Protonation mechanism and location of rate-determining steps for the Ascaris suum nicotinamide adenine dinucleotide-malic enzyme reaction from isotope effects and pH studies

The pH dependence of the kinetic parameters and the primary deuterium isotope effects with nicotinamide adenine dinucleotide (NAD) and also thionicotinamide adenine dinucleotide (thio-NAD) as the nucleotide substrates were determined in order to obtain information about the chemical mechanism and location of rate-determining steps for the Ascaris suum NAD-malic enzyme reaction. The maximum velocity with thio-NAD as the nucleotide is pH-independent from pH 4.2 to 9.6, while with NAD, V decreases below a pK of 4.8. V/K for both nucleotides decreases below a pK of 5.6 and above a pK of 8.9. Both the tartronate pKi and V/Kmalate decrease below a pK of 4.8 and above a pK of 8.9. Oxalate is competitive vs. malate above pH 7 and noncompetitive below pH 7 with NAD as the nucleotide. The oxalate Kis increases from a constant value above a pK of 4.9 to another constant value above a pK of 6.7. The oxalate Kii also increases above a pK of 4.9, and this inhibition is enhanced by NADH. In the presence of thio-NAD the inhibition by oxalate is competitive vs. malate below pH 7. For thio-NAD, both DV and D(V/K) are pH-independent and equal to 1.7. With NAD as the nucleotide, DV decreases to 1.0 below a pK of 4.9, while D(V/KNAD) and D(V/Kmalate) are pH-independent. Above pH 7 the isotope effects on V and the V/K values for NAD and malate are equal to 1.45, the pH-independent value of DV above pH 7. From the above data, the following conclusions can be made concerning the mechanism for this enzyme. Substrates bind to only the correctly protonated form of the enzyme. Two enzyme groups are necessary for binding of substrates and catalysis. Both NAD and malate are released from the Michaelis complex at equal rates which are equal to the rate of NADH release from E-NADH above pH 7. Below pH 7 NADH release becomes more rate-determining as the pH decreases until at pH 4.0 it completely limits the overall rate of the reaction.     

中国的炭疽杆菌DNA分型及其地理分布

炭疽广泛分布于中国各地,特别是西部地区,并经常造成人畜疾病,在一项合作研究中,用多位点VNTR分析（MLVA）对从1952-1998年自中国主要地理流行区域分离的病人,病畜和土壤等来源的炭疽杆菌进行了基因分型,MLVA分析结果揭示了21种新的基因型,其等位基因组合在以前世界范围分离物的研究中未曾发现,此外,分离物的分群显示,A3b组是地理上最广泛分布的基因组,说明该组可能是中国的“地方流行株”。而来自古丝绸之路重要贸易中心新疆的大量分离株其基因型特别分散。     

MicroRNAs in systemic rheumatic diseases

MicroRNAs (miRNAs) are endogenous, non-coding, single-stranded RNAs about 21 nucleotides in length. miRNAs have been shown to regulate gene expression and thus influence a wide range of physiological and pathological processes. Moreover, they are detected in a variety of sources, including tissues, serum, and other body fluids, such as saliva. The role of miRNAs is evident in various malignant and nonmalignant diseases, and there is accumulating evidence also for an important role of miRNAs in systemic rheumatic diseases. Abnormal expression of miRNAs has been reported in autoimmune diseases, mainly in systemic lupus erythematosus and rheumatoid arthritis. miRNAs can be aberrantly expressed even in the different stages of disease progression, allowing miRNAs to be important biomarkers, to help understand the pathogenesis of the disease, and to monitor disease activity and effects of treatment. Different groups have demonstrated a link between miRNA expression and disease activity, as in the case of renal flares in lupus patients. Moreover, miRNAs are emerging as potential targets for new therapeutic strategies of autoimmune disorders. Taken together, recent data demonstrate that miRNAs can influence mechanisms involved in the pathogenesis, relapse, and specific organ involvement of autoimmune diseases. The ultimate goal is the identification of a miRNA target or targets that could be manipulated through specific therapies, aiming at activation or inhibition of specific miRNAs responsible for the development of disease.     

Frequent coexistence of anti-topoisomerase I and anti-U1RNP autoantibodies in African American patients associated with mild skin involvement: a retrospective clinical study

Introduction  

The presence of anti-topoisomerase I (topo I) antibodies is a classic scleroderma (SSc) marker presumably associated with a unique clinical subset. Here the clinical association of anti-topo I was reevaluated in unselected patients seen in a rheumatology clinic setting.     

Tunable mechanical stability and deformation response of a resilin-based elastomer

Resilin, the highly elastomeric protein found in specialized compartments of most arthropods, possesses superior resilience and excellent high-frequency responsiveness. Enabled by biosynthetic strategies, we have designed and produced a modular, recombinant resilin-like polypeptide bearing both mechanically active and biologically active domains to create novel biomaterial microenvironments for engineering mechanically active tissues such as blood vessels, cardiovascular tissues, and vocal folds. Preliminary studies revealed that these recombinant materials exhibit promising mechanical properties and support the adhesion of NIH 3T3 fibroblasts. In this Article, we detail the characterization of the dynamic mechanical properties of these materials, as assessed via dynamic oscillatory shear rheology at various protein concentrations and cross-linking ratios. Simply by varying the polypeptide concentration and cross-linker ratios, the storage modulus G' can be easily tuned within the range of 500 Pa to 10 kPa. Strain-stress cycles and resilience measurements were probed via standard tensile testing methods and indicated the excellent resilience (>90%) of these materials, even when the mechanically active domains are intercepted by nonmechanically active biological cassettes. Further evaluation, at high frequencies, of the mechanical properties of these materials were assessed by a custom-designed torsional wave apparatus (TWA) at frequencies close to human phonation, indicating elastic modulus values from 200 to 2500 Pa, which is within the range of experimental data collected on excised porcine and human vocal fold tissues. The results validate the outstanding mechanical properties of the engineered materials, which are highly comparable to the mechanical properties of targeted vocal fold tissues. The ease of production of these biologically active materials, coupled to their outstanding mechanical properties over a range of compositions, suggests their potential in tissue regeneration applications.     

Polysaccharide-poly(ethylene glycol) star copolymer as a scaffold for the production of bioactive hydrogels

The production of polysaccharide-derivatized surfaces, polymers, and biomaterials has been shown to be a useful strategy for mediating the biological properties of materials, owing to the importance of polysaccharides for the sequestration and protection of bioactive proteins in vivo. We have therefore sought to combine the benefits of polysaccharide derivatization of polymers with unique opportunities to use these polymers for the production of bioactive, noncovalently assembled hydrogels. Accordingly, we report the synthesis of a heparin-modified poly(ethylene glycol) (PEG) star copolymer that can be used in the assembly of bioactive hydrogel networks via multiple strategies and that is also competent for the delivery of bioactive growth factors. A heparin-decorated polymer, synthesized by the reaction of thiol end-terminated four-arm star PEG (M(n) = 10 000) with maleimide functionalized low molecular weight heparin (LMWH, M(r) = 3000), has been characterized via (1)H NMR spectroscopy and size-exclusion chromatography; results indicate attachment of the LMWH with at least 73% efficiency. Both covalently and noncovalently assembled hydrogels can be produced from the PEG-LMWH conjugate. Viscoelastic noncovalently assembled hydrogels have been formed on the basis of the interaction of the PEG-LMWH with a PEG polymer bearing multiple heparin-binding peptide motifs. The binding and release of therapeutically important proteins from the assembled hydrogels have also been demonstrated via immunochemical assays, which demonstrate the slow release of basic fibroblast growth factor (bFGF) as a function of matrix erosion. The combination of these results suggests the opportunities for producing polymer-polysaccharide conjugates that can assemble into novel hydrogel networks on the basis of peptide-saccharide interactions and for employing these materials in delivery applications.     

Conformation and assembly of polypeptide scaffolds in templating the synthesis of silica: an example of a polylysine macromolecular "switch"

Although the role of polycationic macromolecules in catalyzing the synthesis of silica structures is well established, detailed understanding of the mechanisms behind the production of silica structures of controlled morphologies remains unclear. In this study, we have used both poly-L-lysine (PLL) and/or poly-D-lysine (PDL) for silica synthesis to investigate mechanisms controlling inorganic morphologies. The formation of both spherical silica particles and hexagonal plates was observed. The formation of hexagonal plates was suggested, via circular dichroic spectroscopy (CD), to result from the assembly of helical polylysine molecules. We confirm that the formation of PLL helices is a prerequisite to the hexagonal silica synthesis. In addition, we present for the first time that the handedness of the helicity of the macromolecule does not affect the formation of hexagonal silica. We also show, by using two different silica precursors, that the precursor does not have a direct effect on the formation of hexagonal silica plates. Furthermore, when polylysine helices were converted to beta-sheet structure, only silica particles were obtained, thus suggesting that the adoption of a helical conformation by PLL is required for the formation of hexagonally organized silica. These results demonstrate that the change in polylysine conformation can act as a "switch" in silica structure formation and suggest the potential for controlling morphologies and structures of inorganic materials via control of the conformation of soft macromolecular templates.     

Species loss of stoneflies (Plecoptera) in the Czech Republic during the 20th century

1. Rapid expansion and intensification of anthropogenic activities in the 20th century has caused profound changes in freshwater assemblages. Unfortunately, knowledge of the extent and causes of species loss (SL) is limited due to the lack of reliable historical data. An unusual data set allows us to compare changes in the most sensitive of aquatic insect orders, the Plecoptera, at some 170 locations in the Czech Republic between two time periods, 1955–1960 and 2006–2010. Historical data (1890–1911) on assemblages of six lowland rivers allow us to infer even earlier changes. 2. Regional stonefly diversity decreased in the first half of the 20th century. Streams at lower altitudes lost a substantial number of species, which were never recovered. In the second half of the century, large‐scale anthropogenic pressure caused SL in all habitats, leading to a dissimilarity of contemporary and previous assemblages. The greatest changes were found at sites affected by organic pollution and a mixture of organic pollution and channelisation or impoundment. Colonisation of new habitats was observed in only three of the 80 species evaluated. 3. Species of moderate habitat specialisation and tolerance to organic pollution were most likely to be lost. Those with narrow specialisations in protected habitats were present in both historical and contemporary collections. 4. Contemporary assemblages are the consequence of more than a 100 years of anthropogenic impacts. In particular, streams at lower altitude and draining intensively exploited landscapes host a mere fragment of the original species complement. Most stonefly species are less frequently present than before, although their assemblages remain almost intact in near‐natural mountain streams. Our analyses demonstrate dramatic restriction of species ranges and, in some cases, apparent changes in altitudinal preference throughout the area.     

Systems analysis of primary Sjögren's syndrome pathogenesis in salivary glands identifies shared pathways in human and a mouse model

Bone tissue has an exceptional quality to regenerate to native tissue in response to injury. However, the fracture repair process requires mechanical stability or a viable biological microenvironment or both to ensure successful healing to native tissue. An improved understanding of the molecular and cellular events that occur during bone repair and remodeling has led to the development of biologic agents that can augment the biological microenvironment and enhance bone repair. Orthobiologics, including stem cells, osteoinductive growth factors, osteoconductive matrices, and anabolic agents, are available clinically for accelerating fracture repair and treatment of compromised bone repair situations like delayed unions and nonunions. Preclinical and clinical studies using biologic agents like recombinant bone morphogenetic proteins have demonstrated an efficacy similar or better than that of autologous bone graft in acute fracture healing. A lack of standardized outcome measures for comparison of biologic agents in clinical fracture repair trials, frequent off-label use, and a limited understanding of the biological activity of these agents at the bone repair site have limited their efficacy in clinical applications.