should be selectively expressed on the surface of activated T cells, as T cell entry into the central nervous system in EAE depends on the T cells being in an activated state. Thus, the present study examines whether cell surface expression of MPR-300 by T lymphocytes correlates with their state of activation and whether T cells in inflammatory sites express the receptor. Flow cytometric studies showed MPR-300 to be absent from the surface of unstimulated rat T cells isolated from peripheral blood and lymphoid tissues, and T cells resident within the peritoneal cavity. In contrast, MPR-300 was expressed on activated T cells derived from an inflammatory peritoneal exudate. In vitro studies demonstrated transient expression of MPR-300 on the surface of splenic T cells following stimulation with Con A. MPR-300 was also induced on T-cell lines by antigen stimulation. These data demonstrate that T cells in inflammatory sites express MPR-300 on their surface and activation of T lymphocytes induces cell surface expression of MPR-300. Such findings are consistent with the hypothesis that cell surface MPR-300 is required for the entry of T cells into inflammatory sites.     

Automated protein backbone assignment using the projection-decomposition approach

Production of sufficient amounts of human proteins is a frequent bottleneck in structural biology. Here we describe an Escherichia coli-based cell-free system which yields mg-quantities of human proteins in N-terminal fusion constructs with the GB1 domain, which show significantly increased translation efficiency. A newly generated E. coli BL21 (DE3) RIPL-Star strain was used, which contains a variant RNase E with reduced activity and an excess of rare-codon tRNAs, and is devoid of lon and ompT protease activity. In the implementation of the expression system we used freshly in-house prepared cell extract. Batch-mode cell-free expression with this setup was up to twofold more economical than continuous-exchange expression, with yields of 0.2-0.9 mg of purified protein per mL of reaction mixture. Native folding of the proteins thus obtained is documented with 2D [(15)N,(1)H]-HSQC NMR.