Combinatorial Library of Improved Peptide Aptamers,CLIPs to Inhibit RAGE Signal Transduction in Mammalian Cells

Peptide aptamers are small proteins containing a randomized peptide sequence embedded into a stable protein scaffold, such as Thioredoxin. We developed a robust method for building a Combinatorial Library of Improved Peptide aptamers (CLIPs) of high complexity, containing ≥3×10¹⁰ independent clones, to be used as a molecular tool in the study of biological pathways. The Thioredoxin scaffold was modified to increase solubility and eliminate aggregation of the peptide aptamers. The CLIPs was used in a yeast two-hybrid screen to identify peptide aptamers that bind to various domains of the Receptor for Advanced Glycation End products (RAGE). NMR spectroscopy was used to identify interaction surfaces between the peptide aptamers and RAGE domains. Cellular functional assays revealed that in addition to directly interfering with known binding sites, peptide aptamer binding distal to ligand sites also inhibits RAGE ligand-induced signal transduction. This finding underscores the potential of using CLIPs to select allosteric inhibitors of biological targets.     

Molecular Chaperone Mediated Late-Stage Neuroprotection in the SOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective loss of motor neurons in the spinal cord, brain stem, and motor cortex. Mutations in superoxide dismutase (SOD1) are associated with familial ALS and lead to SOD1 protein misfolding and aggregation. Here we show that the molecular chaperone, HSJ1 (DNAJB2), mutations in which cause distal hereditary motor neuropathy, can reduce mutant SOD1 aggregation and improve motor neuron survival in mutant SOD1 models of ALS. Overexpression of human HSJ1a (hHSJ1a) in vivo in motor neurons of SOD1^G93A transgenic mice ameliorated disease. In particular, there was a significant improvement in muscle force, increased motor unit number and enhanced motor neuron survival. hHSJ1a was present in a complex with SOD1^G93A and led to reduced SOD1 aggregation at late stages of disease progression. We also observed altered ubiquitin immunoreactivity in the double transgenic animals, suggesting that ubiquitin modification might be important for the observed improvements. In a cell model of SOD1^G93A aggregation, HSJ1a preferentially bound to mutant SOD1, enhanced SOD1 ubiquitylation and reduced SOD1 aggregation in a J-domain and ubiquitin interaction motif (UIM) dependent manner. Collectively, the data suggest that HSJ1a acts on mutant SOD1 through a combination of chaperone, co-chaperone and pro-ubiquitylation activity. These results show that targeting SOD1 protein misfolding and aggregation in vivo can be neuroprotective and suggest that manipulation of DnaJ molecular chaperones might be useful in the treatment of ALS.     

Metabolic Response of Candida albicans to Phenylethyl Alcohol under Hyphae-Inducing Conditions

Phenylethyl alcohol was one of the first quorum sensing molecules (QSMs) identified in C. albicans. This extracellular signalling molecule inhibits the hyphal formation of C. albicans at high cell density. Little is known, however, about the underlying mechanisms by which this QSM regulates the morphological switches of C. albicans. Therefore, we have applied metabolomics and isotope labelling experiments to investigate the metabolic changes that occur in C. albicans in response to phenylethyl alcohol under defined hyphae-inducing conditions. Our results showed a global upregulation of central carbon metabolism when hyphal development was suppressed by phenylethyl alcohol. By comparing the metabolic changes in response to phenylethyl alcohol to our previous metabolomic studies, we were able to short-list 7 metabolic pathways from central carbon metabolism that appear to be associated with C. albicans morphogenesis. Furthermore, isotope-labelling data showed that phenylethyl alcohol is indeed taken up and catabolised by yeast cells. Isotope-labelled carbon atoms were found in the majority of amino acids as well as in lactate and glyoxylate. However, isotope-labelled carbon atoms from phenylethyl alcohol accumulated mainly in the pyridine ring of NAD⁺/NADH and NADP^−/NADPH molecules, showing that these nucleotides were the main products of phenylethyl alcohol catabolism. Interestingly, two metabolic pathways where these nucleotides play an important role, nitrogen metabolism and nicotinate/nicotinamide metabolism, were also short-listed through our previous metabolomics works as metabolic pathways likely to be closely associated with C. albicans morphogenesis.     

Chiroptical Properties of Bilirubin-Serum Albumin Binding Sites

Although the interactions between bilirubin and serum albumin are among the most studied serum albumin-ligand interactions, the binding-site location and the participation of bilirubin-serum albumin complexes in pathological and physiological processes are under debate. In this article, we have benefited from the chiral structure of bilirubin and used CD spectroscopy to characterize the structure of bilirubin bound to human and bovine serum albumins. We determined that in a phosphate buffer at pH 7.8 there are three binding sites in both human and bovine serum albumins. While the primary binding sites in human and bovine serum albumins bind bilirubin with P- and M-helical conformations, respectively, the secondary binding sites in both albumins bind bilirubin in the P-helical conformation. We have shown that the bonding of bilirubin to the serum albumin matrix is a more favorable process than the self-association of bilirubin under the studied conditions, with a maximum of three bound bilirubins per serum albumin molecule. Although bilirubin bound to the primary binding site has attracted the most attention, the presented results have documented the impact of the secondary binding sites which are relevant in the displacement reactions between BR and drugs and in the phenomena where bilirubin plays antioxidant, antimutagenic, and anti-inflammatory roles. Chirality 00:000000, 2013. © 2013 Wiley Periodicals, Inc.     

Redox regulation of morphology,cell stiffness,and lectin-induced aggregation of human platelets

Redox regulation and carbohydrate recognition are potent molecular mechanisms which can contribute to platelet aggregation in response to various stimuli. The purpose of this study is to investigate the relationship between these mechanisms and to examine whether cell surface glycocalyx and cell stiffness of human platelets are sensitive to the redox potential formed by glutathione. To this end, human platelets were treated with different concentrations (0.05 μM to 6 mM) and ratios of reduced or oxidized glutathione (GSH or GSSG), and platelet morphological, mechanical, and functional properties were determined using conventional light microscopy, atomic force microscopy, and lectin-induced cell aggregation analysis. It was found that lowering the glutathione redox potential changed platelet morphology and increased platelet stiffness as well as modulated nonuniformly platelet aggregation in response to plant lectins with different carbohydrate-binding specificity including wheat germ agglutinin, Sambucus nigra agglutinin, and Canavalia ensiformis agglutinin. Extracellular redox potential and redox buffering capacity of the GSSG/2GSH couple were shown to control the availability of specific lectin-binding glycoligands on the cell surface, while the intracellular glutathione redox state affected the general functional ability of platelets to be aggregated independently of the type of lectins. Our data provide the first experimental evidence that glutathione as a redox molecule can affect the mechanical stiffness of human platelets and induce changes of the cell surface glycocalyx, which may represent a new mechanism of redox regulation of intercellular contacts.     

Semiquantitative proteomic analysis of the human spliceosome via a novel two-dimensional gel electrophoresis method

More than 200 proteins associate with human spliceosomes, but little is known about their relative abundances in a given spliceosomal complex. Here we describe a novel two-dimensional (2D) electrophoresis method that allows separation of high-molecular-mass proteins without in-gel precipitation and thus without loss of protein. Using this system coupled with mass spectrometry, we identified 171 proteins altogether on 2D maps of stage-specific spliceosomal complexes. By staining with a fluorescent dye with a wide linear intensity range, we could quantitate and categorize proteins as present in high, moderate, or low abundance. Affinity-purified human B, B(act), and C complexes contained 69, 63, and 72 highly/moderately abundant proteins, respectively. The recruitment and release of spliceosomal proteins were followed based on their abundances in A, B, B(act), and C spliceosomal complexes. Staining with a phospho-specific dye revealed that approximately one-third of the proteins detected in human spliceosomal complexes by 2D gel analyses are phosphorylated. The 2D gel electrophoresis system described here allows for the first time an objective view of the relative abundances of proteins present in a particular spliceosomal complex and also sheds additional light on the spliceosome's compositional dynamics and the phosphorylation status of spliceosomal proteins at specific stages of splicing.     

Expression of humanized anti-Her2/neu single-chain IgG1-like antibody in mammary glands of transgenic mice

A system for production of single-chain antibody in mammary glands of mice was developed on the basis of a hybrid gene constructed from the coding sequence of anti-Her2/neu single-chain antibody inserted into the first exon of the sheep beta-lactoglobulin gene. Lines of transgenic mice were obtained that expressed humanized single-chain anti-Her2/neu IgG1-like antibody in their milk. These antibodies interact with Her2/neu antigen with high affinity (K_d = 0.4 nM). The expression level of the transgene depended on its integration site in the genome but not on the copy number. The transgene had no toxic effect on the mice and was stably inherited, at least for two generations. The results reveal new opportunities of producing single-chain antibodies in the milk of animals.     

The development of marine biofilms on two commercial non-biocidal coatings: a comparison between silicone and fluoropolymer technologies

The antimicrobial performance of two fouling-release coating systems, Intersleek 700? (IS700; silicone technology), Intersleek 900? (IS900; fluoropolymer technology) and a tie coat (TC, control surface) was investigated in a short term (10 days) field experiment conducted at a depth of ca 0.5 m in the Marina Bandar Rawdha (Muscat, Oman). Microfouling on coated glass slides was analyzed using epifluorescence microscopy and adenosine-5'-triphosphate (ATP) luminometry. All the coatings developed biofilms composed of heterotrophic bacteria, cyanobacteria, seven species of diatoms (2 species of Navicula, Cylindrotheca sp., Nitzschia sp., Amphora sp., Diploneis sp., and Bacillaria sp.) and algal spores (Ulva sp.). IS900 had significantly thinner biofilms with fewer diatom species, no algal spores and the least number of bacteria in comparison with IS700 and the TC. The ATP readings did not correspond to the numbers of bacteria and diatoms in the biofilms. The density of diatoms was negatively correlated with the density of the bacteria in biofilms on the IS900 coating, and, conversely, diatom density was positively correlated in biofilms on the TC. The higher antifouling efficacy of IS900 over IS700 may lead to lower roughness and thus lower fuel consumption for those vessels that utilise the IS900 fouling-release coating.     

Application of a new amidophosphite ligand to Rh‐catalyzed asymmetric hydrogenation of β‐dehydroamino acid derivatives in supercritical carbon dioxide: Activation effect of protic co‐solvents

New chiral amidophosphite ligand was synthesized and tested in the Rh‐catalyzed asymmetric hydrogenation of (Z)‐β‐(acylamino)acrylates in protic solvents and supercritical carbon dioxide (scCO₂) The catalytic performance is affected greatly by the acidity of the solvents. Better enantioselectivity (up to 88% ee) was achieved in scCO₂ containing 1,1,1,3,3,3‐hexafluoro‐2‐propanol, compared to neat protic solvents. Chirality, 2011. © 2011 Wiley‐Liss, Inc.