scholarly journals Characterization of Genipin-Modified Dentin Collagen

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Hiroko Nagaoka ◽  
Hideaki Nagaoka ◽  
Ricardo Walter ◽  
Lee W. Boushell ◽  
Patricia A. Miguez ◽  
...  
Keyword(s):  
Amino Acid ◽  
Lysyl Oxidase ◽  
Biomechanical Properties ◽  
Biochemical Properties ◽  
Cross Linking ◽  
Dependent Manner ◽  
Cross Link ◽  
Concentration Dependent Manner ◽  
Cross Links ◽  
Dentin Collagen

Application of biomodification techniques to dentin can improve its biochemical and biomechanical properties. Several collagen cross-linking agents have been reported to strengthen the mechanical properties of dentin. However, the characteristics of collagen that has undergone agent-induced biomodification are not well understood. The objective of this study was to analyze the effects of a natural cross-linking agent, genipin (GE), on dentin discoloration, collagen stability, and changes in amino acid composition and lysyl oxidase mediated natural collagen cross-links. Dentin collagen obtained from extracted bovine teeth was treated with three different concentrations of GE (0.01%, 0.1%, and 0.5%) for several treatment times (0–24 h). Changes in biochemical properties of NaB3H4-reduced collagen were characterized by amino acid and cross-link analyses. The treatment of dentin collagen with GE resulted in a concentration- and time-dependent pigmentation and stability against bacterial collagenase. The lysyl oxidase-mediated trivalent mature cross-link, pyridinoline, showed no difference among all groups while the major divalent immature cross-link, dehydro-dihydroxylysinonorleucine/its ketoamine in collagen treated with 0.5% GE for 24 h, significantly decreased compared to control (P< 0.05). The newly formed GE-induced cross-links most likely involve lysine and hydroxylysine residues of collagen in a concentration-dependent manner. Some of these cross-links appear to be reducible and stabilized with NaB3H4.

scholarly journals Native cross-links in collagen fibrils induce resistance to human synovial collagenase

1979 ◽  
Vol 181 (3) ◽  
pp. 639-645 ◽  
Author(s):  
C A Vater ◽  
E D Harris ◽  
R C Siegel
Keyword(s):  
Lysyl Oxidase ◽  
Collagen Fibrils ◽  
Bone Collagen ◽  
Collagen Molecule ◽  
Cross Linking ◽  
Pathological Conditions ◽  
Insoluble Material ◽  
Induce Resistance ◽  
Cross Links

A model system consisting of highly purified lysyl oxidase and reconstituted lathyritic chick bone collagen fibrils was used to study the effect of collagen cross-linking on collagen degradation by mammalian collagenase. The results indicate that synthesis of approx. 0.1 Schiff-base cross-link per collagen molecule results in a 2–3-fold resistance to human synovial collagenase when compared with un-cross-linked controls or samples incubated in the presence of beta-aminopropionitrile to inhibit cross-linking. These results confirm previous studies utilizing artificially cross-linked collagens, or collagens isolated as insoluble material after cross-linking in vivo, and suggest that increased resistance to collagenase may be one of the earliest effects of cross-linking in vivo. The extent of intermolecular cross-linking among collagen fibrils may provide a mechanism for regulating the rate of collagen catabolism relative to synthesis in normal and pathological conditions.

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 Two DD-Carboxypeptidases fromMycobacterium smegmatisAffect Cell Surface Properties through Regulation of Peptidoglycan Cross-Linking and Glycopeptidolipids

2018 ◽  
Vol 200 (14) ◽  
Author(s):  
Satya Deo Pandey ◽  
Shilpa Pal ◽  
Ganesh Kumar N ◽  
Ankita Bansal ◽  
Sathi Mallick ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mycobacterium Smegmatis ◽  
Biofilm Formation ◽  
Cross Linking ◽  
Surface Lipid ◽  
Cross Link ◽  
Content Type ◽  
Murine Macrophages ◽  
Cross Links ◽  
Link Formation

ABSTRACTDuring the peptidoglycan (PG) maturation of mycobacteria, the glycan strands are interlinked by both 3-3 (between twomeso-diaminopimelic acids [meso-DAPs]) and 4-3 cross-links (betweend-Ala andmeso-DAP), though there is a predominance (60 to 80%) of 3-3 cross-links. Thedd-carboxypeptidases (dd-CPases) act on pentapeptides to generate tetrapeptides that are used byld-transpeptidases as substrates to form 3-3 cross-links. Therefore,dd-CPases play a crucial role in mycobacterial PG cross-link formation. However, the physiology ofdd-CPases in mycobacteria is relatively unexplored. In this study, we deleted twodd-CPase genes,msmeg_2433andmsmeg_2432, both individually and in combination, fromMycobacterium smegmatismc2155. Though the singledd-CPase gene deletions had no significant impact on the mycobacterial physiology, many interesting functional alterations were observed in the double-deletion mutant,viz., a predominance in PG cross-link formation was shifted from 3-3 cross-links to 4-3, cell surface glycopeptidolipid (GPL) expression was reduced, and susceptibility to β-lactams and antitubercular agents was enhanced. Moreover, the survival rate of the double mutant within murine macrophages was higher than that of the parent. Interestingly, the complementation with any one of thedd-CPase genes could restore the wild-type phenotype. In a nutshell, we infer that the altered ratio of 4-3 to 3-3 PG cross-links might have influenced the expression of surface GPLs, colony morphology, biofilm formation, drug susceptibility, and subsistence of the cells within macrophages.IMPORTANCEThe glycan strands in mycobacterial peptidoglycan (PG) are interlinked by both 3-3 and 4-3 cross-links. Thedd-CPases generate tetrapeptides by acting on the pentapeptides, andld-transpeptidases use tetrapeptides as substrates to form 3-3 cross-links. In this study, we showed that simultaneous deletions of twodd-CPases alter the nature of PG cross-linking from 3-3 cross-links to 4-3 cross-links. The deletions subsequently decrease the expression of glycopeptidolipids (significant surface lipid present in many nontuberculous mycobacteria, includingMycobacterium smegmatis) and affect other physiological parameters, like cell morphology, growth rate, biofilm formation, antibiotic susceptibility, and survival within murine macrophages. Thus, unraveling the physiology ofdd-CPases might help us design antimycobacterial therapeutics in the future.

Chemical and biochemical properties of abasic site adducts and 6-thioguanine DNA cross-links in DNA

2017 ◽  
Author(s):  
◽  
Calvin D. Lewis
Keyword(s):  
Excision Repair ◽  
Biochemical Properties ◽  
Macromolecular Complex ◽  
Abasic Site ◽  
Cross Link ◽  
Dna Lesion ◽  
Phosphate Backbone ◽  
Cross Links ◽  
Ap Sites ◽  
Cellular Dna

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] DNA is a macromolecular complex, composed of the nucleotides adenine, thymine, guanine and cytosine interconnected by a phosphate backbone, that contains the genetic code for living organisms and viruses. Spontaneous and enzymatic hydrolysis of the glycosidic bonds that hold the coding nucleobases to the 2-deoxyribose-phosphate backbone of DNA results in the production of abasic (Ap) sites. These lesions are abundant in cellular DNA, and cellular Ap-containing DNA is damaging and may lead to cellular destruction if left unrepaired. Thus, efficient cellular DNA repair mechanisms that repair Ap sites have evolved in DNA containing organisms. The studies in this report examine the interaction between small molecules or naturally occurring DNA residues with Ap sites in duplex DNA. Experiments provide evidence that hydralazine binds to and forms a stable DNA lesion in single- and double-stranded DNA. Also, the hydralazine-DNA lesion is found to be a poor substrate for mammalian base excision repair enzymes such as Ap endonuclease and 8-oxoguanine DNA glycosylase. In addition, these studies provide preliminary evidence that hydralazine may potentiate the cytotoxicity of temozolomide in U87 cells. The investigation of the formation of cross-links between canonical DNA residues deoxyadenosine (dA) and deoxyguanosine (dG) with Ap sites is also explored. These experiments suggest that sequence effects contribute majorly to the cross-link yield in both dA- and dG-Ap site cross-links, especially when comparing central versus terminal cross-link locations. Here, this manuscript provides novel studies involving the interaction between DNA analog 6-thioguanine and opposing DNA bases in duplex oligonucleotide DNA.

Lysyl oxidases: from enzyme activity to extracellular matrix cross-links

2019 ◽  
Vol 63 (3) ◽  
pp. 349-364 ◽  
Author(s):  
Sylvain D. Vallet ◽  
Sylvie Ricard-Blum
Keyword(s):  
Extracellular Matrix ◽  
Molecular Mechanisms ◽  
Catalytic Domain ◽  
Lysyl Oxidase ◽  
Dimensional Structure ◽  
Cross Linking ◽  
Copper Binding ◽  
Molecular Features ◽  
Ecm Proteins ◽  
Cross Links

Abstract The lysyl oxidase family comprises five members in mammals, lysyl oxidase (LOX) and four lysyl oxidase like proteins (LOXL1-4). They are copper amine oxidases with a highly conserved catalytic domain, a lysine tyrosylquinone cofactor, and a conserved copper-binding site. They catalyze the first step of the covalent cross-linking of the extracellular matrix (ECM) proteins collagens and elastin, which contribute to ECM stiffness and mechanical properties. The role of LOX and LOXL2 in fibrosis, tumorigenesis, and metastasis, including changes in their expression level and their regulation of cell signaling pathways, have been extensively reviewed, and both enzymes have been identified as therapeutic targets. We review here the molecular features and three-dimensional structure/models of LOX and LOXLs, their role in ECM cross-linking, and the regulation of their cross-linking activity by ECM proteins, proteoglycans, and by inhibitors. We also make an overview of the major ECM cross-links, because they are the ultimate molecular readouts of LOX/LOXL activity in tissues. The recent 3D model of LOX, which recapitulates its known structural and biochemical features, will be useful to decipher the molecular mechanisms of LOX interaction with its various substrates, and to design substrate-specific inhibitors, which are potential antifibrotic and antitumor drugs.

scholarly journals Cross-linking modifications of HDL apoproteins by oxidized phospholipids: structural characterization, in vivo detection, and functional implications

2020 ◽  
Vol 295 (7) ◽  
pp. 1973-1984
Author(s):  
Detao Gao ◽  
Mohammad Z. Ashraf ◽  
Lifang Zhang ◽  
Niladri Kar ◽  
Tatiana V. Byzova ◽  
...  
Keyword(s):  
Density Lipoprotein ◽  
Cross Linking ◽  
Oxidized Phospholipids ◽  
Cross Link ◽  
In Vivo Detection ◽  
Lysine Residues ◽  
Cross Links ◽  
Human Atheroma

Apolipoprotein A-I (apoA-I) is cross-linked and dysfunctional in human atheroma. Although multiple mechanisms of apoA-I cross-linking have been demonstrated in vitro, the in vivo mechanisms of cross-linking are not well-established. We have recently demonstrated the highly selective and efficient modification of high-density lipoprotein (HDL) apoproteins by endogenous oxidized phospholipids (oxPLs), including γ-ketoalkenal phospholipids. In the current study, we report that γ-ketoalkenal phospholipids effectively cross-link apoproteins in HDL. We further demonstrate that cross-linking impairs the cholesterol efflux mediated by apoA-I or HDL3 in vitro and in vivo. Using LC-MS/MS analysis, we analyzed the pattern of apoprotein cross-linking in isolated human HDL either by synthetic γ-ketoalkenal phospholipids or by oxPLs generated during HDL oxidation in plasma by the physiologically relevant MPO-H2O2-NO2− system. We found that five histidine residues in helices 5–8 of apoA-I are preferably cross-linked by oxPLs, forming stable pyrrole adducts with lysine residues in the helices 3–4 of another apoA-I or in the central domain of apoA-II. We also identified cross-links of apoA-I and apoA-II with two minor HDL apoproteins, apoA-IV and apoE. We detected a similar pattern of apoprotein cross-linking in oxidized murine HDL. We further detected oxPL cross-link adducts of HDL apoproteins in plasma and aorta of hyperlipidemic LDLR−/− mice, including cross-link adducts of apoA-I His-165–apoA-I Lys-93, apoA-I His-154–apoA-I Lys-105, apoA-I His-154–apoA-IV Lys-149, and apoA-II Lys-30–apoE His-227. These findings suggest an important mechanism that contributes to the loss of HDL's atheroprotective function in vivo.

scholarly journals Thyrotropin cross-links to the thyrotropin receptor through both the α and β subunits

1986 ◽  
Vol 235 (3) ◽  
pp. 879-882 ◽  
Author(s):  
P R Buckland ◽  
C R Rickards ◽  
R D Howells ◽  
B R Smith
Keyword(s):  
Tsh Receptor ◽  
Alpha Subunit ◽  
Receptor Interaction ◽  
Cross Linking ◽  
Thyrotropin Receptor ◽  
Cross Link ◽  
Receptor Binding Site ◽  
Form Part ◽  
Cross Links

We have recently shown that the beta subunit of thyrotropin (TSH) can be cross-linked to the TSH receptor [Buckland, Strickland, Pierce & Rees Smith (1985) Endocrinology (Baltimore) 116, 2122-2124; Buckland, Strickland & Rees Smith (1985) Biochem. Soc. Trans. 13, 942-943]. We failed, however, to cross-link the alpha subunit to the receptor, leaving the role of this subunit in the TSH-TSH-receptor interaction uncertain. We now report the successful cross-linking of the TSH alpha subunit to the receptor by the use of two different cross-linking reagents. Our studies suggest therefore that both subunits of TSH form part of the hormone's receptor-binding site.

A New Mechanism of Platelet Activation and Oxidative Death Induced by ADAMTS-18 and Regulating Bleeding Time.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 133-133
Author(s):  
Zongdong Li ◽  
Michael Nardi ◽  
Ruimin Pan ◽  
Herman Yee ◽  
Simon Karpatkin
Keyword(s):  
Amino Acid ◽  
Platelet Activation ◽  
Bleeding Time ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Activation Mechanism ◽  
Dependent Manner ◽  
Cell Secretion ◽  
Concentration Dependent Manner ◽  
Buffer Control

Abstract Anti-platelet integrin GPIIIa49-66 Ab obtained from HIV-ITP patients (or raised in rabbits) induces complement-independent platelet oxidative fragmentation and death by activating platelet 12-lipoxygenase (generation of 12(S)-HETE) and NADPH oxidase (JCI, 113:973, 2004). Platelet oxidative fragmentation is measured by flow cytometry of generated microparticles as well as intracellular DCFH oxidation. We now report that oxidative fragmentation in human platelets is preceded by Ca++ flux and P-selectin activation, n=6. However, the activation mechanism is different from classic platelet activation in that it is not inhibited by PGE1 or dibutryl cyclic AMP and is operative with Gαq−/− mouse platelets, whereas under these conditions, thrombin-induced platelet activation is completely inhibited, n=5–6. We chose to identify putative physiologic ligands that behave similarly to the GPIIIa49-66 Ab, and are therefore capable of regulating platelet reactive oxygen species (ROS) as well as arterial thrombus formation. The GPIIIa49-66 platelet peptide was used as bait to screen a 7-mer peptide phage display library. A peptide was found with 70% homology at the C-terminal position of ADAMTS-18, an ‘orphan’ disintegrin and metalloproteinase with thrombospondin (TSR)-like motifs, with unknown substrate. We have found it present in HUVEC as well as human pulmonary artery endothelial cells, on fixed sections of pathology specimens employing immunohistochemistry with a specific rabbit Ab raised against a C-terminal 18 mer peptide ADAMTS-18 (no staining with preimmune Ab). Recombinant ADAMTS-18 was produced in HEK 293 T cells and shown to induce ROS and oxidative platelet fragmentation in an identical kinetic fashion as anti-GPIIIa49-66 Ab. HUVEC ADAMTS-18 activity could be inhibited by a human scFv Ab raised against its C-terminal 18 mer peptide, as well as the ADAMTS-18 peptide itself, but not by a rabbit Ab against the N-terminal domain or an irrelevant peptide. Endothelial cell secretion and activation of ADAMTS-18 was optimally induced with 0.5 u/ml thrombin at 2 – 4 hrs, n=3–4. The truncated 385 amino acid C-terminal rADAMTS-18 fragment containing the 4 TSR motifs (produced in E.coli) had full activity at (<0.3 uM) whereas the C-terminal 66 amino acid fragment not containing the 18-mer binding site was inactive at 65 fold higher concentration, n=4. The physiologic significance of ADAMTS-18 was supported by demonstrating its secretion into plasma following iv injection of 4–16 u/ml thrombin into mice. Wild type mice have no detectable ADAMTS-18 in their plasma, with a sensitive ELISA assay (1 ng detectability). Thrombin stimulated mice secrete ADAMTS-18 in a concentration dependent manner. Platelet aggregates produced ex vivo with ADP and fibrinogen were destroyed with ADAMTS-18 as documented by LDH release at 1, 2 and 4 hrs of 83, 241 and 260 fold respectively, of PBS buffer control. In vivo tail vein bleeding time was shortened 4.5 fold with 1 hr prior infusion of 25 ug of a polyclonal rabbit IgG against ADAMTS-18, but not with preimmune IgG, n=10. Thus, a new mechanism is proposed for platelet activation, ROS release, death and platelet thrombus regulation, via platelet membrane oxidative fragmentation induced by thrombin-induced secretion and activation of ADAMTS-18.

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