PspA adopts an ESCRT-III-like fold and remodels bacterial membranes

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An affibody-adalimumab hybrid blocks combined IL-6 and TNF-triggered serum amyloid A secretion in vivo

In inflammatory disease conditions, the regulation of the cytokine system is impaired, leading to tissue damages. Here, we used protein engineering to develop biologicals suitable for blocking a combination of inflammation driving cytokines by a single construct. From a set of interleukin (IL)-6-binding affibody molecules selected by phage display, five variants with a capability of blocking the interaction between complexes of soluble IL-6 receptor α (sIL-6Rα) and IL-6 and the co-receptor gp130 were identified. In cell assays designed to analyze any blocking capacity of the classical or the alternative (trans) signaling IL-6 pathways, one variant, Z_{IL-6_13} with an affinity (K_D) for IL-6 of ～500 pM, showed the best performance. To construct fusion proteins (“AffiMabs”) with dual cytokine specificities, Z_{IL-6_13} was fused to either the N- or C-terminus of both the heavy and light chains of the anti-tumor necrosis factor (TNF) monoclonal antibody adalimumab (Humira®). One AffiMab construct with Z_{IL-6_13} positioned at the N-terminus of the heavy chain, denoted Z_{IL-6_13}-HC_Ada, was determined to be the most optimal, and it was subsequently evaluated in an acute Serum Amyloid A (SAA) model in mice. Administration of the AffiMab or adalimumab prior to challenge with a mix of IL-6 and TNF reduced the levels of serum SAA in a dose-dependent manner. Interestingly, the highest dose (70 mg/kg body weight) of adalimumab only resulted in a 50% reduction of SAA-levels, whereas the corresponding dose of the Z_{IL-6_13}-HC_Ada AffiMab with combined IL-6/TNF specificity, resulted in SAA levels below the detection limit.     

Male-sterile chicory cybrids obtained by intergeneric protoplast fusion

Male-sterile chicory plants were obtained by fusion of chicory mesophyll protoplasts and hypocotyl protoplasts derived from male-sterile sunflower plants. The protoplasts of both species were fused by the PEG method and the products were selected manually and cultivated at very low density in a liquid medium. Three to twenty percent of the heterokaryocytes divided and evolved into microcalli, then into calli where budding could be induced. The mitochondrial genome of ten male-sterile or totally sterile plants was studied. Restriction endonuclease profiles of mitochondrial DNA and molecular hybridization with specific genes of the mitochondrial genome used as probes indicated that mitochondrial DNA rearrangement had occurred between sunflower and chicory and the intensity of the rearrangements correlated with the degree of sterility of the different plants.     

Development of baking yeast from Nigerian palm-wine yeasts

Two local strains of Saccharomyces cerevisiae, Nk, and Nk₂, showed leavening activities of 103% and 102% of that of a commercial yeast strain on wheat flour and of 114% and 113% on composite dough with 40% (w/v) maize substitution. Yeast fusion products Nk/Ng, Nk/Nk₁ and Nk/Nk₂ showed activities of 104% to 113% on wheat flour and 111% to 131% on the composite dough. These advantages were maintained in the yeasts' baking properties and organoleptic qualities. The fusion products also showed enhanced osmotic tolerance, as indicated by good growth in 3%, 6% and 10% (w/v) NaCl and 50% (w/v) glucose. Viability of the fusion products, preserved by drying at 30 to 35°C, dropped to between 62% and 72% after 3 months.A.O. Ejiofor was and N. Okafor and E.N. Ugwueze are with the Department of Applied Microbiology and Brewing, Nnamdi Azikiwe University, P.M.B. 5025, Awka, Nigeria. A.J. Ejiofor is now with the Abteilung Biovertahrenstechnik, Gesellschaft für Biotechnologische Forschung, Mascheroder Weg 1, D-38124 Braunschweig, Germany     

Regulation of the Saccharomyces cerevisiae Srs2 helicase during the mitotic cell cycle, meiosis and after irradiation

The expression of theSRS2 gene, which encodes a DNA helicase involved in DNA repair inSaccharomyces cerevisiae, was studied using anSRS2-lacZ fusion integrated at the chromosomalSRS2 locus. It is shown here that this gene is expressed at a low level and is tightly regulated. It is cell-cycle regulated, with induction probably being coordinated with that of the DNA-synthesis genes, which are transcribed at the G1-S boundary. It is also induced by DNA-damaging agents, but only during the G2 phase of the cell cycle; this distinguishes it from a number of other repair genes, which are inducible throughout the cycle. During meiosis, the expression ofSRS2 rises at a time nearly coincident with commitment to recombination. Sincesrs2 null mutants are radiation sensitive essentially when treated in G1, the mitotic regulation pattern described here leads us to postulate that either secondary regulatory events limit Srs2 activity to G1 cells or Srs2 functions in a repair mechanism associated with replication.     

The Relativistic Quantum Defect Orbital Method and some of its Applications

Justification of a simple and reliable relativistic procedure for the prediction of a large body of transition probability data is made. The main features of the Relativistic Quantum Defect Orbital (RQDO) formalism are described, and several examples of the data yielded by this method are presented in tabular and graph forms.     

Differences in 3D vs. 2D analysis in lumbar spinal fusion simulations

Lumbar interbody fusion is currently the gold standard in treating patients with disc degeneration or segmental instability. Despite it having been used for several decades, the non-union rate remains high. A failed fusion is frequently attributed to an inadequate mechanical environment after instrumentation. Finite element (FE) models can provide insights into the mechanics of the fusion process. Previous fusion simulations using FE models showed that the geometries and material of the cage can greatly influence the fusion outcome. However, these studies used axisymmetric models which lacked realistic spinal geometries. Therefore, different modeling approaches were evaluated to understand the bone-formation process.Three FE models of the lumbar motion segment (L4–L5) were developed: 2D, Sym-3D and Nonsym-3D. The fusion process based on existing mechano-regulation algorithms using the FE simulations to evaluate the mechanical environment was then integrated into these models. In addition, the influence of different lordotic angles (5, 10 and 15°) was investigated. The volume of newly formed bone, the axial stiffness of the whole segment and bone distribution inside and surrounding the cage were evaluated.In contrast to the Nonsym-3D, the 2D and Sym-3D models predicted excessive bone formation prior to bridging (peak values with 36 and 9% higher than in equilibrium, respectively). The 3D models predicted a more uniform bone distribution compared to the 2D model.The current results demonstrate the crucial role of the realistic 3D geometry of the lumbar motion segment in predicting bone formation after lumbar spinal fusion.     

Lipids in biological membrane fusion

The results reviewed suggest that membrane fusion in diverse biological fusion reactions involves formation of some specific intermediates: stalks and pores. Energy of these intermediates and, consequently, the rate and extent of fusion depend on the propensity of the corresponding monolayers of membranes to bend in the required directions.Proteins and peptides can control the bending energy of membrane monolayers in a number of ways. Monolayer lipid composition may be altered by different phospholipases [50, 85, 90], flipases and translocases [4, 50]. Proteins and peptides can change monolayer spontaneous curvature or hydrophobic void energy by direct interaction with membrane lipids [20, 32, 111]. Proteins may also provide some barriers for lipid diffusion in the plane of the monolayer [83, 141]. If diffusion of lipids at some specific membrane sites (e.g., in the vicinity of fusion protein) is somehow hindered, the energy of the bent fusion intermediates would reflect the elastic properties of these particular sites rather than the spontaneous curvature of the whole monolayers. Proteins may deform membranes while bringing them locally into close contact. The alteration of the geometric (external) curvature will certainly change the elastic energy of the initial state and, thus affect the energetic barriers of the formation of the intermediates [143]. In addition, the area and the energy of the stalk can be reduced by preliminary bending of the contacting membranes [111]. The possible effects of proteins and polymers on local elastic properties and local shapes of the membranes have been recently analyzed [22, 39, 45, 63]. These studies may provide a good basis for future development of theoretical models of protein-mediated fusion.     

Comparison of Different Standard Methods to Evaluate the Total Concentrations of Heavy Metals in Waste Rock

The mining industry is a very rapidly increasing production sector in Finland. Many new mines have been opened recently and others are in the planning phase. Mining legislation has been recently revised and compilation of an ID-directive (Industrial Emission Directive, 2010) required to implement environmental law revision is in progress. The aim of this study was to compare different digestion methods recommended by the Finnish Environmental Administration for threshold and guideline value determinations in the Finnish PIMA Decree (214/2007) and Government Decree (591/2006) concerning recovery of certain wastes in earth construction. Altogether six methods were tested, of which five were microwave-assisted acid digestion methods: ISO 11466, EPA Method 3051A (aqua region), EPA Method 3051A (HNO₃), EPA Method 3051A (HNO₃/HCl), and SFS-EN 13656, while one was a fusion method with lithium tetraborate (ASTM C 1391 - 95). The results showed that concentrations of heavy metals (As, Cd, Co, Cu, Ni, Pb, Sb, V, and Zn) can be determined reproducibly with standard methods, but the chemical method used can have a significant impact on the results. This conclusion is very important, because the measured heavy metal values can affect the classification and processing of waste materials, and thereby can significantly impact the cost of treatment.