Probing protein dynamics by nuclear magnetic resonance

Proteins are dynamic molecules that often undergo conformational changes while performing their specific functions, such as target recognition, ligand binding and catalysis. NMR spectroscopy is uniquely suited to study protein dynamics, because site-specific information can be obtained for motions that span a broad range of time scales. The information obtained from NMR dynamics experiments has provided insights into specific structural changes or conformational energetics associated with molecular function. In the last decade, a number of new advancements in NMR methodologies have further extended our ability to characterize protein dynamics. Here, we present an overview of current NMR technology that is used to monitor the dynamic properties of proteins.     

First report of an anti-tumor, anti-fungal, anti-yeast and anti-bacterial hemolysin from Albizia lebbeck seeds

A monomeric 5.5-kDa protein with hemolytic activity toward rabbit erythrocytes was isolated from seeds of Albizia lebbeck by using a protocol that involved ion-exchange chromatography on Q-Sepharose and SP-Sepharose, hydrophobic interaction chromatography on Phenyl-Sepharose, and gel filtration on Superdex 75. It was unadsorbed on both Q-Sepharose and SP-Sepharose, but adsorbed on Phenyl-Sepharose. Its hemolytic activity was fully preserved in the pH range 0-14 and in the temperature range 0-100 °C, and unaffected in the presence of a variety of metal ions and carbohydrates. The hemolysin reduced viability of murine splenocytes and inhibited proliferation of MCF-7 breast cancer cells and HepG2 hepatoma cells with an IC₅₀ of 0.21, 0.97, and 1.37 μM, respectively. It impeded mycelial growth in the fungi Rhizoctonia solani with an IC₅₀ of 39 μM but there was no effect on a variety of other filamentous fungi, including Fusarium oxysporum, Helminthosporium maydis, Valsa mali and Mycosphaerella arachidicola. Lebbeckalysin inhibited growth of Escherichia coli with an IC₅₀ of 0.52 μM.     

Enhancement of excretory production of an exoglucanase from Escherichia coli with phage shock protein A (PspA) overexpression

Production of recombinant proteins by excretory expression has many advantages over intracellular expression in Escherichia coli. Hyperexpression of a secretory exoglucanase, Exg, of Cellulomonas fimi was previously shown to saturate the SecYEG pathway and result in dramatic cell death of E. coli. In this study, we demonstrated that overexpression of the PspA in the JM101(pM1VegGcexL-pspA) strain enhanced excretion of Exg to 1.65 U/ml using shake-flask cultivation, which was 80% higher than the highest yield previously obtained from the optimized JM101(pM1VegGcexL) strain. A much higher excreted Exg activity of 4.5 U/ml was further achieved with high cell density cultivation using rich media. Furthermore, we showed that the PspA overexpression strain enjoyed an elevated critical value (CV), which was defined as the largest quotient between the intracellular unprocessed precursor and its secreted mature counterpart that was still tolerable by the host cells prior to the onset of cell death, improving from the previously determined CV of 20/80 to the currently achieved CV of 45/55 for Exg. The results suggested that the PspA overexpression strain might tolerate a higher level of precursor Exg making use of the SecYEG pathway for secretion. The reduced lethal effect might be attributable to the overexpressed PspA, which was postulated to be able to reduce membrane depolarization and damage. Our findings introduce a novel strategy of the combined application of metabolic engineering and construct optimization to the attainment of the best possible E. coli producers for secretory/excretory production of recombinant proteins, using Exg as the model protein.     

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.     

An ana2/ctp/mud complex regulates spindle orientation in Drosophila neuroblasts

Drosophila neural stem cells, larval brain neuroblasts (NBs), align their mitotic spindles along the apical/basal axis during asymmetric cell division (ACD) to maintain the balance of self-renewal and differentiation. Here, we identified a protein complex composed of the tumor suppressor anastral spindle 2 (Ana2), a dynein light-chain protein Cut up (Ctp), and Mushroom body defect (Mud), which regulates mitotic spindle orientation. We isolated two ana2 alleles that displayed spindle misorientation and NB overgrowth phenotypes in larval brains. The centriolar protein Ana2 anchors Ctp to centrioles during ACD. The centriolar localization of Ctp is important for spindle orientation. Ana2 and Ctp localize Mud to the centrosomes and cell cortex and facilitate/maintain the association of Mud with Pins at the apical cortex. Our findings reveal that the centrosomal proteins Ana2 and Ctp regulate Mud function to?orient the mitotic spindle during NB asymmetric division.     

Human mesenchymal stem cell-conditioned medium improves cardiac function following myocardial infarction

Recent studies suggest that the therapeutic effects of stem cell transplantation following myocardial infarction (MI) are mediated by paracrine factors. One of the main goals in the treatment of ischemic heart disease is to stimulate vascular repair mechanisms. Here, we sought to explore the therapeutic angiogenic potential of mesenchymal stem cell (MSC) secretions. Human MSC secretions were collected as conditioned medium (MSC-CM) using a clinically compliant protocol. Based on proteomic and pathway analysis of MSC-CM, an in vitro assay of HUVEC spheroids was performed identifying the angiogenic properties of MSC-CM. Subsequently, pigs were subjected to surgical left circumflex coronary artery ligation and randomized to intravenous MSC-CM treatment or non-CM (NCM) treatment for 7 days. Three weeks after MI, myocardial capillary density was higher in pigs treated with MSC-CM (645 ± 114 vs 981 ± 55 capillaries/mm(2); P = 0.021), which was accompanied by reduced myocardial infarct size and preserved systolic and diastolic performance. Intravenous MSC-CM treatment after myocardial infarction increases capillary density and preserves cardiac function, probably by increasing myocardial perfusion.     

Characterisation of recombinant epithiospecifier protein and its over-expression in Arabidopsis thaliana

Epithiospecifier protein (ESP) is a protein that catalyses formation of epithionitriles during glucosinolate hydrolysis. In vitro assays with a recombinant ESP showed that the formation of epithionitriles from alkenylglucosinolates is ESP and ferrous ion dependent. Nitrile formation in vitro however does not require ESP but only the presence of Fe(II) and myrosinase. Ectopic expression of ESP in Arabidopsis thaliana Col-5 under control of the strong viral CaMV 35S promoter altered the glucosinolate product profile from isothiocyanates towards the corresponding nitriles.