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Crosslinks
Kinds of Crosslinks Terms modified by Crosslinks Selected AbstractsDynamically Restructuring Hydrogel Networks Formed with Reversible Covalent Crosslinks,ADVANCED MATERIALS, Issue 18 2007C. Roberts Dynamically restructuring hydrogel networks are formed from two water-soluble polymers by functionalization with phenylboronic acid (PBA) and salicylhydroxamic acid (SHA) moieties that interact through pH-sensitive and reversible covalent crosslinks (see figure). The networks can be tuned to display a wide range of pH-responsive properties, which can potentially be exploited for drug delivery systems in biological environments where similar acidic changes occur. [source] Investigations of Miscibility in Interpenetrated Systems of Polyurethane and Polystyrene Obtained at Room TemperatureMACROMOLECULAR SYMPOSIA, Issue 1 2004Jean-Michel Widmaier Abstract Interpenetrating polymer systems based on crosslinked polyurethane (PU) and polystyrene (PS) were prepared at room temperature by a one-shot (in situ) method, starting from an initial homogeneous mixture of reagents via non interfering mechanisms. Both polymerizations were performed either simultaneously or one after the other. Crosslinks and/or covalent bonds between components were deliberately introduced by the addition of appropriate monomers, in order to tailor the degree of microphase separation. Depending on the formation process, transluscent or transparent films were obtained, despite the difference in refractive index of the components. The maximum of miscibility, taken as from the glass transition criterion, was obtained for sequential tightly graft interpenetrating networks. [source] The polypeptide chain release factor eRF1 specifically contacts the s4UGA stop codon located in the A site of eukaryotic ribosomesFEBS JOURNAL, Issue 10 2001Laurent Chavatte It has been shown previously [Brown, C.M. & Tate, W.P. (1994) J. Biol. Chem.269, 33164,33170.] that the polypeptide chain release factor RF2 involved in translation termination in prokaryotes was able to photocrossreact with mini-messenger RNAs containing stop signals in which U was replaced by 4-thiouridine (s4U). Here, using the same strategy we have monitored photocrosslinking to eukaryotic ribosomal components of 14-mer mRNA in the presence of , and 42-mer mRNA in the presence of tRNAAsp (tRNAAsp gene transcript). We show that: (a) both 14-mer and 42-mer mRNAs crossreact with ribosomal RNA and ribosomal proteins. The patterns of the crosslinked ribosomal proteins are similar with both mRNAs and sensitive to ionic conditions; (b) the crosslinking patterns obtained with 42-mer mRNAs show characteristic modification upon addition of tRNAAsp providing evidence for appropriate mRNA phasing onto the ribosome. Similar changes are not detected with the 14-mer pairs; (c) when eukaryotic polypeptide chain release factor 1 (eRF1) is added to the ribosome·tRNAAsp complex it crossreacts with the 42-mer mRNA containing the s4UGA stop codon located in the A site, but not with the s4UCA sense codon; this crosslink involves the N-terminal and middle domains of eRF1 but not the C domain which interacts with eukaryotic polypeptide chain release factor 3 (eRF3); (d) addition of eRF3 has no effect on the yield of eRF1,42-mer mRNA crosslinking and eRF3 does not crossreact with 42-mer mRNA. These experiments delineate the in vitro conditions allowing optimal phasing of mRNA on the eukaryotic ribosome and demonstrate a direct and specific contact of ,core' eRF1 and s4UGA stop codon within the ribosomal A site. [source] Covalently crosslinked complexes of bovine adrenodoxin with adrenodoxin reductase and cytochrome P450sccFEBS JOURNAL, Issue 6 2001Edman degradation of complexes of the steroidogenic hydroxylase system, Mass spectrometry NADPH-dependent adrenodoxin reductase, adrenodoxin and several diverse cytochromes P450 constitute the mitochondrial steroid hydroxylase system of vertebrates. During the reaction cycle, adrenodoxin transfers electrons from the FAD of adrenodoxin reductase to the heme iron of the catalytically active cytochrome P450 (P450scc). A shuttle model for adrenodoxin or an organized cluster model of all three components has been discussed to explain electron transfer from adrenodoxin reductase to P450. Here, we characterize new covalent, zero-length crosslinks mediated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide between bovine adrenodoxin and adrenodoxin reductase, and between adrenodoxin and P450scc, respectively, which allow to discriminate between the electron transfer models. Using Edman degradation, mass spectrometry and X-ray crystallography a crosslink between adrenodoxin reductase Lys27 and adrenodoxin Asp39 was detected, establishing a secondary polar interaction site between both molecules. No crosslink exists in the primary polar interaction site around the acidic residues Asp76 to Asp79 of adrenodoxin. However, in a covalent complex of adrenodoxin and P450scc, adrenodoxin Asp79 is involved in a crosslink to Lys403 of P450scc. No steroidogenic hydroxylase activity could be detected in an adrenodoxin ,P450scc complex/adrenodoxin reductase test system. Because the acidic residues Asp76 and Asp79 belong to the binding site of adrenodoxin to adrenodoxin reductase, as well as to the P450scc, the covalent bond within the adrenodoxin,P450scc complex prevents electron transfer by a putative shuttle mechanism. Thus, chemical crosslinking provides evidence favoring the shuttle model over the cluster model for the steroid hydroxylase system. [source] PKR, a cognitive decline biomarker, can regulate translation via two consecutive molecular targets p53 and Redd1 in lymphocytes of AD patientsJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 8b 2009Milena Damjanac Abstract In Alzheimer's disease (AD), the control of translation is dysregulated, precisely, two opposite pathways: double-stranded RNA-dependent protein kinase (PKR) is up-regulated and mammalian target of rapamycin (mTOR) is down-regulated. These biochemical alterations were found at the periphery in lymphocytes of AD patients and were significantly correlated with cognitive and memory test scores. However, the molecular crosslink between these two opposite signalling pathways remains unknown. The tumour suppressor p53 and Redd1 (regulated in development and DNA damage response) could be two downstream targets of active PKR to explain the breakdown of translation in AD patients. In this study, the protein and gene levels of p53 and Redd1 were assayed in lymphocytes of AD patients and in age-matched controls by Western blotting and RT-PCR. Furthermore, correlations were analysed with both the level of active PKR and the Mini Mental State Examination score (MMSE). The results show that the gene and protein levels of p53 and Redd1 were significantly increased about 1.5-fold for both gene and Redd1 protein and 2.3-fold for active p53 in AD lymphocytes compared to age-matched controls. Furthermore, statistical correlations between proteins and genes suggest that active PKR could phosphorylate p53 which could induce the transcription of Redd1 gene. No correlations were found between MMSE scores and levels of p53 or Redd1, contrary to active PKR levels. PKR represents a cognitive decline biomarker able to dysregulate translation via two consecutive targets p53 and Redd1 in AD lymphocytes. [source] Chemistry and Reactions of Reactive Oxygen Species in FoodsJOURNAL OF FOOD SCIENCE, Issue 9 2005Eunok Choe ABSTRACT: Reactive oxygen species (ROS) is formed enzymatically, chemically, photochemically, and by irradiation of food. It is also formed by the decomposition and the inter-reactions of ROS. The hydroxy radical is the most reactive ROS and then followed by singlet oxygen. Reactions of ROS with food components produce undesirable volatile compounds and carcinogens, destroy essential nutrients, and change the functionalities of proteins, lipids, and carbohydrates. Lipid oxidation by ROS produces low-molecular-weight volatile aldehydes, alcohols, and hydrocarbons. ROS causes crosslink or cleavage of proteins. ROS produces low-molecular-weight carbonyl compounds from carbohydrates. Vitamins are easily oxidized by ROS, especially singlet oxygen. The singlet oxygen reaction rate was the highest in ,-carotene followed by tocopherol, riboflavin, vitamin D, and ascorbic acid. [source] Synthesis and study of a gramicidin B mutant possessing a ditryptophan crosslinkJOURNAL OF PEPTIDE SCIENCE, Issue 9 2002Alice L. Presley Bodnar Abstract Recent studies of peptide dimers linked by Trp-Trp (ditryptophan) crosslinks suggest that the crosslinks can reinforce antiparallel ,-structure. Depending on environment, gramicidins A, B and C form either helical ion channels with parallel ,-structure or non-functional pores with antiparallel ,-structure. In the channel conformation of the gramicidins Trp9 and Trp15 are close in space, but in the pore conformation Trp9 and Trp15 are far apart. We hypothesized that a ditryptophan crosslink between Trp9 and Trp15 could pre-organize gramicidin in an active conformation. To test the potential for pre-organization, an intramolecular ditryptophan crosslink was formed between Trp9 and Trp15 in a W13F mutant of gramicidin B. Photooxidative conditions were shown to generate ditryptophan crosslinks in low yields. While not preparatively useful, photooxidative tryptophan crosslinking may have implications for protein aging processes like cataract formation. The ditryptophan crosslink in the gramicidin B mutant substantially lowered the antibiotic activity of the gramicidin B mutant, unlike the ditryptophan crosslink in the antibiotic X-indolicidin. The biaryl chromophore generated diagnostic Cotton effects in the CD spectrum that revealed the absolute stereochemistry of the biaryl chromophore, but the biaryl chromophore obscured diagnostic features below 220 nm. However, changes in peptide conformation were reflected in changes in the biaryl region of the CD spectrum above 240 nm. Copyright © 2002 European Peptide Society and John Wiley & Sons, Ltd. [source] Dithiocarbamate mediated controlled/living free radical polymerization of methyl acrylate under 60Co ,-ray irradiation: Conjugation effect of N -groupJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 22 2004Daoben Hua Abstract The free radical polymerizations of methyl acrylate have been studied under ,-ray irradiation in the presence of the dithiocarbamates with different N -groups. The results indicate that the conjugation structure of the N -group of dithiocarbamate plays an important role in living free radical polymerization. The polymerizations reveal good living characteristics in the presence of dithiocarbamates (benzyl 1H-imidazole-1-carbodithioate, benzyl 1H-pyrrole-1-carbodithioate, benzyl 1H-indole-1-carbodithioate, and benzyl 9H-carbazole-9-carbodithioate) with N -aryl group. In contrast, the polymerization with benzyl N,N -diethyldithiocarbamate cannot be controlled, and the obtained polymer has a broad molecular weight distribution or even crosslink occurs. Moreover, polymerization rate is influenced by the conjugation structure of the N -group of dithiocarbamate, and the aromatic polycyclic structure of the N -group leads to slow polymerization. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 5670,5677, 2004 [source] Grafting of maleic anhydride onto linear polyethylene: A Monte Carlo studyJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 22 2004Yutian Zhu Abstract Monte Carlo simulation was used to study the graft of maleic anhydride (MAH) onto linear polyethylene (PE-g-MAH) initiated by dicumyl peroxide (DCP). Simulation results revealed that major MAH monomers attached onto PE chains as branched graft at higher MAH content. However, at extremely low MAH content, the fraction of bridged graft was very close to that of branched graft. This conclusion was somewhat different from the conventional viewpoint, namely, the fraction of bridged graft was always much lower than that of branched graft under any condition. Moreover, the results indicated that the grafting degree increased almost linearly to MAH and DCP concentrations. On the other hand, it was found that the amount of grafted MAH dropped sharply with increasing the length of grafted MAH, indicating that MAH monomers were mainly attached onto the PE chain as single MAH groups or very short oligomers. With respect to the crosslink of PE, the results showed that the fraction of PE-(MAH)n -PE crosslink structure increased continuously, and hence the fraction of PE-PE crosslink decreased with increasing MAH concentration. Finally, quantitative relationship among number average molecular weight of the PE, MAH, and DCP contents was given. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 5714,5724, 2004 [source] Photopolymerization and Characteristics of Polyurethane/Organoclay NanocompositesMACROMOLECULAR REACTION ENGINEERING, Issue 3 2007Hailin Tan Abstract Different polymerizable ammonium surfactants were successfully synthesized via Michael-addition and quaternarization reactions. Organoclays containing reactive methacrylate groups were prepared by the cationic exchange process. Intercalated nanocomposites were produced by UV initiated polymerization with 5 wt.-% organophilic clay loading. DMTA tests and tensile analysis implied that a long chain polymerizable modifier could crosslink with the polymer matrix, and mechanical and tensile properties were enhanced dramatically about 80%. Water sorption was not elevated, which was determined by the polymer itself and the hydrophobicity of the modifier, although the water permeation was improved. [source] Isoniazid-resistance conferring mutations in Mycobacterium tuberculosis KatG: Catalase, peroxidase, and INH-NADH adduct formation activitiesPROTEIN SCIENCE, Issue 3 2010Christine E. Cade Abstract Mycobacterium tuberculosis catalase-peroxidase (KatG) is a bifunctional hemoprotein that has been shown to activate isoniazid (INH), a pro-drug that is integral to frontline antituberculosis treatments. The activated species, presumed to be an isonicotinoyl radical, couples to NAD+/NADH forming an isoniazid-NADH adduct that ultimately confers anti-tubercular activity. To better understand the mechanisms of isoniazid activation as well as the origins of KatG-derived INH-resistance, we have compared the catalytic properties (including the ability to form the INH-NADH adduct) of the wild-type enzyme to 23 KatG mutants which have been associated with isoniazid resistance in clinical M. tuberculosis isolates. Neither catalase nor peroxidase activities, the two inherent enzymatic functions of KatG, were found to correlate with isoniazid resistance. Furthermore, catalase function was lost in mutants which lacked the Met-Tyr-Trp crosslink, the biogenic cofactor in KatG which has been previously shown to be integral to this activity. The presence or absence of the crosslink itself, however, was also found to not correlate with INH resistance. The KatG resistance-conferring mutants were then assayed for their ability to generate the INH-NADH adduct in the presence of peroxide (t -BuOOH and H2O2), superoxide, and no exogenous oxidant (air-only background control). The results demonstrate that residue location plays a critical role in determining INH-resistance mechanisms associated with INH activation; however, different mutations at the same location can produce vastly different reactivities that are oxidant-specific. Furthermore, the data can be interpreted to suggest the presence of a second mechanism of INH-resistance that is not correlated with the formation of the INH-NADH adduct. [source] Effect of genipin-crosslinked chitin-chitosan scaffolds with hydroxyapatite modifications on the cultivation of bovine knee chondrocytesBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2006Yung-Chih Kuo Abstract Chitin and chitosan were hybridized in various weight percentages by genipin crosslinkage under various prefreezing temperatures to form tissue-engineering scaffolds via lyophilization. In addition, deposition of hydroxyapatite (HA) on the surface of the porous scaffolds was performed by precipitation method to achieve modified chemical compositions for chondrocyte attachments and growths. The experimental results revealed that a lower prefreezing temperature or a higher weight percentage of chitin in the chitin-chitosan scaffolds would yield a smaller pore diameter, a greater porosity, a larger specific surface area, a higher Young's modulus, and a lower extensibility. Moreover, a higher chitin percentage could also result in a higher content of amine groups after crosslink and a lower onset temperature for the phase transition after thermal treatment. A decrease in the prefreezing temperature from ,4°C to ,80°C, an increase in the chitin percentage from 20% to 50%, and an increase in the cycle number of alternate immersion for HA deposition from 1 to 5 generated positive effects on the cell number, the content of glycosaminoglycans, and the collagen level over 28-day cultivation of bovine knee chondrocytes. © 2006 Wiley Periodicals, Inc. [source] Location of crosslinks in chemically stabilized horseradish peroxidase: Implications for design of crosslinksBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2001Anne Marie O'Brien Abstract The bifunctional compound, ethylene-glycol bis(N -hydroxysuccinimidylsuccinate) (EGNHS), stabilizes horseradish peroxidase C (HRP) by reaction with the enzyme's lysine residues. In this study we compare native and modified HRP by proteolytic fragmentation, peptide sequencing, and mass spectroscopy, and identify the sites of modification. Most significantly, EGNHS is shown to form a crosslink between Lys232 and Lys241 of HRP and modifies Lys174 without formation of a crosslink. These findings are in agreement with the lysine side-chain reactivities predicted from the surface accessibility of the amino groups, and the maximal span of 16 Å of the EGNHS crosslinker. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 76: 277,284, 2001. [source] Clinical relevance of the homologous recombination machinery in cancer therapyCANCER SCIENCE, Issue 2 2008Kiyoshi Miyagawa Cancer chemotherapy and radiotherapy kill cancer cells by inducing DNA damage, unless the lesions are repaired by intrinsic repair pathways. DNA double-strand breaks (DSB) are the most deleterious type of damage caused by cancer therapy. Homologous recombination (HR) is one of the major repair pathways for DSB and is thus a potential target of cancer therapy. Cells with a defect in HR have been shown to be sensitive to a variety of DNA-damaging agents, particularly interstrand crosslink (ICL)-inducing agents such as mitomycin C and cisplatin. These findings have recently been applied to clinical studies of cancer therapy. ERCC1, a structure-specific endonuclease involved in nucleotide excision repair (NER) and HR, confers resistance to cisplatin. Patients with ERCC1-negative non-small-cell lung cancer were shown to benefit from adjuvant cisplatin-based chemotherapy. Imatinib, an inhibitor of the c-Abl kinase, has been investigated as a sensitizer in DNA-damaging therapy, because c-Abl activates Rad51, which plays a key role in HR. Furthermore, proteins involved in HR have been shown to repair DNA damage induced by a variety of other chemotherapeutic agents, including camptothecin and gemcitabine. These findings highlight the importance of HR machinery in cancer therapy. (Cancer Sci 2008; 99: 187,194) [source] Photoaffinity Isolation and Identification of Proteins in Cancer Cell Extracts that Bind to Platinum-Modified DNACHEMBIOCHEM, Issue 1 2009Evan R. Guggenheim Dr. Abstract The activity of the anticancer drug cisplatin is a consequence of its ability to bind DNA. Platinum adducts bend and unwind the DNA duplex, creating recognition sites for nuclear proteins. Following DNA damage recognition, the lesions will either be repaired, facilitating cell viability, or if repair is unsuccessful and the Pt adduct interrupts vital cellular functions, apoptosis will follow. With the use of the benzophenone-modified cisplatin analogue Pt-BP6, 25 bp DNA duplexes containing either a 1,2-d(G*pG*) intrastrand or a 1,3-d(G*pTpG*) intrastrand crosslink were synthesized, where the asterisks designate platinated nucleobases. Proteins having affinity for these platinated DNAs were photocrosslinked and identified in cervical, testicular, pancreatic and bone cancer-cell nuclear extracts. Proteins identified in this manner include the DNA repair factors RPA1, Ku70, Ku80, Msh2, DNA ligase III, PARP-1, and DNA,PKcs, as well as HMG-domain proteins HMGB1, HMGB2, HMGB3, and UBF1. The latter strongly associate with the 1,2-d(G*pG*) adduct and weakly or not at all with the 1,3-d(G*pTpG*) adduct. The nucleotide excision repair protein RPA1 was photocrosslinked only by the probe containing a 1,3-d(G*pTpG*) intrastrand crosslink. The affinity of PARP-1 for platinum-modified DNA was established using this type of probe for the first time. To ensure that the proteins were not photocrosslinked because of an affinity for DNA ends, a 90-base dumbbell probe modified with Pt-BP6 was investigated. Photocrosslinking experiments with this longer probe revealed the same proteins, as well as some additional proteins involved in chromatin remodeling, transcription, or repair. These findings reveal a more complete list of proteins involved in the early steps of the mechanism of action of the cisplatin and its close analogue carboplatin than previously was available. [source] Can Copper(II) Mediate Hoogsteen Base-Pairing in a Left-Handed DNA Duplex?CHEMPHYSCHEM, Issue 3 2010A Pulse EPR Study Abstract Pulse EPR spectroscopy is used to investigate possible structural features of the copper(II) ion coordinated to poly(dG-dC),poly(dG-dC) in a frozen aqueous solution, and the structural changes of the polynucleotide induced by the presence of the metal ion. Two different copper species were identified and their geometry explained by a molecular model. According to this model, one species is exclusively coordinated to a single guanine with the N7 nitrogen atom forming a coordinative bond with the copper. In the other species, a guanine and a cytosine form a ternary complex together with the copper ion. A copper crosslink between the N7 of guanine and N3 of cytosine is proposed as the most probable coordination site. Moreover, no evidence was found for an interaction of either copper species with a phosphate group or equatorial water molecules. In addition, circular dichroism (CD) spectroscopy showed that the DNA of the CuII -poly(dG-dC),poly(dG-dC) adducts resembles the left-handed Z-form. These results suggest that metal-mediated Hoogsteen base pairing, as previously proposed for a right-handed DNA duplex, can also occur in a double-stranded left-handed DNA. [source] DNA interstrand crosslinks: Repair, cell signaling, and therapeutic implicationsENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 6 2010Karen M. Vasquez No abstract is available for this article. [source] Mutagenic repair of DNA interstrand crosslinksENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 6 2010Xi Shen Abstract Formation of DNA interstrand crosslinks (ICLs) in chromosomal DNA imposes acute obstruction of all essential DNA functions. For over 70 years bifunctional alkylators, also known as DNA crosslinkers, have been an important class of cancer chemotherapeutic regimens. The mechanisms of ICL repair remains largely elusive. Here, we review a eukaryotic mutagenic ICL repair pathway discovered by work from several laboratories. This repair pathway, alternatively termed recombination-independent ICL repair, involves the incision activities of the nucleotide excision repair (NER) mechanism and lesion bypass polymerase(s). Repair of the ICL is initiated by dual incisions flanking the ICL on one strand of the double helix; the resulting gap is filled in by lesion bypass polymerases. The remaining lesion is subsequently removed by a second round of NER reaction. The mutagenic repair of ICL likely interacts with other cellular mechanisms such as the Fanconi anemia pathway and recombinational repair of ICLs. These aspects will also be discussed. Environ. Mol. Mutagen., 2010. © 2010 Wiley-Liss, Inc. [source] Assessing the link between BACH1/FANCJ and MLH1 in DNA crosslink repairENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 6 2010Sharon B. Cantor Abstract FANCJ (also known as BRIP1 or BACH1) is a DNA helicase that was originally identified by its direct interaction with the hereditary breast cancer protein, BRCA1. Similar to BRCA1, FANCJ function is essential for DNA repair and breast cancer suppression. FANCJ is also mutated in the cancer prone syndrome Fanconi anemia, for which patient cells are characterized by extreme sensitivity to agents that generate DNA interstand crosslinks. Unexpectedly, correction of the interstrand crosslink sensitivity of FANCJ-null patient cells did not require the FANCJ/BRCA1 interaction. Instead, FANCJ binding to the mismatch repair protein, MLH1 was required. Given this finding, we address the role of FANCJ and MLH1 in DNA crosslink processing and how their functions could be linked in checkpoint and/or recombination pathways. We speculate that after DNA crosslink processing and repair, the FANCJ/MLH1 interaction is critical for recovery and restart of replication. These ideas are considered and summarized in this review. Environ. Mol. Mutagen., 2010. © 2010 Wiley-Liss, Inc. [source] Strategies for DNA interstrand crosslink repair: Insights from worms, flies, frogs, and slime moldsENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 6 2010Mitch McVey Abstract DNA interstrand crosslinks (ICLs) are complex lesions that covalently link both strands of the DNA double helix and impede essential cellular processes such as DNA replication and transcription. Recent studies suggest that multiple repair pathways are involved in their removal. Elegant genetic analysis has demonstrated that at least three distinct sets of pathways cooperate in the repair and/or bypass of ICLs in budding yeast. Although the mechanisms of ICL repair in mammals appear similar to those in yeast, important differences have been documented. In addition, mammalian crosslink repair requires other repair factors, such as the Fanconi anemia proteins, whose functions are poorly understood. Because many of these proteins are conserved in simpler metazoans, nonmammalian models have become attractive systems for studying the function(s) of key crosslink repair factors. This review discusses the contributions that various model organisms have made to the field of ICL repair. Specifically, it highlights how studies performed with C. elegans, Drosophila, Xenopus, and the social amoeba Dictyostelium serve to complement those from bacteria, yeast, and mammals. Together, these investigations have revealed that although the underlying themes of ICL repair are largely conserved, the complement of DNA repair proteins utilized and the ways in which each of the proteins is used can vary substantially between different organisms. Environ. Mol. Mutagen., 2010. © 2010 Wiley-Liss, Inc. [source] The influence of strenuous exercise on collagen characteristics of articular cartilage in Thoroughbreds age 2 yearsEQUINE VETERINARY JOURNAL, Issue 6 2000P. A. J. BRAMA Summary In order to assess the influence of strenuous exercise on collagen characteristics of articularcartilage, the response of the collagen network was studied in seven 2-year-old Thoroughbreds subjected to strenuous exercise compared to 7 nontrained individuals. After 13 weeks, the animals were subjected to euthanasia, fetlock joints of the forelimbs were scored macroscopically after Indian Ink staining, and articular cartilage from different locations of the articular surface of the proximal first phalanx was sampled and analysed for water content, collagen content, hydroxylysine content and amount of hydroxylysylpyridinoline (HP) crosslinks. Gross lesions were significantly more severe in the exercised than in the nonexercised group. In the control animals, the characteristic site-specific differences in collagen parameters were found as described earlier, but in the strenuously exercised animals this physiological biochemical heterogeneity had disappeared. In the exercised animals, an increase in water content and a sharp decrease in HP crosslinking was found that was correlated with the presence of wear lines. It is concluded that the strenuous exercise provoked significant alterations in the characteristics of the collagen network of the articular cartilage of the fetlock joint which were suggestive of microdamage and loosening of the collagen network. The collagen component of cartilage, in contrast to the proteoglycan component, is known to have a very limited capacity for repairand remodelling due to an extremely low turnover rate. Therefore, alterations within the articular collagen network might be expected to play an important role in the pathophysiology of degenerative joint disorders. [source] Covalently crosslinked complexes of bovine adrenodoxin with adrenodoxin reductase and cytochrome P450sccFEBS JOURNAL, Issue 6 2001Edman degradation of complexes of the steroidogenic hydroxylase system, Mass spectrometry NADPH-dependent adrenodoxin reductase, adrenodoxin and several diverse cytochromes P450 constitute the mitochondrial steroid hydroxylase system of vertebrates. During the reaction cycle, adrenodoxin transfers electrons from the FAD of adrenodoxin reductase to the heme iron of the catalytically active cytochrome P450 (P450scc). A shuttle model for adrenodoxin or an organized cluster model of all three components has been discussed to explain electron transfer from adrenodoxin reductase to P450. Here, we characterize new covalent, zero-length crosslinks mediated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide between bovine adrenodoxin and adrenodoxin reductase, and between adrenodoxin and P450scc, respectively, which allow to discriminate between the electron transfer models. Using Edman degradation, mass spectrometry and X-ray crystallography a crosslink between adrenodoxin reductase Lys27 and adrenodoxin Asp39 was detected, establishing a secondary polar interaction site between both molecules. No crosslink exists in the primary polar interaction site around the acidic residues Asp76 to Asp79 of adrenodoxin. However, in a covalent complex of adrenodoxin and P450scc, adrenodoxin Asp79 is involved in a crosslink to Lys403 of P450scc. No steroidogenic hydroxylase activity could be detected in an adrenodoxin ,P450scc complex/adrenodoxin reductase test system. Because the acidic residues Asp76 and Asp79 belong to the binding site of adrenodoxin to adrenodoxin reductase, as well as to the P450scc, the covalent bond within the adrenodoxin,P450scc complex prevents electron transfer by a putative shuttle mechanism. Thus, chemical crosslinking provides evidence favoring the shuttle model over the cluster model for the steroid hydroxylase system. [source] Specific transcriptional responses induced by 8-methoxypsoralen and UVA in yeastFEMS YEAST RESEARCH, Issue 6 2007Michèle Dardalhon Abstract Treatment of eukaryotic cells with 8-methoxypsoralen plus UVA irradiation (8-MOP/UVA) induces pyrimidine monoadducts and interstrand crosslinks and initiates a cascade of events leading to cytotoxic, mutagenic and carcinogenic responses. Transcriptional activation plays an important part in these responses. Our previous study in Saccharomyces cerevisiae showed that the repair of these lesions involves the transient formation of DNA double-strand breaks and the enhanced expression of landmark DNA damage response genes such as RAD51, RNR2 and DUN1, as well as the Mec1/Rad53 kinase signaling cascade. We have now used DNA microarrays to examine genome-wide transcriptional changes produced after induction of 8-MOP/UVA photolesions. We found that 128 genes were strongly induced and 29 genes strongly repressed. Modifications in gene expression concern numerous biological processes. Compared to other genotoxic treatments, c. 42% of the response genes were specific to 8-MOP/UVA treatment. In addition to common DNA damage response genes and genes induced by environmental stresses, a large fraction of 8-MOP/UVA response genes correspond to membrane-related functions. [source] The combination of a hydroxy-functional organophosphorus oligomer and melamine-formaldehyde as a flame retarding finishing system for cottonFIRE AND MATERIALS, Issue 2 2005Charles Q. Yang Abstract In previous research, it was found that melamine-formaldehyde resin can be used as a binder for a hydroxy-functional organophosphorus flame retarding agent (FR) on cotton. The role that trimethylol melamine (TMM) plays in this flame retarding system was studied. When TMM is applied to cotton, it forms crosslinks between cellulose molecules. When TMM is applied to cotton in the presence of FR, it reacts with FR to form a crosslinked polymeric network in addition to reacting with cotton. The formation of the crosslinked network improves the laundering durability of FR and also increases the fabric stiffness. The number of crosslinks among cotton cellulose formed by TMM decreases as the FR concentration in the system is increased. TMM also functions as a nitrogen provider to enhance the flame retarding performance of FR due to phosphorus,nitrogen synergism. Therefore, the amount of TMM used in a FR/TMM formula plays the most critical role in determining the effectiveness of this flame retarding system. The finish bath pH also plays a significant role in influencing the performance of the flame retarding system on cotton. The optimum pH was found to be around 4. Copyright © 2004 John Wiley & Sons, Ltd. [source] Modulation of Viscoelasticity and HIV Transport as a Function of pH in a Reversibly Crosslinked HydrogelADVANCED FUNCTIONAL MATERIALS, Issue 18 2009Julie I. Jay Abstract Materials that respond to physiological stimuli are important in developing advanced biomaterials for modern therapies. The reversibility of covalent crosslinks formed by phenylboronate (PBA) and salicylhydroxamate (SHA) has been exploited to provide a pH-responsive gel for application to the vaginal tract. Dynamic rheology reveals that the gel frequency-dependent viscoelastic properties are modulated by pH. At pH 4.8 the viscous component dominates throughout most of the frequency range. As the pH increases, the characteristic relaxation time continues to increase while the G,Plateau levels off above pH 6. At pH 7.5, the elastic component dominates throughout the frequency sweep and is predominately independent of frequency. Particle tracking assesses the transport of both fluorescently labeled HIV-1 and 100-nm latex particles in the PBA,SHA crosslinked gel as a function of pH. At pH 4.8 the ensemble-averaged mean squared displacement at lag times greater than three seconds reveals that transport of the HIV-1 and 100-nm particles becomes significantly impeded by the matrix, exhibiting diffusion coefficients less than 0.0002,µm2 s,1. This pH-responsive gel thus displays properties that have the potential to significantly reduce the transport of HIV-1 to susceptible tissues and thus prevent the first stage of male-to-female transmission of HIV-1. [source] Deletion of Brca2 exon 27 causes hypersensitivity to DNA crosslinks, chromosomal instability, and reduced life span in miceGENES, CHROMOSOMES AND CANCER, Issue 4 2003Greg Donoho The Brca2 tumor-suppressor gene contributes to genomic stability, at least in part by a role in homologous recombinational repair. BRCA2 protein is presumed to function in homologous recombination through interactions with RAD51. Both exons 11 and 27 of Brca2 code for domains that interact with RAD51; exon 11 encodes eight BRC motifs, whereas exon 27 encodes a single, distinct interaction domain. Deletion of all RAD51-interacting domains causes embryonic lethality in mice. A less severe phenotype is seen with BRAC2 truncations that preserve some, but not all, of the BRC motifs. These mice can survive beyond weaning, but are runted and infertile, and die very young from cancer. Cells from such mice show hypersensitivity to some genotoxic agents and chromosomal instability. Here, we have analyzed mice and cells with a deletion of only the RAD51-interacting region encoded by exon 27. Mice homozygous for this mutation (called brca2lex1) have a shorter life span than that of control littermates, possibly because of early onsets of cancer and sepsis. No other phenotype was observed in these animals; therefore, the brca2lex1 mutation is less severe than truncations that delete some BRC motifs. However, at the cellular level, the brca2lex1 mutation causes reduced viability, hypersensitivity to the DNA interstrand crosslinking agent mitomycin C, and gross chromosomal instability, much like more severe truncations. Thus, the extreme carboxy-terminal region encoded by exon 27 is important for BRCA2 function, probably because it is required for a fully functional interaction between BRCA2 and RAD51. © 2003 Wiley-Liss, Inc. [source] Scaffold-Mediated 2D Cellular Orientations for Construction of Three Dimensionally Engineered Tissues Composed of Oriented Cells and Extracellular MatricesADVANCED FUNCTIONAL MATERIALS, Issue 7 2009Hiroaki Yoshida Abstract Various hydrogels, such as poly(, -glutamic acid) (, -PGA), gelatin (GT), alginic acid (Alg), and agarose (Aga), with 3D interconnected and oriented fibrous pores (OP gels) are prepared for 3D polymeric cellular scaffolds by using silica fiber cloth (SC) as template. After the preparation of these hydrogels with the SC templates, the latter are subsequently removed by washing with hydrofluoric acid solution. Scanning electron microscopy (SEM) clearly shows OP structures in the hydrogels. These various types of OP gels are successfully prepared in this way, independently of the crosslinking mechanism, such as chemical (, -PGA or GT), coordinate-bonded (Alg), or hydrogen-bonded (Aga) crosslinks. SEM, confocal laser scanning microscopy, and histological evaluations clearly demonstrate that mouse L929 fibroblast cells adhere to and extend along these OP structures on/in , -PGA hydrogels during 3D cell culture. The L929 cells that adhere on/in the oriented hydrogel are viable and proliferative. Furthermore, 3D engineered tissues, composed of the oriented cells and extracellular matrices (ECM) produced by the cells, are constructed in vitro by subsequent decomposition of the hydrogel with cysteine after 14 days of cell culture. This novel technology to fabricate 3D-engineered tissues, consisting of oriented cells and ECM, will be useful for tissue engineering. [source] One-Step Process for Creating Triple-Shape Capability of AB Polymer NetworksADVANCED FUNCTIONAL MATERIALS, Issue 1 2009Marc Behl Abstract Triple-shape polymers can move from a first shape (A) to a second shape (B) and from there to a third shape (C), where both shape changes are induced by temperature increases. This triple-shape capability is obtained for multiphase polymer networks after application of a complex thermomechanical programming process, which consists of two steps; these steps create shapes (B) and (A), while shape (C) is defined by the covalent crosslinks of the polymer network. Here, the creation of the triple-shape capability for an AB polymer network system by a simple one-step process similar to a conventional dual-shape programming process is reported. The polymer networks are based on poly(, -caprolactone) (PCL) and poly(cyclohexyl methacrylate); favorable compositions for obtaining a triple shape effect have a PCL content between 35 and 60 wt%. This finding substantially facilitates handling of the triple-shape technology and is an important step toward the realization of potential applications in which more than one shape change is required. [source] Dynamic Hydrogels: Switching of 3D Microenvironments Using Two-Component Naturally Derived Extracellular Matrices (Adv. Mater.ADVANCED MATERIALS, Issue 6 20106/2010) The front cover image depicts a two-component extracellular matrix (ECM) in which one component acts as a stable structural element (which supports cell attachment and migration) and another component gels or dissolves reversibly (a modulatory component). Samuel K. Sia and co-workers show on p. 686 that by dynamically adding or removing crosslinks in the modulatory component, properties of the composite ECM, such as the ability of cells to migrate and the rate of diffusive transport, can be altered. [source] Dynamically Restructuring Hydrogel Networks Formed with Reversible Covalent Crosslinks,ADVANCED MATERIALS, Issue 18 2007C. Roberts Dynamically restructuring hydrogel networks are formed from two water-soluble polymers by functionalization with phenylboronic acid (PBA) and salicylhydroxamic acid (SHA) moieties that interact through pH-sensitive and reversible covalent crosslinks (see figure). The networks can be tuned to display a wide range of pH-responsive properties, which can potentially be exploited for drug delivery systems in biological environments where similar acidic changes occur. [source] | ||||||||||||||||||||||||||||||||||