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Cleavage
Kinds of Cleavage Terms modified by Cleavage Selected AbstractsFAULT-RELATED SOLUTION CLEAVAGE IN EXPOSED CARBONATE RESERVOIR ROCKS IN THE SOUTHERN APENNINES, ITALYJOURNAL OF PETROLEUM GEOLOGY, Issue 2 2001A. Billi The deformation associated with a number of kilometre-scale strike-slip fault zones which cut through outcropping carbonate rocks in the Southern Apennines was investigated at regional and outcrop scales. These faults trend roughly east-west and were studied at the Gargano Promontory on the Adriatic Coast (in the Apulian foreland) and in the Matese Mountains, about 120 km to the west (within the Apenninic fold-and-thrust belt). The fault zones are 200,300 m wide and typically comprise a core surrounded by a damage zone. Within fault cores, fault rocks (gouges and cataclasites) typically occur along master slip planes; in damage zones, secondary slip planes and solution cleavage are the most important planar discontinuities. The protolith carbonates surrounding the fault zone at Gargano show little deformation, but they are fractured in the Matese Mountains as a result of an earlier thrust phase. Cleavage surfaces in the damage zone of the studied faults are interpreted to be fault-propagation structures. Our field data indicate that cleavage-fault intersection lines are parallel to the normals of fault slip-vectors. The angle between a fault plane and the associated cleavage was found to be fairly constant (c. 40") at different scales of observation. Finally, the spacing of the solution cleavage surfaces appeared in general to be regular (with a mean of about 22 mm), although it was found to decrease slightly near a fault plane. These results are intended to provide a basis for predicting the architecture of fault zones in buried carbonate reservoirs using seismic reflection and borehole data. [source] Cathepsin X cleaves the ,2 cytoplasmic tail of LFA-1 inducing the intermediate affinity form of LFA-1 and ,-actinin-1 bindingEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 11 2009Zala Jevnikar Abstract The motility of T cells depends on the dynamic spatial regulation of integrin-mediated adhesion and de-adhesion. Cathepsin X, a cysteine protease, has been shown to regulate T-cell migration by interaction with lymphocyte function associated antigen-1 (LFA-1). LFA-1 adhesion to the ICAM-1 is controlled by the association of actin-binding proteins with the cytoplasmic tail of the ,2 chain of LFA-1. Cleavage by cathepsin X of the amino acid residues S769, E768 and A767 from the C-terminal of the ,2 cytoplasmic tail of LFA-1 is shown to promote binding of the actin-binding protein ,-actinin-1. Furthermore, cathepsin X overexpression reduced LFA-1 clustering and induced an intermediate affinity LFA-1 conformation that is known to associate with ,-actinin-1. Increased levels of intermediate affinity LFA-1 resulted in augmented cell spreading due to reduced attachment of T cells to the ICAM-1-coated surface. Gradual cleavage of LFA-1 by cathepsin X enables the transition between intermediate and high affinity LFA-1, an event that is crucial for effective T-cell migration. [source] A novel form of NF-,B is induced by Leishmania infection: Involvement in macrophage gene expressionEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 4 2008David Abstract Leishmania spp. are obligate intracellular parasites that inhabit the phagolysosomes of macrophages. Manipulation of host cell signaling pathways and gene expression by Leishmania is critical for Leishmania's survival and resultant pathology. Here, we show that infection of macrophages with Leishmania promastigotes in vitro causes specific cleavage of the NF-,B p65RelA subunit. Cleavage occurs in the cytoplasm and is dependent on the Leishmania protease gp63. The resulting fragment, p35RelA, migrates to the nucleus, where it binds DNA as a heterodimer with NF-,B p50. Importantly, induction of chemokine gene expression (MIP-2/CXCL2, MCP-1/CCL2, MIP-1,/CCL3, MIP-1,/CCL4) by Leishmania is NF-,B dependent, which implies that p35RelA/p50 dimers are able to activate transcription, despite the absence of a recognized transcriptional transactivation domain. NF-,B cleavage was observed following infection with a range of pathogenic species, including L.,donovani, L.,major, L.,mexicana, and L.,(Viannia) braziliensis, but not the non-pathogenic L.,tarentolae or treatment with IFN-,. These results indicate a novel mechanism by which a pathogen can subvert a macrophage's regulatory pathways to alter NF-,B activity. [source] Cleavage of CO by Mo[N(R)Ar]3 ComplexesEUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 23 2007Gemma Christian Abstract The reaction of MoL3 [L = NH2 and N(tBu)Ar] with CO was explored using DFT in order to rationalize why CO cleavage is not observed experimentally for this system in contrast to the corresponding N2 reaction which results in spontaneous cleavage of the N,N bond. The binding of CO to MoL3 was found to be both kinetically and thermodynamically favored over the binding of N2, with the formation of the encounter complex, L3Mo,CO, calculated to be without barrier and exothermic. While the overall reaction to form the C,MoL3 and O,MoL3 products was calculated to be energetically favorable, both the encounter complex and intermediate dimer, L3Mo,CO,MoL3, were found to be lower in energy than the products, with the final C,O cleavage step calculated to be endothermic by 169 kJ,mol,1 and 163 kJ,mol,1 for L = NH2 and N(tBu)Ar, respectively. The unfavorable CO cleavage step can be attributed to the fact that Mo does not possess the optimum d-electron configuration to sufficiently stabilise the carbide and oxide products relative to the CO-bridged intermediate dimer.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007) [source] Optimization of the Azobenzene Scaffold for Reductive Cleavage by Dithionite; Development of an Azobenzene Cleavable Linker for Proteomic ApplicationsEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 23 2010Geoffray Leriche Abstract In this paper we conducted an extensive reactivity study to determine the key structural features that favour the dithionite-triggered reductive cleavage of the azo,arene group. Our stepwise investigation allowed identification of a highly reactive azo,arene structure 25 bearing a carboxylic acid at the ortho position of the electron-poor arene and an ortho - O -alkyl-resorcinol as the electron-rich arene. Based on this 2-(2,-alkoxy-4,-hydroxyphenylazo)benzoic acid (HAZA) scaffold, the orthogonally protected difunctional azo,arene cleavable linker 26 was designed and synthesized. Selective linker deprotection and derivatization was performed by introducing an alkyne reactive group and a biotin affinity tag. This optimized azo,arene cleavable linker led to a total cleavage in less than 10 s with only 1 mM dithionite. Similar results were obtained in biological media. [source] A Practical Method for Selective Cleavage of a tert -Butoxycarbamoyl N -Protective Group from N,N -Diprotected ,-Amino Acid Derivatives Using Montmorillonite K-10,EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 30 2007J. Nicolás Hernández Abstract A new, practical, and mild procedure for the selective cleavage of a tert -butoxycarbonyl group (Boc) in N -Boc- N -acyl-diprotected amines is described. When applied to ,-amino acids, complete integrity of the stereochemistry was observed. The use of N,N -di-Boc-,-amino-,- and ,-hydroxy esters provided both ,- and ,-lactones in very good yields. The method is based on the use of Montmorillonite K-10 either in CH2Cl2 at room temperature or in toluene at 65 °C and is compatible with the presence of a large range of functional and other protecting groups in the substrates. In most cases virtually pure samples are obtained after filtration and removal of solvents. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007) [source] Utilization of the Versatility of Sulfur in C,C Bond Formation and Cleavage: Synthesis of ABC Taxoid SkeletonsEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 20 2007Subhash P. Chavan Abstract A practical and convenient five-step protocol is described to access the ABC ring system of Taxol by utilizing the versatility of the sulfur atom in its various oxidation states viz., condensation/Pummerer cyclization/coupling/annulation/fragmentation. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007) [source] A Microwave-Assisted Heck Reaction in Poly(ethylene glycol) for the Synthesis of BenzazepinesEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 1 2007Valérie Declerck Abstract The Heck reaction of alkylated 2-(trimethylsilyl)ethanesulfonyl (SES)-protected ,-amino esters provides benzazepines in good yields. Good selectivity towards cyclisation was obtained when the reaction was performed in PEG 3400 as the solvent under microwave activation. Cleavage of the SES group with HF provides the corresponding free benzazepine. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007) [source] Reinvestigation of the Mechanism of gem -Diacylation: Chemoselective Conversion of Aldehydes to Various gem -Diacylates and Their Cleavage under Acidic and Basic ConditionsEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 2 2005Veerababurao Kavala Abstract The mechanism of gem -diacylate formation has been studied extensively using tetrabutylammonium tribromide (TBATB) as the catalyst. The reaction proceeds by a nucleophilic attack of an anhydride on an aldehydic carbonyl group, nucleophilic attack of the hemiacylate intermediate on a second molecule of the anhydride, followed by an intermolecular attack of a second acetate group to regenerate the anhydride. gem -Diacylates of various aliphatic and aromatic aldehydes were obtained directly from the reaction of a variety of aliphatic and aromatic acid anhydrides in the presence of a catalytic quantity of tetrabutylammonium tribromide (TBATB) under solvent-free conditions. A significant electronic effect was observed during its formation as well as deprotection to the corresponding aldehyde. Chemoselective gem -diacylation of the aromatic aldehyde containing an electron-donating group has been achieved in the presence of an aldehyde containing an electron-withdrawing group. Deprotection of the gem -diacylate to the parent carbonyl compound can be accomplished in methanol in presence of the same catalyst. Here again, chemoselective deprotection of the gem -diacylate of a substrate containing an electrondonating group has been achieved in the presence of a substrate containing an electron-withdrawing group. Both the acid and base stability order of the various gem -diacylates examined follow a similar order. The stability order determined from the present study is: gem -dibenzoate > gemdipivalate > gem -diisobutyrate > gem -diacetate > gem -dipropionate. All the gem -diacylals are more stable under basic conditions than acidic condition. No correlation was found between the stability order and the pKa's of the corresponding acids; rather, the stability order is directly related to the steric crowding around the carbonyl carbon. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005) [source] Facile Cleavage of Si,C Bonds during the Sol-Gel Hydrolysis of Aminomethyltrialkoxysilanes , A New Method for the Methylation of Primary AminesEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 12 2004Augustin Adima Abstract The reaction of chloromethyltriethoxysilane with (1R,2R)-bis(methylamino)cyclohexane (1) afforded the corresponding bis-silylated compound 2. The sol-gel hydrolysis of 2 did not give the expected bridged silsesquioxane owing to quantitative Si,C-bond cleavage. Instead, silica and (1R,2R)-bis(dimethylamino)cyclohexane (3) were obtained. This reaction was exploited to propose a new route for the methylation of amines. Such methylation reaction of amines could be extended to other amines and provides a new method for the selective monomethylation of primary amines. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004) [source] Soluble LDL-R are formed by cell surface cleavage in response to phorbol estersFEBS JOURNAL, Issue 3 2004Michael J. Begg A 140-kDa soluble form of the low density lipoprotein (LDL) receptor has been isolated from the culture medium of HepG2 cells and a number of other cell types. It is produced from the 160-kDa mature LDL receptor by a proteolytic cleavage, which is stimulated in the presence of 4,-phorbol 12-myristate 13-acetate (PMA), leading to the release of a soluble fragment that constitutes the bulk of the extracellular domain of the LDL receptor. By labeling HepG2 cells with [35S]methionine and chasing in the presence of PMA, we demonstrated that up to 20% of LDL-receptors were released into the medium in a 2-h period. Simultaneously, the level of labeled cellular receptors was reduced by 30% in those cells treated with PMA compared to untreated cells, as was the total number of cell surface LDL-receptors assayed by the binding of 125I-labeled antibody to whole cells. To determine if endocytosis was required for cleavage, internalization-defective LDL-receptors were created by mutagenesis or deletion of the NPXY internalization signal, transfected into Chinese hamster ovary cells, and assayed for cleavage in the presence and absence of PMA. Cleavage was significantly greater in the case of the mutant receptors than for wild-type receptors, both in the absence and presence of PMA. Similar results were seen in human skin fibroblasts homozygous for each of the internalization-defective LDL receptor phenotypes. LDL receptor cleavage was inhibited by the hydoxamate-based inhibitor TAPI, indicating the resemblance of the LDL receptor cleavage mechanism to that of other surface released membrane proteins. [source] ORF6 from the clavulanic acid gene cluster of Streptomyces clavuligerus has ornithine acetyltransferase activityFEBS JOURNAL, Issue 8 2002Nadia J. Kershaw The clinically used beta-lactamase inhibitor clavulanic acid is produced by fermentation of Streptomyces clavuligerus. The orf6 gene of the clavulanic acid biosynthetic gene cluster in S. clavuligerus encodes a protein that shows sequence homology to ornithine acetyltransferase (OAT), the fifth enzyme of the arginine biosynthetic pathway. Orf6 was overexpressed in Escherichia coli (at ,,15% of total soluble protein by SDS/PAGE analysis) indicating it was not toxic to the host cells. The recombinant protein was purified (to >,95% purity) by a one-step technique. Like other OATs it was synthesized as a precursor protein which underwent autocatalytic internal cleavage in E. coli to generate , and , subunits. Cleavage was shown to occur between the alanine and threonine residues in a KGXGMXXPX--(M/L)AT (M/L)L motif conserved within all identified OAT sequences. Gel filtration and native electrophoresis analyses implied that the ORF6 protein was an ,2,2 heterotetramer and direct evidence for this came from mass spectrometric analyses. Although anomalous migration of the , subunit was observed by standard SDS/PAGE analysis, which indicated the presence of two bands (as previously observed for other OATs), mass spectrometric analyses did not reveal any evidence for post-translational modification of the , subunit. Extended denaturation with SDS before PAGE resulted in observation of a single major , subunit band. Purified ORF6 was able to catalyse the reversible transfer of an acetyl group from N -acetylornithine to glutamate, but not the formation of N -acetylglutamate from glutamate and acetyl-coenzyme A, nor (detectably) the hydrolysis of N -acetylornithine. Mass spectrometry also revealed the reaction proceeds via acetylation of the , subunit. [source] Characterization of the NAD+ binding site of Candida boidinii formate dehydrogenase by affinity labelling and site-directed mutagenesisFEBS JOURNAL, Issue 22 2000Nikolas E. Labrou The 2,,3,-dialdehyde derivative of ADP (oADP) has been shown to be an affinity label for the NAD+ binding site of recombinant Candida boidinii formate dehydrogenase (FDH). Inactivation of FDH by oADP at pH 7.6 followed biphasic pseudo first-order saturation kinetics. The rate of inactivation exhibited a nonlinear dependence on the concentration of oADP, which can be described by reversible binding of reagent to the enzyme (Kd = 0.46 mm for the fast phase, 0.45 mm for the slow phase) prior to the irreversible reaction, with maximum rate constants of 0.012 and 0.007 min,1 for the fast and slow phases, respectively. Inactivation of formate dehydrogenase by oADP resulted in the formation of an enzyme,oADP product, a process that was reversed after dialysis or after treatment with 2-mercaptoethanol (> 90% reactivation). The reactivation of the enzyme by 2-mercaptoethanol was prevented if the enzyme,oADP complex was previously reduced by NaBH4, suggesting that the reaction product was a stable Schiff's base. Protection from inactivation was afforded by nucleotides (NAD+, NADH and ADP) demonstrating the specificity of the reaction. When the enzyme was completely inactivated, approximately 1 mol of [14C]oADP per mol of subunit was incorporated. Cleavage of [14C]oADP-modified enzyme with trypsin and subsequent separation of peptides by RP-HPLC gave only one radioactive peak. Amino-acid sequencing of the radioactive tryptic peptide revealed the target site of oADP reaction to be Lys360. These results indicate that oADP inactivates FDH by specific reaction at the nucleotide binding site, with negative cooperativity between subunits accounting for the appearance of two phases of inactivation. Molecular modelling studies were used to create a model of C. boidinii FDH, based on the known structure of the Pseudomonas enzyme, using the modeller 4 program. The model confirmed that Lys360 is positioned at the NAD+ -binding site. Site-directed mutagenesis was used in dissecting the structure and functional role of Lys360. The mutant Lys360,Ala enzyme exhibited unchanged kcat and Km values for formate but showed reduced affinity for NAD+. The molecular model was used to help interpret these biochemical data concerning the Lys360,Ala enzyme. The data are discussed in terms of engineering coenzyme specificity. [source] Facile Synthesis of Diastereoisomerically and Optically Pure 2-Substituted Hexahydro-1H -pyrrolizin-3-onesHELVETICA CHIMICA ACTA, Issue 8 2005Romain Siegrist We report a short synthetic route that provides optically active 2-substituted hexahydro-1H -pyrrolizin-3-ones in four steps from commercially available Boc (tert -but(oxy)carbonyl))-protected proline. Diastereoisomers (,)- 11 and (,)- 12 were assembled from the proline-derived aldehyde (,)- 8 and ylide 9via a Wittig reaction and subsequent catalytic hydrogenation (Scheme,3). Cleavage of the Boc protecting group under acidic conditions, followed by intramolecular cyclization, afforded the desired hexahydro-1H -pyrrolizinones (,)- 1 and (+)- 13. Applying the same protocol to ylide 19 afforded hexahydro-1H -pyrrolizinones (,)- 25 and (,)- 26 (Scheme,5). The absolute configuration of the target compounds was determined by a combination of NMR studies (Figs.,1 and 2) and X-ray crystallographic analysis (Fig.,3). [source] Effective High-Pressure Cleavage of Sterically Hindered Steroid EstersHELVETICA CHIMICA ACTA, Issue 6 2004Wojciech Kroszczy A simple and effective method to deprotect of sterically hindered steroid esters is described. Deprotection was carried out in MeOH in the presence of a catalytic amount of Et3N under high-pressure conditions. Enzymatic, anionite, and high-pressure methods are compared. [source] Multiple cleavage sites for polymeric immunoglobulin receptorIMMUNOLOGY, Issue 4 2004Masatake Asano Summary Human polymeric immunoglobulin receptor (pIgR) was expressed in baby hamster kidney (BHK) cells using a recombinant vaccinia virus transfection system. Cleavage of pIgR on the cell surface was partially inhibited by the proteinase inhibitor, leupeptin. We addressed the question whether some particular regions of pIgR could affect the efficient cleavage of this molecule, with the following results: (1) a mutant lacking the entire cytoplasmic region resulted in release of secretory component (SC) into the culture supernatant much faster than wild-type; (2) a pIgR mutant lacking the entire extracellular domain 6, the region containing the susceptible cleavage sites, could be cleaved and released as a mutant SC. The transport kinetics of this mutant between endoplasmic reticulum (ER) and Golgi or Golgi and the cell surface was equivalent to wild-type pIgR. Our results indicate that although the main cleavage site is in domain 6, at least one other cleavage site may exist. [source] Cleavage and conformational changes of tau protein follow phosphorylation during Alzheimer's diseaseINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 2 2008Siddhartha Mondragón-Rodríguez Summary Phosphorylation, cleavage and conformational changes in tau protein all play pivotal roles during Alzheimer's disease (AD). In an effort to determine the chronological sequence of these changes, in this study, using confocal microscopy, we compared phosphorylation at several sites (Ser199/202/396/404/422 -Thr205 and the second repeat domain), cleavage of tau (D421) and the canonical conformational Alz-50 epitope. While all of these posttranslational modifications are found in neurofibrillary tangles (NFTs) at all stages of the disease, we found significantly higher numbers of phospho-tau positive NFTs when compared with cleaved tau (P = 0.006 in Braak III; P = 0.002 in Braak IV; P = 0.012 in Braak V) or compared with the Alz-50 epitope (P < 0.05). Consistent with these findings, in a double transgenic mice model (Tet/GSK-3,/VLW) overexpressing the enzyme glycogen synthase kinase-3, (GSK-3,) and tau with a triple FTDP-17 mutation (VLW) with AD-like neurodegeneration, phosphorylation at sites Ser199/202 -Thr205 was greater than truncated tau. Taken together, these data strongly support the notion that the conformational changes and truncation of tau occur after the phosphorylation of tau. We propose two probable pathways for the pathological processing of tau protein during AD, either phosphorylation and cleavage of tau followed by the Alz-50 conformational change or phosphorylation followed by the conformational change and cleavage as the last step. [source] Efficient Biocatalytic Cleavage and Recovery of Organic Substrates Supported on Soluble PolymersADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 6 2007Dario Pasini Abstract The applicability of novel solution-phase supports in combination with enzymes for biocatalytic transformations is reported. Ex novo designed styrene-based copolymers, bearing a phenylacetic residue in variable loadings and linked as a pendant group to the macromolecular backbone, through a spacer of variable length, have been synthesized and characterized. These derivatives are compatible and can be used as soluble supports in combination with immobilized penicillin G acylase (PGA , EC 3.5.1.11) for the biocatalytic cleavage of the covalently anchored organic substrate in quantitative yields, in water or water/dimethylformamide solvent mixtures, with recovery of the immobilized enzyme with negligible losses in activity. [source] Enantioselective Enzymatic Cleavage of N -Benzyloxycarbonyl GroupsADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 6-7 2003Ramesh Abstract A new enzymatic process for the enantioselective cleavage of N -benzyloxycarbonyl (Cbz) groups from protected amino acids and related compounds has been developed. The Cbz-deprotecting enzyme was isolated from cell extracts of Sphingomonas paucimobilis SC 16113 and purified to homogeneity. The purified protein has a molecular weight of 155,000 daltons and a subunit size of 44,000 daltons. [source] Cleavage of p130Cas in anoikisJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2004Lin Wei Abstract p130Cas is a multifunctional signaling adaptor protein. It integrates and relays signals generated from a variety of extracellular stimuli and regulates a number of cellular activities including cell death. In this study, we analyzed the regulation and function of p130Cas in anoikis, a type of apoptosis caused by disruption of cell-matrix interactions. We found that p130Cas was specifically cleaved during anoikis in anoikis-sensitive epithelial cells, but not in anoikis-resistant tumor cells. There is a close correlation between p130Cas cleavage and anoikis. Furthermore, we found that the cleavage of p130Cas, as well as another focal adhesion component FAK, is different from that of caspase substrate PARP and spectrin. Although caspases and calpain were found to be involved in the cleavage of p130Cas, there appear to be other unidentified proteases that are mainly responsible for the cleavage of p130Cas, particularly at the early stage of anoikis. Overexpression of the p130Cas cleavage product induced apoptosis. Taken together, these data suggest that there are novel proteases involved in the cleavage of p130Cas during anoikis, which may be functionally involved in the onset of anoikis. p130Cas may have a dual role in the regulation of anoikis. On one hand, it mediates a survival signal from cell-matrix interactions when cells are attached to the extracellular matrix. On the other hand, it participates in executing cell death when cell-matrix interactions are disrupted. These observations provide new insights into the understanding of the function of p130Cas and the molecular mechanism of anoikis. © 2003 Wiley-Liss, Inc. [source] Matrix-assisted laser desorption/ionization collision-induced dissociation of linear single oligomers of nylon-6JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 10 2001Renata Murgasova Abstract Matrix-assisted laser desorption/ionization, collision induced-dissociation (MALDI-CID) has been used to obtain structural information for linear single oligomers of nylon-6. The effects of matrix and cationization agent in MALDI-CID analysis have been investigated. Fragmentation mechanisms are proposed for the series of ions that are observed in the MALDI-CID spectra of the hexamer, octamer and dodecamer. Fragmentation processes observed in the MALDI-CID spectra include cleavage of the end groups followed by dissociation of the m/z 113 unit. Cleavage of the oligamide chain occurs at the amide linkage, as well as at adjacent bonds. For the four matrices and three cationization agents investigated, 2,5-dihydroxybenzoic acid and sodium chloride showed the best performance for MALDI-CID analysis of the dodecamer. In addition, yields of the fragment ions in MALDI-CID spectra were found to be dependent on the chain length distribution. Copyright © 2001 John Wiley & Sons, Ltd. [source] Transient forebrain ischemia modulates focal adhesion kinase (FAK)-mediated signal transduction in gerbil hippocampusJOURNAL OF NEUROCHEMISTRY, Issue 2003M. Ziemka-Na Focal adhesion kinase (FAK) is thought to play a major role in conveying survival signals from extracellular matrix (ECM). Phosphorylated FAK may interact with other nonreceptor kinases such as Src, and adaptor molecule Cas, perhaps providing a pathway by which ECM may regulate cell viability. In the present study the expression and tyrosine phosphorylation of FAK, Src and Cas after 5 min of global ischemia were investigated. The primary activation/phosphorylation of FAK, observed during first 6 h after ischemic injury, was followed by its profound down-regulation. At 72 h of reperfusion the level of phosphorylated FAK decrease to about 50% of the control. The decrease of FAK phosphorylation coincides with its proteolytic degradation. Cleavage of FAK coincided temporally with the loss of Src and Cas. Ischemia-induced proteolytic processing of the investigated proteins may lead to the interruption of ECM-derived signals and compromise neuronal survival. Acknowledgements:, Sponsored by SCSR 4P05A 08619 and Med. Res. Ctr. [source] A novel prohormone processing site in Aplysia californica: the Leu,Leu ruleJOURNAL OF NEUROCHEMISTRY, Issue 6 2002Amanda B. Hummon Abstract Neuropeptides are a complex set of signaling molecules produced through enzymatic cleavages from longer prohormone sequences. The most common cleavage sites in prohormones are basic amino acid residues; however, processing is observed at non-basic sites. Cleavage at Leu,Leu sequences has been observed in three Aplysia californica prohormones. To further investigate this unusual event, native and non-native synthetic peptides containing Leu,Leu residues are incubated with homogenates of Aplysia californica ganglia and the resulting products monitored with MALDI MS. Cleavage near and between Leu,Leu residues is observed in the abdominal and buccal ganglia homogenates, confirming the presence of an unidentified peptidase. In addition, fractions from an HPLC separation of buccal ganglia homogenates also produce cleavages at Leu,Leu residues. Products resulting from cleavage at Leu,Leu sites are observed and are produced in larger amounts in acidic and neutral pH ranges, and cleavage is inhibited by the addition of EDTA, suggesting a metal is required for activity. [source] Cleavage of platelet endothelial cell adhesion molecule-1 (PECAM-1) in platelets exposed to high shear stressJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 11 2004Y. Naganuma Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a 130 kDa transmembrane glycoprotein that belongs to the immunoglobulin superfamily and is expressed on the surface of endothelial cells, platelets, and other blood cells. Although the importance of this adhesion molecule in various cell,cell interactions is established, its functional role in platelets remains to be elucidated. In this study, we examined whether PECAM-1 underwent changes in platelets exposed to high shear stress. Platelet PECAM-1 was cleaved under high shear stress and was released into the extracellular fluid as a fragment with an approximate molecular weight of 118 kDa. The cleavage was inhibited by an anti-VWF MoAb, but not by recombinant VWF A1 domains. These findings suggest that the GPIb,VWF interaction is involved in PECAM-1 cleavage under high shear stress, and that the cleavage is independent of GPIb clustering by VWF multimers. Furthermore, EGTA or calpeptin inhibited PECAM-1 cleavage. This finding provides evidence for the involvement of calpain in PECAM-1 cleavage. Flow-cytometric analysis revealed that PECAM-1 expression on the platelet surface was decreased under high shear stress. This reduction occurred exclusively in a specific population of platelets, which corresponded to platelet-derived microparticles (PMP). In conclusion, PECAM-1 cleavage under high shear stress is closely related to the activation of calpain and the process of PMP formation mediated by the GPIb,VWF interaction. [source] Cleavage of mRNAs and role of tmRNA system under amino acid starvation in Escherichia coliMOLECULAR MICROBIOLOGY, Issue 2 2008Xia Li Summary We have shown previously that ribosome stalling during translation caused by various reasons leads to mRNA cleavage, resulting in non-stop mRNAs that are eliminated in a tmRNA-dependent manner. Amino acid starvation is a physiological condition in which ribosome stalling is expected to occur more frequently. Here we demonstrate that mRNA cleavage is induced by amino acid starvation, resulting in accumulation of truncated mRNAs in cells lacking tmRNA. The truncated mRNAs are eliminated in wild-type cells, indicating that the tmRNA system rapidly degrade the truncated mRNAs. The cleavage pattern of model mRNAs in which serine codons were replaced with threonine codons indicated that mRNA cleavage occurs near serine codons in response to serine starvation. Cells lacking all of the five known toxin loci were proficient in mRNA cleavage, showing that toxin,antitoxin systems are not responsible for the cleavage. A mild serine starvation caused a significant growth inhibition in cells lacking tmRNA but not in wild-type cells. The ribosome-mediated mRNA cleavage along with the tmRNA system is an important mechanism that enables cells to adapt to amino acid starvation conditions. [source] Molecular targets of botulinum toxin at the mammalian neuromuscular junctionMOVEMENT DISORDERS, Issue S8 2004Dorothy D. Whelchel MS Abstract The molecular targets of botulinum neurotoxins (BoNTs) are SNARE (soluble N -ethylmaleimide-sensitive factor- attachment protein- receptor) proteins necessary for neurotransmitter release. BoNT are powerful therapeutic agents in the treatment of numerous neurological disorders. The goals of this study were to (1) assess toxin diffusion by measuring substrate cleavage in adjacent and distant muscles, and (2) characterize the clinical course using SNARE protein chemistry. A small volume of BoNT/A was injected unilaterally into the mouse gastrocnemius muscle. Motor impairment was limited to the toxin-treated limb. No systemic illness or deaths occurred. At five time points, a subset of mice were killed, and muscles from both hindlimbs, and the diaphragm, were collected. Protein samples were examined for changes in SNAP-25 (synaptosomal-associated protein of Mr = 25 kDa) using immunochemistry. SNAP-25 cleavage product was noted in the toxin-treated limb as early as 1 day postinjection and continued through day 28. Onset and peak levels of substrate cleavage corresponded to the onset and peak clinical response. Cleavage was observed in adjacent and distant muscles, demonstrating that substrate cleavage is a sensitive indicator of toxin diffusion. Significant increases in full-length SNAP-25 and vesicle-associated membrane protein II were evident early in the impaired limb and continued through day 28. The increased SNARE protein most likely originates from nerve terminal sprouts. © 2004 Movement Disorder Society [source] Cleavage of the iron-methionine bond in c-type cytochromes: Crystal structure of oxidized and reduced cytochrome c2 from Rhodopseudomonas palustris and its ammonia complexPROTEIN SCIENCE, Issue 1 2002Silvano Geremia Abstract The three-dimensional structures of the native cytochrome c2 from Rhodopseudomonas palustris and of its ammonia complex have been obtained at pH 4.4 and pH 8.5, respectively. The structure of the native form has been refined in the oxidized state at 1.70 Ĺ and in the reduced state at 1.95 Ĺ resolution. These are the first high-resolution crystal structures in both oxidation states of a cytochrome c2 with relatively high redox potential (+350 mV). The differences between the two oxidation states of the native form, including the position of internal water molecules, are small. The unusual six-residue insertion Gly82-Ala87, which precedes the heme binding Met93, forms an isolated 310 -helix secondary structural element not previously observed in other c-type cytochromes. Furthermore, this cytochrome shows an external methionine residue involved in a strained folding near the exposed edge of the heme. The structural comparison of the present cytochrome c2 with other c-type cytochromes has revealed that the presence of such a residue, with torsion angles , and , of approximately ,140 and ,130°, respectively, is a typical feature of this family of proteins. The refined crystal structure of the ammonia complex, obtained at 1.15 Ĺ resolution, shows that the sulphur atom of the Met93 axial ligand does not coordinate the heme iron atom, but is replaced by an exogenous ammonia molecule. This is the only example so far reported of an X-ray structure with the heme iron coordinated by an ammonia molecule. The detachment of Met93 is accompanied by a very localized change in backbone conformation, involving mainly the residues Lys92, Met93, and Thr94. Previous studies under typical denaturing conditions, including high-pH values and the presence of exogenous ligands, have shown that the detachment of the Met axial ligand is a basic step in the folding/unfolding process of c-type cytochromes. The ammonia adduct represents a structural model for this important step of the unfolding pathway. Factors proposed to be important for the methionine dissociation are the strength of the H-bond between the Met93 and Tyr66 residues that stabilizes the native form, and the presence in this bacterial cytochrome c2 of the rare six-residue insertion in the helix 310 conformation that increases Met loop flexibility. [source] Synergistic Effect of Porcine Follicular Fluid and Dibutyryl Cyclic Adenosine Monophosphate on Development of Parthenogenetically Activated Oocytes from Pre-Pubertal GiltsREPRODUCTION IN DOMESTIC ANIMALS, Issue 5 2010AB Nascimento Contents This study investigated the effect of porcine follicular fluid (PFF) and dibutyryl cyclic adenosine monophosphate (dbcAMP) during in vitro maturation (IVM) of porcine oocytes on meiotic maturation, fertilization and embryo development, and compared the effect of supplementing the embryo culture media with PFF or foetal bovine serum (FBS) on embryo development. Oocytes from pre-pubertal gilts were IVM for 44 h, and parthenogenetically activated or in vitro -fertilized. Embryos were cultured in porcine zygote medium (PZM3) for 7 days. Cleavage and blastocyst rates were evaluated at 48 h and 7 days of culture. The supplementation of the IVM medium with 25% PFF and 1 mm dbcAMP for the first 22 h resulted in more (p < 0.05) embryos developing to the blastocyst stage as compared with the inclusion of dbcAMP alone. The dbcAMP + PFF combination increased (p < 0.05) the average number of nuclei per blastocyst as compared with either of these components alone or in its absence. A synergistic effect of dbcAMP + PFF during IVM was also reflected in the capacity of oocytes to regulate sperm penetration and prevent polyspermy, as twice as many oocytes from the control group were penetrated by more than one sperm as compared with those matured in the presence of both dbcAMP and PFF. The supplementation of PZM3 with 10% FBS from days 5 to 7 of culture significantly improved the total cell quantity in embryos derived either from control or dbcAMP + PFF matured oocytes. There was no effect on the total cell quantity when FBS was replaced by the same concentration of PFF. These studies showed that dbcAMP, PFF and FBS can improve both the quantity (57.3% vs 41.5%) and quality (74.8 vs 33.3 nuclei) of porcine blastocysts derived from oocytes recovered of pre-pubertal gilts. [source] Consequences of Nitric Oxide Synthase Inhibition During Bovine Oocyte Maturation on Meiosis and Embryo DevelopmentREPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2010KRL Schwarz Contents The importance of nitric oxide synthase (NOS) in bovine oocyte maturation was investigated. Oocytes were in vitro matured with the NOS inhibitor Nw - l -nitro-arginine methyl-ester (10,7, 10,5 and 10,3 m l -NAME) and metaphase II (MII) rates and embryo development and quality were assessed. The effect of l -NAME (10,7 m) during pre-maturation and/or maturation on embryo development and quality was also assessed. l -NAME decreased MII rates (78,82%, p < 0.05) when compared with controls without l -NAME (96%). Cleavage (77,88%, p > 0.05), Day 7 blastocyst rates (34,42%, p > 0.05) and total cell numbers in blastocysts were similar for all groups (146,171 cells, p > 0.05). Day 8 blastocyst TUNEL positive cells (3,4 cells) increased with l -NAME treatment (p < 0.05). For oocytes cultured with l -NAME during pre-maturation and/or maturation, Day 8 blastocyst development (26,34%) and Day 9 hatching rates (15,22%) were similar (p > 0.05) to controls pre-matured and matured without NOS inhibition (33 and 18%, respectively), while total cell numbers (Day 9 hatched blastocysts) increased (264,324 cells, p < 0.05) when compared with the controls (191 cells). TUNEL positive cells increased when NOS was inhibited only during the maturation period (8 cells, p < 0.05) when compared with the other groups (3,4 cells). NO may be involved in meiosis progression to MII and its deficiency during maturation increases apoptosis in embryos produced in vitro. Nitric oxide synthase inhibition during pre-maturation and/or maturation affects embryo quality. [source] Transformation of organic molecules on the low-valent {M(Ph2PCH2CH2PPh2)2} moiety derived from trans -[M(N2)2(Ph2PCH2CH2PPh2)2] or related complexes (M = MO, W)THE CHEMICAL RECORD, Issue 5 2001Hidetake Seino Abstract A zero-valent {M(Ph2PCH2CH2PPh2)2} moiety (M = Mo, W) generated in situ by dissociation of the N2 ligands in trans -[M(N2)2(Ph2PCH2CH2PPh2)2] can activate ,-accepting organic molecules including isocyanides and nitriles, which undergo the electrophilic attack caused by a strong ,-donation from a zero-valent metal center. Cleavage of a variety of C,X bonds (X = H, C, N, O, P, halogen) also occurs at their electron-rich sites through oxidative addition to form reactive intermediates, which subsequently degradate to yield smaller molecules either bound to or dissociated from the metal center. The mechanism is substantiated unambiguously by isolation of numerous intermediate stages. © 2001 John Wiley & Sons, Inc. and The Japan Chemical Journal Forum Chem Rec 1:349,361, 2001 [source] | ||||||||||||||||||||||||||||||||||||||||||||||