EXPERIMENTAL VALIDATION OF THE CHARLESBY AND HORIKX MODELS APPLIED TO DE-VULCANIZATION OF SULFUR AND PEROXIDE VULCANIZATES OF NR AND EPDM

2016 ◽  
Vol 89 (4) ◽  
pp. 671-688 ◽  
Author(s):  
M. A. L. Verbruggen ◽  
L. van der Does ◽  
W. K. Dierkes ◽  
J. W. M. Noordermeer
Keyword(s):  
Experimental Validation ◽  
Main Chain ◽  
Sol Gel ◽  
Chain Scission ◽  
Cross Linking ◽  
Cross Link ◽  
Practical Reality ◽  
Cross Links ◽  
The Cross ◽  
Theoretical Considerations

ABSTRACT The theoretical model developed by Charlesby to quantify the balance between cross-links creation of polymers and chain scission during radiation cross-linking and further modifications by Horikx to describe network breakdown from aging were merged to characterize the balance of both types of scission on the development of the sol content during de-vulcanization of rubber networks. There are, however, disturbing factors in these theoretical considerations vis-à-vis practical reality. Sulfur- and peroxide-cured NR and EPDM vulcanizates were de-vulcanized under conditions of selective cross-link and random main-chain scissions. Cross-link scission was obtained using thiol-amine reagents for selective cleavage of sulfur cross-links. Random main-chain scission was achieved by heating peroxide vulcanizates of NR with diphenyldisulfide, a method commonly employed for NR reclaiming. An important factor in the analyses of these experiments is the cross-linking index. Its value must be calculated using the sol fraction of the cross-linked network before de-vulcanization to obtain reliable results. The values for the cross-linking index calculated with sol-gel data before de-vulcanization appear to fit the experimentally determined modes of network scission during de-vulcanization very well. This study confirms that the treatment of de-vulcanization data with the merged Charlesby and Horikx models can be used satisfactorily to characterize the de-vulcanization of NR and EPDM vulcanizates.

scholarly journals Chemistry of collagen cross-linking: biochemical changes in collagen during the partial mineralization of turkey leg tendon

1997 ◽  
Vol 322 (2) ◽  
pp. 535-542 ◽  
Author(s):  
Lynda KNOTT ◽  
John F. TARLTON ◽  
Allen J. BAILEY
Keyword(s):  
Collagen Matrix ◽  
Cross Linking ◽  
Cross Link ◽  
Lysine Residues ◽  
Collagen Mineralization ◽  
Cross Links ◽  
The Cross ◽  
Calcified Tissues ◽  
Turkey Leg Tendon ◽  
Collagen Cross Linking

With age, the proximal sections of turkey leg tendons become calcified, and this phenomenon has led to their use as a model for collagen mineralization. Mineralizing turkey leg tendon was used in this study to characterize further the composition and cross-linking of collagen in calcified tissues. The cross-link profiles of mineralizing collagen are significantly different from those of other collagenous matrices with characteristically low amounts of hydroxylysyl-pyridinoline and the presence of lysyl-pyridinoline and pyrrolic cross-links. However, the presence of the immature cross-link precursors previously reported in calcifying tissues was not supported in the present study, and was found to be due to the decalcification procedure using EDTA. Analysis of tendons from young birds demonstrated differences in the cross-link profile which indicated a higher level of hydroxylation of specific triple-helical lysines involved in cross-linking of the proximal tendon. This may be related to later calcification, suggesting that this part of the tendon is predestined to be calcified. The minimal changes in lysyl hydroxylation in both regions of the tendon with age were in contrast with the large changes in the cross-link profile, indicating differential hydroxylation of the helical and telopeptide lysine residues. Changes with age in the collagen matrix, its turnover and thermal properties in both the proximal and distal sections of the tendon clearly demonstrate that a new and modified matrix is formed throughout the tendon, and that a different type of matrix is formed at each site.

FANCD2 Localizes to Cross-Linked Induced Nuclear Foci That Are Distinct From Those to Which αaII Spectrin and the Cross-Link Repair Protein, XPF, Co-Localize, Indicating An Involvement of These Proteins in Different Steps of the DNA Interstrand Cross-Link Repair Process.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3196-3196
Author(s):  
Pan Zhang ◽  
Deepa Sridharan ◽  
Michael Acosta ◽  
Muriel Lambert
Keyword(s):  
Time Course ◽  
Repair Process ◽  
Myelogenous Leukemia ◽  
Cross Linking ◽  
Cross Link ◽  
Repair Protein ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
Nuclear Foci ◽  
The Cross

Abstract Abstract 3196 Poster Board III-133 The hereditary bone marrow failure disorder, Fanconi anemia (FA), is characterized by a markedly increased incidence of acute myelogenous leukemia, diverse congenital abnormalities and a defect in ability to repair DNA interstrand cross-links. We have previously shown that in FA cells there is a deficiency in the structural protein nonerythroid a spectrin (aSpII), which is involved in repair of DNA interstrand cross-links and binds to cross-linked DNA. aSpII co-localizes in nuclear foci with FANCA and the cross-link repair protein, XPF, after normal human cells are damaged with a DNA interstrand cross-linking agent. One of the FA proteins which is thought to play an important role in the repair of DNA interstrand cross-links is FANCD2, which is known to form nuclear foci after cross-link damage. The present study was undertaken in order to get a better understanding of the relationship between aSpII and FANCD2, whether they interact with each other during the DNA repair process and co-localize in damage-induced nuclear foci. Immunofluorescence microscopy was carried out to determine whether these proteins co-localized in nuclear foci after cells were damaged with a DNA interstrand cross-linking agent, 8-methylpsoralen plus UVA light (8-MOP) or mitomycin C (MMC). Time course measurements showed that FANCD2 foci were first visible at 2 hours after damage and increased up to 16 hours and were still present at 72 hours after damage. This time course of foci formation correlated with levels of monoubiquitination of FANCD2. Measurement of gH2AX foci formation showed that the time course of foci formation was similar to that of FANCD2 measured up to 72 hours post damage. In contrast, aSpII foci were first visible between 8-10 hours after damage. The number of these foci peaked at 16 hours and by 24 hours foci were no longer observed. Co-localization studies showed that there was little co-localization of the FANCD2 and aSpII foci over this time course. This indicates that these two proteins may be involved in different steps in the DNA interstrand cross-link repair process. Based on models that have been proposed for the role of FANCD2 in the repair of DNA interstrand cross-links, we propose that, after DNA damage, FANCD2 localizes at DNA replication forks stalled at sites of interstrand cross-links and aids in the assembly of proteins at this site. This is followed by localization of aSpII and XPF and other proteins involved in the initial incision steps in DNA interstrand cross-link repair where they play a role in the unhooking of the cross-link. FANCD2 is then involved in subsequent steps in the repair process, which involve homologous recombination. Thus two proteins, FANCD2 and aSpII, both of which have been shown to be critical for the DNA interstrand cross-link repair process may be involved in different or distinct steps in this repair process. Deficiencies in these proteins would impact on DNA interstrand cross-link repair and, as we have shown for aIISp, would have an adverse effect on the genomic stability of FA cells. . Disclosures No relevant conflicts of interest to declare.

scholarly journals Targeted in situ cross-linking mass spectrometry and integrative modeling reveal the architectures of Nsp1, Nsp2, and Nucleocapsid proteins from SARS-CoV-2

2021 ◽  
Author(s):  
Moriya Slavin ◽  
Joanna Zamel ◽  
Keren Zohar ◽  
Siona Eliyahu ◽  
Merav Braitbard ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Full Length ◽  
Integrative Model ◽  
Cross Linking ◽  
Cross Link ◽  
Integrative Modeling ◽  
Cellular Context ◽  
Cross Links ◽  
The Cross

AbstractAtomic structures of several proteins from the coronavirus family are still partial or unavailable. A possible reason for this gap is the instability of these proteins outside of the cellular context, thereby prompting the use of in-cell approaches. In situ cross-linking and mass spectrometry (in situ CLMS) can provide information on the structures of such proteins as they occur in the intact cell. Here, we applied targeted in situ CLMS to structurally probe Nsp1, Nsp2, and Nucleocapsid (N) proteins from SARS-CoV-2, and obtained cross-link sets with an average density of one cross-link per twenty residues. We then employed integrative modeling that computationally combined the cross-linking data with domain structures to determine full-length atomic models. For the Nsp2, the cross-links report on a complex topology with long-range interactions. Integrative modeling with structural prediction of individual domains by the AlphaFold2 system allowed us to generate a single consistent all-atom model of the full-length Nsp2. The model reveals three putative metal binding sites, and suggests a role for Nsp2 in zinc regulation within the replication-transcription complex. For the N protein, we identified multiple intra- and inter-domain cross-links. Our integrative model of the N dimer demonstrates that it can accommodate three single RNA strands simultaneously, both stereochemically and electrostatically. For the Nsp1, cross-links with the 40S ribosome were highly consistent with recent cryo-EM structures. These results highlight the importance of cellular context for the structural probing of recalcitrant proteins and demonstrate the effectiveness of targeted in situ CLMS and integrative modeling.

scholarly journals Mutational Analysis of a Conserved Glutamic Acid Required for Self-Catalyzed Cross-Linking of Bacteriophage HK97 Capsids

2008 ◽  
Vol 83 (5) ◽  
pp. 2088-2098 ◽  
Author(s):  
Lindsay E. Dierkes ◽  
Craig L. Peebles ◽  
Brian A. Firek ◽  
Roger W. Hendrix ◽  
Robert L. Duda
Keyword(s):  
Glutamic Acid ◽  
Large Scale ◽  
Mutational Analysis ◽  
Chemical Mechanism ◽  
Proton Acceptor ◽  
Cross Linking ◽  
Side Chains ◽  
Cross Link ◽  
Cross Links ◽  
The Cross

ABSTRACT The capsid of bacteriophage HK97 is stabilized by ∼400 covalent cross-links between subunits which form without any action by external enzymes or cofactors. Cross-linking only occurs in fully assembled particles after large-scale structural changes bring together side chains from three subunits at each cross-linking site. Isopeptide cross-links form between asparagine and lysine side chains on two subunits. The carboxylate of glutamic acid 363 (E363) from a third subunit is found ∼2.4 Å from the isopeptide bond in the partly hydrophobic pocket that contains the cross-link. It was previously reported without supporting data that changing E363 to alanine abolishes cross-linking, suggesting that E363 plays a role in cross-linking. This alanine mutant and six additional substitutions for E363 were fully characterized and the proheads produced by the mutants were tested for their ability to cross-link under a variety of conditions. Aspartic acid and histidine substitutions supported cross-linking to a significant extent, while alanine, asparagine, glutamine, and tyrosine did not, suggesting that residue 363 acts as a proton acceptor during cross-linking. These results support a chemical mechanism, not yet fully tested, that incorporates this suggestion, as well as features of the structure at the cross-link site. The chemically identical isopeptide bonds recently documented in bacterial pili have a strikingly similar chemical geometry at their cross-linking sites, suggesting a common chemical mechanism with the phage protein, but the completely different structures and folds of the two proteins argues that the phage capsid and bacterial pilus proteins have achieved shared cross-linking chemistry by convergent evolution.

Introducing an Innovative Perfluoroelastomer Cross Linking Technology: Achieving Superior Chemical Resistance and Thermal Stability

2021 ◽  
Author(s):  
Jimmy Alvarez ◽  
Christopher J. Bish ◽  
Andres Rodriguez
Keyword(s):  
Thermal Stability ◽  
Chemical Resistance ◽  
Standardized Test ◽  
Test Methods ◽  
Cross Linking ◽  
Cross Link ◽  
Cross Links ◽  
The Cross ◽  
Almost All ◽  
Performance Results

Objectives/Scope Perfluoroelastomers are a class of synthetic elastomers that provide extraordinary resistance to oils, chemicals, and heat. The outstanding thermal stability and excellent corrosion resistance of these materials is dependent on the perfluorinated polymer chain, and the absence of unsaturation. However, the cross-link which is necessary to impart elastomeric properties must also share those stability traits. Unfortunately, designing a suitably inert cross-link is technically difficult to achieve and consequently it has not been possible to provide the ultimate in perfluoroelastomer properties. This paper is a review of the cross-linking chemistry of perfluoroelastomers and highlight a novel and patented cross-link which combines broad chemical resistance and superior thermal stability. Methods, Procedures, Process Several perfluoroelastomers formulations were selected and tested using representative standardized test methods to quantify their performance. The testing included compression set resistance, swell in fluids, and compression stress relaxation. In addition to the testing, chemistry of the cross-link was explained and how it is related to the observed performance results. The novel cross-link, triazole, was identified as an improvement versus existing cross-links. Results, Observations, Conclusions The testing demonstrated that previously available perfluoroelastomer cross-link chemistries display compromises in overall performance. For example, it had not been possible to achieve high thermal stability combined with resistance to almost all fluids. Overall, the data generated showed that the newly developed triazole cross-link system broadens the performance envelope of perfluoroelastomers and helps to alleviate the compromises of the past. Novel/Additive Information The newly discovered triazole cross-link with enhanced chemical and thermal stability enables broader use of perfluoroelastomers in extreme applications.

scholarly journals Targeted in situ cross-linking mass spectrometry and integrative modeling reveal the architectures of three proteins from SARS-CoV-2

2021 ◽  
Vol 118 (34) ◽  
pp. e2103554118
Author(s):  
Moriya Slavin ◽  
Joanna Zamel ◽  
Keren Zohar ◽  
Tsiona Eliyahu ◽  
Merav Braitbard ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Full Length ◽  
Integrative Model ◽  
Cross Linking ◽  
Cross Link ◽  
Integrative Modeling ◽  
Cellular Context ◽  
Cross Links ◽  
The Cross

Atomic structures of several proteins from the coronavirus family are still partial or unavailable. A possible reason for this gap is the instability of these proteins outside of the cellular context, thereby prompting the use of in-cell approaches. In situ cross-linking and mass spectrometry (in situ CLMS) can provide information on the structures of such proteins as they occur in the intact cell. Here, we applied targeted in situ CLMS to structurally probe Nsp1, Nsp2, and nucleocapsid (N) proteins from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and obtained cross-link sets with an average density of one cross-link per 20 residues. We then employed integrative modeling that computationally combined the cross-linking data with domain structures to determine full-length atomic models. For the Nsp2, the cross-links report on a complex topology with long-range interactions. Integrative modeling with structural prediction of individual domains by the AlphaFold2 system allowed us to generate a single consistent all-atom model of the full-length Nsp2. The model reveals three putative metal binding sites and suggests a role for Nsp2 in zinc regulation within the replication–transcription complex. For the N protein, we identified multiple intra- and interdomain cross-links. Our integrative model of the N dimer demonstrates that it can accommodate three single RNA strands simultaneously, both stereochemically and electrostatically. For the Nsp1, cross-links with the 40S ribosome were highly consistent with recent cryogenic electron microscopy structures. These results highlight the importance of cellular context for the structural probing of recalcitrant proteins and demonstrate the effectiveness of targeted in situ CLMS and integrative modeling.

Cross-Linking in Natural-Rubber Vulcanizates

1953 ◽  
Vol 26 (4) ◽  
pp. 741-758 ◽  
Author(s):  
H. E. Adams ◽  
B. L. Johnson
Keyword(s):  
Molecular Weight ◽  
Natural Rubber ◽  
Cross Linking ◽  
Cross Link ◽  
General Validity ◽  
Tetramethylthiuram Disulfide ◽  
Cross Links ◽  
The Cross ◽  
Natural Rubber Vulcanizates ◽  
The Relationship

Abstract Recently, a method for measuring the average number of cross-links per chain of vulcanized polymer has been developed. It is possible to calculate the degree of cross-linking of the vulcanizate from its amount of swelling in a solvent such as benzene. This method was used by Flory to study the effect of primary molecular weight on the cross-linking of Butyl vulcanizates. An evaluation of the general validity of the method was ascertained by using quantitative cross-linking agents (diazodicarboxylates) to prepare vulcanizates of natural rubber and GR-S. Bardwell and Winkler have also used this technique to study the relationship between the degree of cross-linking and the force of retraction at 300 per cent elongation of GR-S latex vulcanized with potassium persulfate. The formation of cross-linking during the vulcanization by sulfur of several polymers has also been investigated. Gee has compared the formation of cross-linking in natural rubber vulcanizates with the amount of combined sulfur. Carbon-to-carbon cross-links were believed to be formed in a nonsulfur tetramethylthiuram disulfide (TMTD) cure. A similar study of Butyl rubber vulcanizates, cured with sulfur-TMTD, indicates that disulfide cross-links are formed. Scott and Magat have estimated that eight sulfur atoms are associated with each cross-link in Russian SK (sodium polybutadiene). This investigation was undertaken to extend Gee's study on the correlation of the cross-linking of natural-rubber vulcanizates with the amount of combined sulfur.

scholarly journals Cross-link analysis of the C-telopeptide domain from type III collagen

1996 ◽  
Vol 318 (2) ◽  
pp. 497-503 ◽  
Author(s):  
W. HENKEL
Keyword(s):  
Sequence Analysis ◽  
Cross Linking ◽  
Type Iii Collagen ◽  
Cross Link ◽  
Type Iii ◽  
C Terminus ◽  
Collagen Iii ◽  
Cross Links ◽  
The Cross ◽  
Helical Region

Several peptides were isolated from tryptic digests of insoluble calf aorta matrix by chromatography. Reductive pyridylethylation of a tryptic 15 kDa pool released fragments deriving from the C-terminus of type III collagen. A 50-residue peptide TC(III) was shown by sequence analysis to be the C-terminal peptide from the α1(III)-chain, containing a helical and non-helical region of equal sizes. The peptide was further digested with collagenase to give ColC(III), comprising the complete C-terminal non-helical region of α1(III) including a hydroxylysine in position 16C. The peptide TC(III)×TN(III) was isolated, demonstrating covalent cross-linking between the C-terminal non-helical region of one type III molecule and the N-terminal helical cross-linking region of another. Its digestion with cyanogen bromide yielded the small fragments α1(III)CB3B* and α1(III)CB3C, confirming TN(III) as an N-terminal helical cross-link site. Sequence analysis of both TC(III)×TN(III) and its collagenase-derived cross-linked peptide ColC(III)×TN(III) established the 4D-staggered alignment of adjacent collagen III molecules. The cross-link structure of both peptides was mainly dihydroxylysinonorleucine with a small amount of hydroxylysinonorleucine, indicating that the lysine residues involved in formation of the cross-links are both hydroxylated. No pyridinoline or histidinohydroxylysinonorleucine cross-links were found within the non-reduced C-telopeptide region of type III collagen.

A Critical Study of Selected Formaldehyde-Cross-linked Cotton Celluloses by Sol-Gel Analysis and by Distention-Index Analysis

1969 ◽  
Vol 39 (3) ◽  
pp. 247-253 ◽  
Author(s):  
G. K. Joarder ◽  
S. P. Rowland
Keyword(s):  
Vapor Phase ◽  
Sol Gel ◽  
Chain Scission ◽  
Quantitative Information ◽  
Critical Study ◽  
Cross Linking ◽  
Fiber Structure ◽  
Vapor Phase Deposition ◽  
Index Analysis ◽  
The Cross

Cotton celluloses selected for pronounced differences in crystallinities and accessibilities have been cross-linked by the vapor-phase deposition of formaldehyde under conditions previously shown to cause different degrees of molecular degradation. The products at various levels of bound formaldehyde have been analyzed for sol and gel fractions and for degree of distention of the gel fraction in cupriethylenediamine hydroxide. The results of these analyses provide quantitative information on the gross uniformity of distribution of cross linkages in the fiber structure of the celluloses, the uniformity of the distribution of cross linkages along the molecular chains of the cellulose, and the ratio of chain scission to molecular cross-linking in the cross-linked compositions. These analyses can now be applied with greater confidence to obtain useful information on cross-linked celluloses, despite the heterogeneity of phases in the cross-linking reactions and the crystallinity of the cellulose.

The cross-linking and degradation of paraffin chains by high-energy radiation

1954 ◽  
Vol 222 (1148) ◽  
pp. 60-74 ◽  
Keyword(s):  
Melting Point ◽  
Physical State ◽  
Main Chain ◽  
High Energy ◽  
Cross Linking ◽  
Cross Link ◽  
High Energy Radiation ◽  
The Cross ◽  
Solid Liquid ◽  
Α Particles

n -Paraffins from C 7 H 16 to C 36 H 74 and polyethylene polymers (Polythene and Winnothene) have been subjected to atomic pile radiation. For the paraffins there is a decrease in the melting-point, until, for a radiation dose R , they no longer melt at temperatures of 160° C or above. At about this same radiation the paraffin is turned into an insoluble gel. The product Rn ρ, where n is the number of carbons per atom, and ρ the density, is approximately constant from heptane (n = 7) to Polythene (n ~ 2000), although an anomaly may occur for Winnothene (n ~ 250). This indicates that the energy required to form a cross-link is approximately independent of chain length. An analysis of published experiments on methane and butane extends this conclusion down to n = 1. The results obtained by earlier workers when paraffinic gases are bombarded with deuterons and α-particles are explained in terms of the cross-linking phenomenon. Solubility measurements give similar values for Rn ρ in the case of Polythene and Winnothene, and show that for every cross-link formed, on the average about 0·35 C—C bonds in the main chain are fractured. Similar values are obtained for methane and butane. The energy absorbed per C—H bond fracture is about 12 eV, and the energy per cross-link is 24 eV. This corresponds to 0·5 % of carbons becoming cross-linked per unit radiation, independent of the physical state (solid, liquid or gaseous) of the irradiated paraffin. The importance of these results, as far as polymerization theory is concerned, is briefly discussed.

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