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Other Proteins (other + protein)
Kinds of Other Proteins Selected AbstractsConstitutive Secretion of Immunoglobulin a and Other Proteins into Lumina of Unstimulated Submandibular Glands in Anaesthetised RatsEXPERIMENTAL PHYSIOLOGY, Issue 1 2003G. B. Proctor Salivary fluid secretion is dependent upon reflex stimuli mediated by autonomic nerves. In order to determine if immunoglobulin A (IgA) and salivary proteins are secreted in the absence of nerve stimulation, small volumes (< 2 µl) of saliva were consecutively collected from the submandibular duct of anaesthetised rats following rest pauses in order to sample the protein contents of the ductal system. Within the first 5 µl of such saliva collected by parasympathetic nerve stimulation, IgA and other salivary proteins reached peak concentrations that were over 20-fold greater than levels in parasympathetically stimulated saliva subsequently collected during a 5 min period of stimulation. Confocal microscopy of TRITC-labelled IgA added to live, acutely isolated submandibular acini indicated that it did not enter the lumina by paracellular leakage. IgG is thought to enter saliva by paracellular leakage but did not accumulate in luminal saliva in the present study. Electrophoresis suggested that the major proteins secreted in the absence of stimulation were the same as those present in subsequently stimulated saliva. It can be concluded that IgA and other major submandibular proteins are secreted into glandular lumina in the absence of nerve stimulation. The functional significance of such unstimulated protein secretion is at present unclear. [source] Proteomic analysis of pancreatic endocrine tumor cell lines treated with the histone deacetylase inhibitor trichostatin APROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 10 2007Daniela Cecconi Abstract Effects of the histone-deacetylases inhibitor trichostatin A (TSA) on the growth of three different human pancreatic endocrine carcinoma cell lines (CM, BON, and QGP-1) have been assessed via dosage-dependent growth inhibition curves. TSA determined strong inhibition of cell growth with similar IC50 values for the different cell lines: 80.5,nM (CM), 61.6,nM (BON), and 86,nM (QGP-1), by arresting the cell cycle in G2/M phase and inducing apoptosis. 2DE and nano-RP-HPLC-ESI-MS/MS analysis revealed 34, 33, and 38 unique proteins differentially expressed after TSA treatment in the CM, BON, and QGP-1 cell lines, respectively. The most important groups of modulated proteins belong to cell proliferation, cell cycle, and apoptosis classes (such as peroxiredoxins 1 and 2, the diablo protein, and HSP27). Other proteins pertain to processes such as regulation of gene expression (nucleophosmin, oncoprotein dek), signal transduction (calcium-calmodulin), chromatin, and cytoskeleton organization (calgizzarin, dynein, and lamin), RNA splicing (nucleolin, HNRPC), and protein folding (HSP70). The present data are in agreement with previous proteomic analyses performed on pancreatic ductal carcinoma cell lines (Cecconi, D. et al.., Electrophoresis 2003; Cecconi, D. et al., J. Proteome Res. 2005) and place histone-deacetylases inhibitors among the potentially most powerful drugs for the treatment of pancreatic tumors. [source] Expression of Na+/HCO3, co-transporter proteins (NBCs) in rat and human skeletal muscleACTA PHYSIOLOGICA, Issue 1 2004J. M. Kristensen Abstract Aim:, Sodium/bicarbonate co-transport (NBC) has been suggested to have a role in muscle pH regulation. We investigated the presence of NBC proteins in rat and human muscle samples and the fibre type distribution of the identified NBCs. Methods and results:, Western blotting of muscle homogenates and sarcolemmal membranes (sarcolemmal giant vesicles) were used to screen for the presence of NBCs. Immunohistochemistry was used for the subcellular localization. The functional test revealed that approximately half of the pH recovery in sarcolemmal vesicles produced from rat muscle is mediated by bicarbonate-dependent transport. This indicates that the NBCs are preserved in the vesicles. The western blotting experiments demonstrated the existence of at least two NBC proteins in skeletal muscle. One NBC protein (approximately 150 kDa) seems to be related to the kidney/pancreas/heart isoform NBC1, whereas the other protein (approximately 200 kDa) is related to the NBC4 isoform. The two NBC proteins represent the electrogenic isoforms named NBCe1 and NBCe2. Membrane fractionation and immunofluorescence techniques confirmed that the two NBCs are located in the sarcolemmal membrane as well as in some internal membranes, probably the T-tubules. The two NBCs localized in muscle have distinct fibre type distributions. Conclusions:, Skeletal muscle possesses two variants of the sodium/bicarbonate co-transporter (NBC) isoforms, which have been called NBCe1 and NBCe2. [source] Evaluation of apoptosis in cytologic specimensDIAGNOSTIC CYTOPATHOLOGY, Issue 9 2010Viktor Shtilbans Ph.D. Abstract A hallmark of neoplasia is dysregulated apoptosis, programmed cell death. Apoptosis is crucial for normal tissue homeostasis. Dysregulation of apoptotic pathways leads to reduced cytocidal responses to chemotherapeutic drugs or radiation and is a frequent contributor to therapeutic resistance in cancer. The literature pertaining to detection of apoptotic pathway constituents in cytologic specimens is reviewed herein. Virtually all methods for detecting apoptosis, including classic cytomorphologic evaluation, TUNEL assay, immunocytochemistry, and gene sequence analysis, may be applied to cytologic samples as well as tissue. Components of both intrinsic and extrinsic apoptotic pathways have been studied, including many reports examining p53 and bcl-2, as well as studies of caspase inhibitory proteins XIAP and survivin, death receptors and ligands such as Fas, Fas-ligand, and TRAIL. p53 undergoes oncogenic alteration more than any other protein; its immunocytochemical detection almost always connotes loss of its physiologic role as an inducer of apoptosis in response to a damaged genome. Several reports establish cytologic sampling as being as useful as tissue sampling. In one respect cytologic sampling is superior to tissue sampling in particular, by allowing clinicians to repeat sampling of the same tumor before and after administration of therapy; a number of reports use this approach to attempt to predict tumor response by assaying the effect of chemotherapy on the induction of apoptosis. Diagn. Cytopathol. 2010;38:685,697. © 2010 Wiley-Liss, Inc. [source] Human antibody response during sepsis against targets expressed by methicillin resistant Staphylococcus aureusFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 2 2000Udo Lorenz Abstract The identification of target structures is a prerequisite for the development of new treatment options, like antibody based therapy, against methicillin resistant Staphylococcus aureus (MRSA). In this study we identified immunodominant structures which were expressed in vivo during sepsis caused by MRSA. Using human sera we compared the immune response of humans with MRSA sepsis with the immune response of normal individuals and asymptomatically colonized individuals. We identified and characterized four staphylococcal specific antigenic structures. One target is a staphylococcal protein of 29 kDa that exhibited 29% identity to secreted protein SceA precursor of Staphylococcus carnosus. The putative function of this protein, which was designated IsaA (immunodominant staphylococcal antigen), is unknown. The second target is an immunodominant protein of 17 kDa that showed no homology to any currently known protein. This immunodominant protein was designated IsaB. The third and fourth antigens are both immunodominant proteins of 10 kDa. One of these proteins showed 100% identity to major cold shock protein CspA of S. aureus and the other protein was identified as the phosphocarrier protein Hpr of S. aureus. The identified immunodominant proteins may serve as potential targets for the development of antibody based therapy against MRSA. [source] Hepatic covalent adduct formation with zomepirac in the CD26-deficient mouseJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 1 2002MIN WANG Abstract Background and Aims: Zomepirac (ZP), a non-steroidal anti-inflammatory drug (NSAID), has been reported to cause immune-mediated liver injury. In vivo, ZP is metabolized to a chemically reactive acyl glucuronide conjugate (ZAG) which can undergo covalent adduct formation with proteins. Such acyl glucuronide-derived drug-protein adducts may be important in the development of immune and toxic responses caused by NSAID. We have shown using immunoabsorptions that the 110 kDa CD26 (dipeptidyl peptidase IV) is one of the hepatic target proteins for covalent modification by ZAG. In the present study, a CD26-deficient mouse strain was used to examine protein targets for covalent modification by ZP/metabolites in the liver. Methods and Results: The CD26-deficient phenotype was confirmed by immunohistochemistry, flow cytometry analysis, RT-PCR, enzyme assay and immunoblotting. Moreover, by using monoclonal antibody immunoblots, CD26 was not detected in the livers of ZP-treated CD26-deficient mice. Immunoblots using a polyclonal antiserum to ZP on liver from ZP-treated mice showed three major sizes of protein bands, in the 70, 110 and 140 kDa regions. Most, but not all, of the anti-ZP immunoreactivity in the 110 kDa region was absent from ZP-treated CD26-deficient mice. Conclusion: These data definitively showed that CD26 was a component of ZP-modified proteins in vivo. In addition, the data suggested that at least one other protein of approximately 110 kDa was modified by covalent adduct formation with ZAG. [source] Study of peptide,sugar non-covalent complexes by infrared atmospheric pressure matrix-assisted laser desorption/ionizationJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 7 2004Christopher E. Von Seggern Abstract Infrared atmospheric pressure matrix-assisted laser desorption/ionization quadrupole ion trap mass spectrometry was applied to the study of siglec binding to oligosaccharide ligands. Peptides were designed to mimic the binding sites of three members of the siglec family: sialoadhesin, MAG and CD22. These peptides were tested for their ability to complex with their carbohydrate ligands 3, -sialyllactose (3,SL) and 6, -sialyllactose (6,SL). All peptides demonstrated the ability to bind to the carbohydrates, with the peptide representing sialoadhesin maintaining its binding specificity for 3,SL in preference to 6,SL. This technique can be used to study other protein,sugar interactions and can be expanded to create high-throughput screening techniques. Copyright © 2004 John Wiley & Sons, Ltd. [source] Denaturing and refolding of protein molecules on surfacesPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 11 2007Bryan Holtz Abstract Keeping protein molecules in the active state on a solid surface is essential to protein microarrays and other protein-based biosensors. Here, we show that the 2-D chemical environment controls the refolding of the denatured green fluorescent proteins tethered to solid surfaces. Refolding occurs readily on the repulsive PEG functionalized surface but is inhibited on the attractive ,NH2 functionalized surface. This result shows the critical importance of the 2-D chemical environment in the maintenance and revival of protein activity on surfaces and opens the door to designing 2-D molecular chaperones for protein folding. [source] Repolarization of the cardiac action potential.ACTA PHYSIOLOGICA, Issue 2010Does an increase in repolarization capacity constitute a new anti-arrhythmic principle? Abstract The cardiac action potential can be divided into five distinct phases designated phases 0,4. The exact shape of the action potential comes about primarily as an orchestrated function of ion channels. The present review will give an overview of ion channels involved in generating the cardiac action potential with special emphasis on potassium channels involved in phase 3 repolarization. In humans, these channels are primarily Kv11.1 (hERG1), Kv7.1 (KCNQ1) and Kir2.1 (KCNJ2) being the responsible ,-subunits for conducting IKr, IKs and IK1. An account will be given about molecular components, biophysical properties, regulation, interaction with other proteins and involvement in diseases. Both loss and gain of function of these currents are associated with different arrhythmogenic diseases. The second part of this review will therefore elucidate arrhythmias and subsequently focus on newly developed chemical entities having the ability to increase the activity of IKr, IKs and IK1. An evaluation will be given addressing the possibility that this novel class of compounds have the ability to constitute a new anti-arrhythmic principle. Experimental evidence from in vitro, ex vivo and in vivo settings will be included. Furthermore, conceptual differences between the short QT syndrome and IKr activation will be accounted for. [source] Cytoplasmic fatty acid-binding protein facilitates fatty acid utilization by skeletal muscleACTA PHYSIOLOGICA, Issue 4 2003J. F. C. Glatz Abstract The intracellular transport of long-chain fatty acids in muscle cells is facilitated to a great extent by heart-type cytoplasmic fatty acid-binding protein (H-FABP). By virtue of the marked affinity of this 14.5-kDa protein for fatty acids, H-FABP dramatically increases their concentration in the aqueous cytoplasm by non-covalent binding, thereby facilitating both the transition of fatty acids from membranes to the aqueous space and their diffusional transport from membranes (e.g. sarcolemma) to other cellular compartments (e.g. mitochondria). Striking features are the relative abundance of H-FABP in muscle, especially in oxidative muscle fibres, and the modulation of the muscular H-FABP content in concert with the modulation of other proteins and enzymes involved in fatty acid handling and utilization. Newer studies with mice carrying a homozygous or heterozygous deletion of the H-FABP gene show that, in comparison with wild-type mice, hindlimb muscles from heterozygous animals have a markedly lowered (,66%) H-FABP content but unaltered palmitate uptake rate, while in hindlimb muscles from homozygous animals (no H-FABP present) palmitate uptake was reduced by 45%. These findings indicate that H-FABP is present in relative excess and plays a substantial, but merely permissive role in fatty acid uptake by skeletal muscles. [source] Native nonmuscle myosin II stability and light chain binding in Drosophila melanogasterCYTOSKELETON, Issue 10 2006Josef D. Franke Abstract Native nonmuscle myosin IIs play essential roles in cellular and developmental processes throughout phylogeny. Individual motor molecules consist of a heterohexameric complex of three polypeptides which, when properly assembled, are capable of force generation. Here, we more completely characterize the properties, relationships and associations that each subunit has with one another in Drosophila melanogaster. All three native nonmuscle myosin II polypeptide subunits are expressed in close to constant stoichiometry to each other throughout development. We find that the stability of two subunits, the heavy chain and the regulatory light chain, depend on one another whereas the stability of the third subunit, the essential light chain, does not depend on either the heavy chain or regulatory light chain. We demonstrate that heavy chain aggregates, which form when regulatory light chain is lacking, associate with the essential light chain in vivo,thus showing that regulatory light chain association is required for heavy chain solubility. By immunodepletion we find that the majority of both light chains are associated with the nonmuscle myosin II heavy chain but pools of free light chain and/or light chain bound to other proteins are present. We identify four myosins (myosin II, myosin V, myosin VI and myosin VIIA) and a microtubule-associated protein (asp/Abnormal spindle) as binding partners for the essential light chain (but not the regulatory light chain) through mass spectrometry and co-precipitation. Using an in silico approach we identify six previously uncharacterized genes that contain IQ-motifs and may be essential light chain binding partners. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source] Ca2+ -dependent in vitro contractility of a precipitate isolated from an extract of the heliozoon Actinophrys solCYTOSKELETON, Issue 2 2006Mikihiko Arikawa Abstract Contraction of axopodia in actinophrid heliozoons (protozoa) is induced by a unique contractile structure, the "contractile tubules structure (CTS)". We have previously shown that a cell homogenate of the heliozoon Actinophrys sol yields a precipitate on addition of Ca2+ that is mainly composed of filamentous structures morphologically identical to the CTS. In this study, to further characterize the nature of the CTS in vitro, biochemical and physiological properties of the precipitate were examined. SDS-PAGE analysis showed that the Ca2+ -induced precipitate was composed of many proteins, and that no proteins in the precipitate showed any detectable changes in electrophoretic mobility on addition of Ca2+. Addition of extraneous proteins such as bovine serum albumin to the cell homogenate resulted in cosedimentation of the proteins with the Ca2+ -induced precipitate, suggesting that the CTS has a high affinity for other proteins that are not related to precipitate formation. Appearance and disappearance of the precipitate were repeatedly induced by alternating addition of Ca2+ and EGTA, and its protein composition remained unchanged even after repeated cycles. When adhered to a glass surface, the precipitate showed Ca2+ -dependent contractility with a threshold of 10,100 nM, and this contractility was not inhibited by colchicine or cytochalasin B. The precipitate repeatedly contracted and relaxed with successive addition and removal of Ca2+, indicating that the contraction was controlled by Ca2+ alone with no need for any other energy supply. From our characterization of the precipitate, we concluded that its Ca2+ -dependent formation and contraction are associated with the unique contractile organelle, the "contractile tubules structure". Cell Motil. Cytoskeleton 2006. © 2005 Wiley-Liss, Inc. [source] Identification and characterization of nucleoplasmin 3 as a histone-binding protein in embryonic stem cellsDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 5 2008Natsuki Motoi Embryonic stem (ES) cells are thought to have unique chromatin structures responsible for their capacity for self-renewal and pluripotency. To examine this possibility, we sought nuclear proteins in mouse ES cells that specifically bind to histones using a pull-down assay with synthetic peptides of histone H3 and H4 tail domain as baits. Nuclear proteins preferentially bound to the latter. We identified 45 proteins associated with the histone H4 tail and grouped them into four categories: 10 chromatin remodeling proteins, five histone chaperones, two histone modification-related proteins, and 28 other proteins. mRNA expression levels of 20 proteins selected from these 45 proteins were compared between undifferentiated and retinoic acid (RA)-induced differentiated ES cells. All of the genes were similarly expressed in both states of ES cells, except nucleoplasmin 3 (NPM3) that was expressed at a higher level in the undifferentiated cells. NPM3 proteins were localized in the nucleoli and nuclei of the cells and expression was decreased during RA-induced differentiation. When transfected with NPM3 gene, ES cells significantly increased their proliferation compared with control cells. The present study strongly suggests that NPM3 is a chromatin remodeling protein responsible for the unique chromatin structure and replicative capacity of ES cells. [source] Gene expression profiles of lens regeneration and development in Xenopus laevisDEVELOPMENTAL DYNAMICS, Issue 9 2009Erica L. Malloch Abstract Seven hundred and thirty-four unique genes were recovered from a cDNA library enriched for genes up-regulated during the process of lens regeneration in the frog Xenopus laevis. The sequences represent transcription factors, proteins involved in RNA synthesis/processing, components of prominent cell signaling pathways, genes involved in protein processing, transport, and degradation (e.g., the ubiquitin/proteasome pathway), matrix metalloproteases (MMPs), as well as many other proteins. The findings implicate specific signal transduction pathways in the process of lens regeneration, including the FGF, TGF-beta, MAPK, Retinoic acid, Wnt, and hedgehog signaling pathways, which are known to play important roles in eye/lens development and regeneration in various systems. In situ hybridization revealed that the majority of genes recovered are expressed during embryogenesis, including in eye tissues. Several novel genes specifically expressed in lenses were identified. The suite of genes was compared to those up-regulated in other regenerating tissues/organisms, and a small degree of overlap was detected. Developmental Dynamics 238:2340,2356, 2009. © 2009 Wiley-Liss, Inc. [source] ,Lipoproteins, glycoxidation and diabetic angiopathy'DIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue 5 2004Alicia J. Jenkins Abstract The chronic vascular complications of diabetes (nephropathy, retinopathy and accelerated atherosclerosis) are a major cause of morbidity and premature mortality. In spite of the more widespread availability of intensive diabetes management, approximately one in three people with diabetes develop aggressive complications and over 70% die of atherosclerosis-related diseases. Genetic and acquired factors are likely to be contributory. Potential mediators of vascular damage may include the interrelated processes of lipoprotein abnormalities, glycation, oxidation and endothelial dysfunction. Lipoprotein abnormalities encompass alterations in lipid concentrations, lipoprotein composition and subclass distribution and lipoprotein-related enzymes. Nonenzymatic glycation and oxidative damage to lipoproteins, other proteins and to vascular structures may also be deleterious. As atherosclerosis is a chronic condition commencing in youth, and because clinical events may be silent in diabetes, surrogate measures of vascular disease are important for early identification of diabetic patients with or at high risk of vascular damage, and for monitoring efficacy of interventions. The increasing array of biochemical assays for markers and mediators of vascular damage, noninvasive measures of vascular health, and therapeutic options should enable a reduction in the excessive personal and economic burden of vascular disease in type 1 and type 2 diabetes. Copyright © 2004 John Wiley & Sons, Ltd. [source] Direct Electrochemistry of Cytochrome c at Gold Electrode Modified with Fumed SilicaELECTROANALYSIS, Issue 20 2005Hongjun Chen Abstract Direct electrochemistry of horse heart cytochrome c (cytc) has been obtained at a gold electrode constructed by self-assembling fumed silica particles (FSPs) onto a silane monolayer. A pair of well-defined and nearly symmetrical redox peaks of cytc is obtained at the FSPs film modified gold electrode. Cyclic voltammetry (CV) and tapping-mode atomic force microscopy (AFM) are used to characterize the FSPs film modified electrode, showing that the FSPs can provide a favorable microenvironment for cytc and facilitate the direct electron transfer between the cytc and the gold electrode, which may propose an approach to realize the direct electrochemistry of other proteins. [source] Suppression of the mouse double minute 4 gene causes changes in cell cycle control in a human mesothelial cell line responsive to ultraviolet radiation exposureENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 9 2009Melisa Bunderson-Schelvan Abstract The TP53 tumor suppressor gene is the most frequently inactivated gene in human cancer identified to date. However, TP53 mutations are rare in human mesotheliomas, as well as in many other types of cancer, suggesting that aberrant TP53 function may be due to alterations in its regulatory pathways. Mouse double minute 4 (MDM4) has been shown to be a key regulator of TP53 activity, both independently as well as in concert with its structural homolog, Mouse Double Minute 2 (MDM2). The purpose of this study was to characterize the effects of MDM4 suppression on TP53 and other proteins involved in cell cycle control before and after ultraviolet (UV) exposure in MeT5a cells, a nonmalignant human mesothelial line. Short hairpin RNA (shRNA) was used to investigate the impact of MDM4 on TP53 function and cellular transcription. Suppression of MDM4 was confirmed by Western blot. MDM4 suppressed cells were analyzed for cell cycle changes with and without exposure to UV. Changes in cell growth as well as differences in the regulation of direct transcriptional targets of TP53, CDKN1A (cyclin-dependent kinase 1,, p21) and BAX, suggest a shift from cell cycle arrest to apoptosis upon increasing UV exposure. These results demonstrate the importance of MDM4in cell cycle regulation as well as a possible role inthe pathogenesis of mesothelioma-type cancers. Environ. Mol. Mutagen. 2009. © 2009 Wiley-Liss, Inc. [source] Association of heavy metals with metallothionein and other proteins in hepatic cytosol of marine mammals and seabirdsENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 8 2004Tokutaka Ikemoto Abstract Distribution of Cu, Zn, Cd, Ag, Hg, and Se were determined in hepatocytosol of northern fur seals (Callorhinus ursinus), black-footed albatrosses (Diomedea nigripes), and Dall's porpoises (Phocoenoides dalli). Copper, Zn, and Cd were accumulated preferentially in metallothionein (MT) fraction and their contents in MT fraction increased with the amounts in the hepatocytosol. Silver was bound to both high-molecular-weight substances (HMWS) and MT in the hepatocytosol for all three species, whereas the distribution of Ag in the cytosol was different among the three species. In northern fur seals, Ag mainly was bound to MT, whereas it mainly was associated with HMWS in Dall's porpoises. In contrast, Ag was distributed almost equally in both HMWS and MT for black-footed albatrosses. Mercury content in HMWS and Se content in HMWS and low-molecular-weight substances (LMWS) increased with their contents in hepatocytosol for all the three species. A significant positive correlation was found between Se and Hg contents in high-molecular weight (HMW) fraction in cytosol. The molar ratio of Hg and Se was close to unity in HMW fraction of the specimens with high Hg concentration in cytosol, implying that the Hg,Se complex was bound to the HMWS. Analysis of metals in the hepatocytosol by high-performance liquid chromatography/inductively coupled plasma,mass spectrometry (HPLC/ICP-MS) suggests that multiple isoforms of MT are present in hepatocytosol of the three species and that the metal profiles in hepatocytosols are different among the species. To our knowledge, this is the first report on the association of Ag with HMWS and MT in hepatocytosol of marine mammals and seabirds. Also, distribution and interaction of Hg and Se were investigated for the first time in hepatocytosol of the higher trophic marine animals. [source] From heparins to factor Xa inhibitors and beyondEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 2005S. Alban Abstract Despite some disadvantages, unfractionated heparin (UFH) and oral anticoagulants have been the only anticoagulants for prophylaxis and therapy of thromboembolic disorders for several decades. Based on the increasing knowledge of the structure and pharmacology of heparin, low molecular weight heparins (LMWH) have been developed in the 1980s. Compared to UFH, their advantages are mainly based on their reduced nonspecific binding to proteins and cells resulting in improved pharmacokinetics. In 1991, LMWH were declared as the most efficient prophylaxis in high-risk patients. Although the use of LMWH is increasing and they are today also applied for therapy and in other indications like acute coronary syndrome, they are considered not optimal concerning efficacy and safety. With the approval of fondaparinux for the prevention of venous thromboembolic disease in high-risk orthopedic patients, there might be a paradigm shift in the field of anticoagulants. Fondaparinux, a synthetic, chemically defined pentasaccharide, is the first selective inhibitor of factor Xa. By its highly specific binding to antithrombin, it selectively inhibits factor Xa and consequently prevents thrombin generation. In contrast to UFH and LMWH, it does not bind to any other cells and other proteins than antithrombin. This leads to a favourable linear pharmacokinetic profile, allowing once-daily subcutaneous application of a fixed dose without monitoring in thromboembolism prophylaxis. In addition to the evaluation of fondaparinux for further indications, chemical modifications of this pentasaccharide such as the long-acting idraparinux are currently under investigation. [source] T cell costimulation by the hepatitis C virus envelope protein E2 binding to CD81 is mediated by LckEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 2 2003Elisabetta Soldaini Abstract Binding of the hepatitis C virus (HCV) envelope protein E2 to CD81 provides a costimulatory signal for human T cells. This phenomenon may play a role in liver damage and autoimmune manifestations associated with HCV infection. Here we show that cross-linking of CD81 by HCV E2 induced a calcium flux in T cells that depends on Lck since it was blocked by PP1 and absent in Lck-deficient Jurkat T cells. In wild-type Jurkat cells, Lck was activated by CD81 cross-linking, and CD81, like Lck, was found in lipid rafts. Indeed, the integrity of the raft compartment was required for the induction of a calcium flux by E2, since methyl-,-cyclodextrin abolished this response. A requirement for TCR/CD3 expression was indicated by the absence of a calcium flux following E2 stimulation of TCR/CD3-deficient Jurkat cells. CD81 cross-linking increased and prolonged the anti-CD3-induced tyrosine phosphorylation of TCR, and of other proteins, indicating that the CD81-mediated signal converges with the TCR/CD3 signaling cascade at its most upstream step. In conclusion, we propose that the costimulatory effects of HCV E2 on T cells depend on CD81 cross-linking that activates Lck through raft aggregation and thus leads to enhanced TCR signaling. [source] Mass spectrometric characterization of the covalent modification of the nitrogenase Fe-protein in Azoarcus sp.FEBS JOURNAL, Issue 13 2009Nitrogenase Fe-protein modification was analyzed in the endophytic ,-proteobacterium Azoarcus sp. BH72. Application of modern MS techniques localized the modification in the peptide sequence and revealed it to be an ADP-ribosylation on Arg102 of one subunit of nitrogenase Fe-protein. A double digest with trypsin and endoproteinase Asp-N was necessary to obtain an analyzable peptide because the modification blocked the trypsin cleavage site at this residue. Furthermore, a peptide extraction protocol without trifluoroacetic acid was crucial to acquire the modified peptide, indicating an acid lability of the ADP-ribosylation. This finding was supported by the presence of a truncated version of the original peptide with Arg102 exchanged by ornithine. Site-directed mutagenesis verified that the ADP-ribosylation occurred on Arg102. With our approach, we were able to localize a labile modification within a large peptide of 31 amino acid residues. The present study provides a method suitable for the identification of so far unknown protein modifications on nitrogenases or other proteins. It represents a new tool for the MS analysis of protein mono-ADP-ribosylations. [source] Studies on structural and functional divergence among seven WhiB proteins of Mycobacterium tuberculosis H37RvFEBS JOURNAL, Issue 1 2009Md. Suhail Alam The whiB -like genes (1-7) of Mycobacterium tuberculosis are involved in cell division, nutrient starvation, pathogenesis, antibiotic resistance and stress sensing. Although the biochemical properties of WhiB1, WhiB3 and WhiB4 are known, there is no information about the other proteins. Here, we elucidate in detail the biochemical and biophysical properties of WhiB2, WhiB5, WhiB6 and WhiB7 of M. tuberculosis and present a comprehensive comparative study on the molecular properties of all WhiB proteins. UV,Vis spectroscopy has suggested the presence of a redox-sensitive [2Fe,2S] cluster in each of the WhiB proteins, which remains stably bound to the proteins in the presence of 8 m urea. The [2Fe,2S] cluster of each protein was oxidation labile but the rate of cluster loss decreased under reducing environments. The [2Fe,2S] cluster of each WhiB protein responded differently to the oxidative effect of air and oxidized glutathione. In all cases, disassembly of the [2Fe,2S] cluster was coupled with the oxidation of cysteine-thiols and the formation of two intramolecular disulfide bonds. Both CD and fluorescence spectroscopy revealed that WhiB proteins are structurally divergent members of the same family. Similar to WhiB1, WhiB3 and WhiB4, apo WhiB5, WhiB6 and WhiB7 also reduced the disulfide of insulin, a model substrate. However, the reduction efficiency varied significantly. Surprisingly, WhiB2 did not reduce the insulin disulfide, even though its basic properties were similar to those of others. The structural and functional divergence among WhiB proteins indicated that each WhiB protein is a distinguished member of the same family and together they may represent a novel redox system for M. tuberculosis. [source] Identification of substrates for transglutaminase in Physarum polycephalum, an acellular slime mold, upon cellular mechanical damageFEBS JOURNAL, Issue 11 2007Fumitaka Wada Transglutaminases are Ca2+ -dependent enzymes that post-translationally modify proteins by crosslinking or polyamination at specific polypeptide-bound glutamine residues. Physarum polycephalum, an acellular slime mold, is the evolutionarily lowest organism expressing a transglutimase whose primary structure is similar to that of mammalian transglutimases. We observed transglutimase reaction products at injured sites in Physarum macroplasmodia upon mechanical damage. With use of a biotin-labeled primary amine, three major proteins constituting possible transglutimase substrates were affinity-purified from the damaged slime mold. The purified proteins were Physarum actin, a 40 kDa Ca2+ -binding protein with four EF-hand motifs (CBP40), and a novel 33 kDa protein highly homologous to the eukaryotic adenine nucleotide translocator, which is expressed in mitochondria. Immunochemical analysis of extracts from the damaged macroplasmodia indicated that CBP40 is partly dimerized, whereas the other proteins migrated as monomers on SDS/PAGE. Of the three proteins, CBP40 accumulated most significantly around injured areas, as observed by immunofluoresence. These results suggested that transglutimase reactions function in the response to mechanical injury. [source] R120G ,B-crystallin promotes the unfolding of reduced ,-lactalbumin and is inherently unstableFEBS JOURNAL, Issue 3 2005Teresa M. Treweek ,-Crystallin is the principal lens protein which, in addition to its structural role, also acts as a molecular chaperone, to prevent aggregation and precipitation of other lens proteins. One of its two subunits, ,B-crystallin, is also expressed in many nonlenticular tissues, and a natural missense mutation, R120G, has been associated with cataract and desmin-related myopathy, a disorder of skeletal muscles [Vicart P, Caron A, Guicheney P, Li Z, Prevost MC, Faure A, Chateau D, Chapon F, Tome F, Dupret JM, Paulin D & Fardeau M (1998) Nat Genet20, 92,95]. In the present study, real-time 1H-NMR spectroscopy showed that the ability of R120G ,B-crystallin to stabilize the partially folded, molten globule state of ,-lactalbumin was significantly reduced in comparison with wild-type ,B-crystallin. The mutant showed enhanced interaction with, and promoted unfolding of, reduced ,-lactalbumin, but showed limited chaperone activity for other target proteins. Using NMR spectroscopy, gel electrophoresis, and MS, we observed that, unlike the wild-type protein, R120G ,B-crystallin is intrinsically unstable in solution, with unfolding of the protein over time leading to aggregation and progressive truncation from the C-terminus. Light scattering, MS, and size-exclusion chromatography data indicated that R120G ,B-crystallin exists as a larger oligomer than wild-type ,B-crystallin, and its size increases with time. It is likely that removal of the positive charge from R120 of ,B-crystallin causes partial unfolding, increased exposure of hydrophobic regions, and enhances its susceptibility to proteolysis, thus reducing its solubility and promoting its aggregation and complexation with other proteins. These characteristics may explain the involvement of R120G ,B-crystallin with human disease states. [source] The conformational stability of the Streptomyces coelicolor histidine-phosphocarrier proteinFEBS JOURNAL, Issue 11 2004Characterization of cold denaturation, protein interactions Thermodynamic parameters describing the conformational stability of the histidine-containing phosphocarrier protein from Streptomyces coelicolor, scHPr, have been determined by steady-state fluorescence measurements of isothermal urea-denaturations, differential scanning calorimetry at different guanidinium hydrochloride concentrations and, independently, by far-UV circular dichroism measurements of isothermal urea-denaturations, and thermal denaturations at fixed urea concentrations. The equilibrium unfolding transitions are described adequately by the two-state model and they validate the linear free-energy extrapolation model, over the large temperature range explored, and the urea concentrations used. At moderate urea concentrations (from 2 to 3 m), scHPr undergoes both high- and low-temperature unfolding. The free-energy stability curves have been obtained for the whole temperature range and values of the thermodynamic parameters governing the heat- and cold-denaturation processes have been obtained. Cold-denaturation of the protein is the result of the combination of an unusually high heat capacity change (1.4 ± 0.3 kcal·mol,1·K,1, at 0 m urea, being the average of the fluorescence, circular dichroism and differential scanning calorimetry measurements) and a fairly low enthalpy change upon unfolding at the midpoint temperature of heat-denaturation (59 ± 4 kcal·mol,1, the average of the fluorescence, circular dichroism and differential scanning calorimetry measurements). The changes in enthalpy (m,Hi), entropy (m,Si) and heat capacity (m,Cpi), which occur upon preferential urea binding to the unfolded state vs. the folded state of the protein, have also been determined. The m,Hi and the m,Si are negative at low temperatures, but as the temperature is increased, m,Hi makes a less favourable contribution than m,Si to the change in free energy upon urea binding. The m,Cpi is larger than those observed for other proteins; however, its contribution to the global heat capacity change upon unfolding is small. [source] Solution structure of the matrix attachment region-binding domain of chicken MeCP2FEBS JOURNAL, Issue 15 2003Björn Heitmann Methyl-CpG-binding protein 2 (MeCP2) is a multifunctional protein involved in chromatin organization and silencing of methylated DNA. MAR-BD, a 125-amino-acid residue domain of chicken MeCP2 (cMeCP2, originally named ARBP), is the minimal protein fragment required to recognize MAR elements and mouse satellite DNA. Here we report the solution structure of MAR-BD as determined by multidimensional heteronuclear NMR spectroscopy. The global fold of this domain is very similar to that of rat MeCP2 MBD and MBD1 MBD (the methyl-CpG-binding domains of rat MeCP2 and methyl-CpG-binding domain protein 1, respectively), exhibiting a three-stranded antiparallel ,-sheet and an ,-helix ,1. We show that the C-terminal portion of MAR-BD also contains an amphipathic helical coil, ,2/,3. The hydrophilic residues of this coil form a surface opposite the DNA interface, available for interactions with other domains of MeCP2 or other proteins. Spectroscopic studies of the complex formed by MAR-BD and a 15-bp fragment of a high-affinity binding site from mouse satellite DNA indicates that the coil is also involved in protein·DNA interactions. These studies provide a basis for discussion of the consequences of six missense mutations within the helical coil found in Rett syndrome cases. [source] Functional reconstitution of the HIV receptors CCR5 and CD4 in liposomesFEBS JOURNAL, Issue 21 2002François Devesa Reconstitution of membrane proteins allows their study in a membrane environment that can be manipulated at will. Because membrane proteins have diverse biophysical properties, reconstitution methods have so far been developed for individual proteins on an ad hoc basis. We developed a postinsertion reconstitution method for CCR5, a G protein coupled receptor, with seven transmembrane ,,helices and small ecto- and endodomains. A His6 -tagged version of CCR5 was expressed in mammalian cells, purified using the detergent N -dodecyl-,- d -maltoside (DDM) and reconstituted into preformed liposomal membranes saturated with DDM, removing the detergent with hydrophobic polystyrene beads. We then attempted to incorporate CD4, a protein with a single transmembrane helix and a large hydrophilic ectodomain into liposomal membranes, together with CCR5. Surprisingly, reconstitution of this protein was also achieved by the method. Both proteins were found to be present together in individual liposomes. The reconstituted CCR5 was recognized by several monoclonal antibodies, recognized its natural ligand, and CD4 bound a soluble form of gp120, a subunit of the HIV fusion protein that uses CD4 as a receptor. Moreover, cells expressing the entire fusion protein of HIV bound to the liposomes, indicating that the proteins were intact and that most of them were oriented right side out. Thus, functional coreconstitution of two widely different proteins can be achieved by this method, suggesting that it might be useful for other proteins. [source] Cloning and characterization of novel snake venom proteins that block smooth muscle contractionFEBS JOURNAL, Issue 11 2002Yasuo Yamazaki In this study, we isolated a 25-kDa novel snake venom protein, designated ablomin, from the venom of the Japanese Mamushi snake (Agkistrodon blomhoffi). The amino-acid sequence of this protein was determined by peptide sequencing and cDNA cloning. The deduced sequence showed high similarity to helothermine from the Mexican beaded lizard (Heloderma horridum horridum), which blocks voltage-gated calcium and potassium channels, and ryanodine receptors. Ablomin blocked contraction of rat tail arterial smooth muscle elicited by high K+ -induced depolarization in the 0.1,1 µm range, but did not block caffeine-stimulated contraction. Furthermore, we isolated three other proteins from snake venoms that are homologous to ablomin and cloned the corresponding cDNAs. Two of these homologous proteins, triflin and latisemin, also inhibited high K+ -induced contraction of the artery. These results indicate that several snake venoms contain novel proteins with neurotoxin-like activity. [source] Capturing Complex Protein Gradients on Biomimetic Hydrogels for Cell-Based AssaysADVANCED FUNCTIONAL MATERIALS, Issue 21 2009Steffen Cosson Abstract A versatile strategy to rapidly immobilize complex gradients of virtually any desired protein on soft poly(ethylene glycol) (PEG) hydrogel surfaces that are reminiscent of natural extracellular matrices (ECM) is reported. A microfluidic chip is used to generate steady-state gradients of biotinylated or Fc-tagged fusion proteins that are captured and bound to the surface in less than 5,min by NeutrAvidin or ProteinA, displayed on the surface. The selectivity and orthogonality of the binding schemes enables the formation of parallel and orthogonal overlapping gradients of multiple proteins, which is not possible on conventional cell culture substrates. After patterning, the hydrogels are released from the microfluidic chip and used for cell culture. This novel platform is validated by conducting single-cell migration experiments using time-lapse microscopy. The orientation of cell migration, as well as the migration rate of primary human fibroblasts, depends on the concentration of an immobilized fibronectin fragment. This technique can be readily applied to other proteins to address a wealth of biological questions with different cell types. [source] Tamo selectively modulates nuclear import in DrosophilaGENES TO CELLS, Issue 4 2003Svetlana Minakhina Background: The NF-,B/Rel pathway functions in the establishment of dorsal-ventral polarity and in the innate humoral and cellular immune response in Drosophila. An important aspect of all NF-,B/Rel pathways is the translocation of the Rel proteins from the cytoplasm to the nucleus, where they function as transcription factors. Results: We have identified a new protein, Tamo, which binds to Drosophila Rel protein Dorsal, but not to Dorsal lacking the nuclear localization sequence. Tamo does not bind to the other Drosophila Rel proteins, Dif and Relish. The Tamo-Dorsal complex forms in the cytoplasm and Tamo also interacts with a cytoplasmically orientated nucleoporin. In addition Tamo binds the Ras family small GTPase, Ran. Tamo functions during oogenesis and, based on phenotypic analysis, controls the levels of nuclear Dorsal in early embryos. It further regulates the accumulation of Dorsal in the nucleus after immune challenge. Conclusions:Tamo has an essential function during oogenesis. Tamo interacts with Dorsal and proteins that are part of the nuclear import machinery. We propose that tamo modulates the levels of import of Dorsal and other proteins. [source] | |||||||||||||||||||||||||||||||