scholarly journals A study of the interaction in vitro between type I collagen and a small dermatan sulphate proteoglycan

1988 ◽  
Vol 251 (3) ◽  
pp. 643-648 ◽  
Author(s):  
N Uldbjerg ◽  
C C Danielsen
Keyword(s):  
Steady State ◽  
Binding Sites ◽  
Type I Collagen ◽  
Enzyme Linked Immunosorbent Assay ◽  
Side Chains ◽  
Type I ◽  
Dermatan Sulphate ◽  
Collagen Fibrillogenesis ◽  
High Affinity ◽  
Sulphate Proteoglycan

The interaction between a small dermatan sulphate proteoglycan isolated from human uterine cervix and collagen type I from human and rat skin was investigated by collagen-fibrillogenesis experiments. Collagen fibrillogenesis was initiated by elevation of temperature and pH after addition of proteoglycan, chondroitinase-digested proteoglycan or isolated side chains, and monitored by turbidimetry. Collagen-associated and unbound proteoglycan was determined by enzyme-linked immunosorbent assay after aggregation was complete. (1) The binding of proteoglycan to collagen could be explained by the presence of two mutually non-interacting binding sites, with Ka1 = 1.3 x 10(8) M-1 and Ka2 = 1.3 x 10(6) M-1. The number of binding sites per tropocollagen molecule was n1 = 0.11 and n2 = 1.1. The 0.1 high-affinity binding site per tropocollagen molecule indicates that the strong interaction between proteoglycan and collagen results from a concerted action of tropocollagen molecules in fibrils. Digestion of the proteoglycan with chondroitinase ABC did not affect these binding characteristics. (2) Proteoglycan did not affect the rate of fibrillogenesis, but increased the steady-state A400 by up to 90%. This increase was directly proportional to the saturation of the high-affinity type of binding sites. Neither isolated core protein nor isolated side chains induced a similar high increase in steady-state A400. (3) Electron micrographs showed that the fibril diameter was affected only to a minor extent, if at all, by the proteoglycan, whereas bundles of laterally aligned fibrils were common in the presence of proteoglycan. (4) Results obtained with human and rat collagen were similar.

RAP1 is required for BAS1/BAS2- and GCN4-dependent transcription of the yeast HIS4 gene

1991 ◽  
Vol 11 (7) ◽  
pp. 3642-3651 ◽  
Author(s):  
C Devlin ◽  
K Tice-Baldwin ◽  
D Shore ◽  
K T Arndt
Keyword(s):  
Steady State ◽  
Binding Site ◽  
Binding Sites ◽  
Binding Activity ◽  
Micrococcal Nuclease ◽  
Mrna Levels ◽  
High Affinity ◽  
Steady State Levels

The major in vitro binding activity to the Saccharomyces cerevisiae HIS4 promoter is due to the RAP1 protein. In the absence of GCN4, BAS1, and BAS2, the RAP1 protein binds to the HIS4 promoter in vivo but cannot efficiently stimulate HIS4 transcription. RAP1, which binds adjacently to BAS2 on the HIS4 promoter, is required for BAS1/BAS2-dependent activation of HIS4 basal-level transcription. In addition, the RAP1-binding site overlaps with the single high-affinity HIS4 GCN4-binding site. Even though RAP1 and GCN4 bind competitively in vitro, RAP1 is required in vivo for (i) the normal steady-state levels of GCN4-dependent HIS4 transcription under nonstarvation conditions and (ii) the rapid increase in GCN4-dependent steady-state HIS4 mRNA levels following amino acid starvation. The presence of the RAP1-binding site in the HIS4 promoter causes a dramatic increase in the micrococcal nuclease sensitivity of two adjacent regions within HIS4 chromatin: one region contains the high-affinity GCN4-binding site, and the other region contains the BAS1- and BAS2-binding sites. These results suggest that RAP1 functions at HIS4 by increasing the accessibility of GCN4, BAS1, and BAS2 to their respective binding sites when these sites are present within chromatin.

scholarly journals Biosynthesis of sulphated macromolecules by rabbit lens epithelium. II. Relationship to basement membrane formation.

1984 ◽  
Vol 99 (3) ◽  
pp. 861-869 ◽  
Author(s):  
J G Heathcote ◽  
R R Bruns ◽  
R W Orkin
Keyword(s):  
Basement Membrane ◽  
Type I Collagen ◽  
Heparan Sulphate ◽  
Significant Proportion ◽  
Type I ◽  
Heparan Sulphate Proteoglycan ◽  
Membrane Formation ◽  
Sulphate Proteoglycan ◽  
Type Iv ◽  
Rabbit Lens

Rabbit lens epithelial cells display a similar "cobblestone" morphology and produce the same complement of sulphated macromolecules (also see Heathcote, J.G., and R.W. Orkin, 1984, J. Cell Biol., 99:852-860) whether grown on plastic or glass, dried films of gelatin or type IV collagen with laminin, or on gels of type I collagen. There was no evidence of basement membrane formation by these cells when they were grown on plastic, glass, or dried films. In contrast, cultures that had been grown on gels deposited a discrete basement membrane that followed the contours of the basal surfaces of the cells and in addition, they secreted amorphous basement membrane-like material that diffused into the interstices of the gel and associated with the collagen fibrils of the gel. A significant proportion (approximately 70%) of the heparan sulphate proteoglycan fraction that was secreted into the culture medium (fraction MI) when the cells were grown on plastic became associated with the cell-gel layer in the gel cultures. Further, when basement membrane was isolated by detergent extraction, greater than 90% of the 35S-labeled material present was in this heparan sulphate proteoglycan.

scholarly journals Salivary Melatonin and the Severity of Attachment Loss: A Case-Control Study

2014 ◽  
Vol 2014 ◽  
pp. 1-4
Author(s):  
Leila Golpasand-Hagh ◽  
Faramarz Zakavi ◽  
Arash Daraeighadikolaei ◽  
Akram Ahangarpour ◽  
Sara Hajati ◽  
...  
Keyword(s):  
Chronic Periodontitis ◽  
Healthy Subjects ◽  
Type I Collagen ◽  
Periodontal Diseases ◽  
Protective Role ◽  
Enzyme Linked Immunosorbent Assay ◽  
Blood Levels ◽  
Melatonin Level ◽  
Type I ◽  
Salivary Melatonin

Background. Melatonin (MT: N-acetyl-5-methoxytryptamine) is a neuroendocrine hormone secreted mainly by the pineal gland in the brain. MT is produced with a circadian rhythm characterized by elevated blood levels during the night. In healthy individuals, maximal secretion of MT occurs between midnight and 2:00 am, whereas the minimal production occurs during the day. MT can be determined by repeated measurement of plasma or salivary MT or urine sulfatoxy-melatonin. Melatonin has powerful antioxidant effects, has an immunomodulatory role, stimulates the synthesis of type I collagen fibers, and promotes bone formation. Melatonin is also secreted in the saliva, although its role in the mouth is not known well. The purpose of this study was to examine the correlation between salivary melatonin level and periodontal diseases. Methods. Fifty subjects by mean age of 40.44±6.38 years were equally divided into 5 groups: 10 healthy subjects, 10 subjects with gingivitis, 10 subjects with localized moderate chronic periodontitis, 10 subjects with generalized moderate chronic periodontitis, and 10 subjects with generalized severe chronic periodontitis. Saliva samples were collected from all the subjects and melatonin levels were determined using an enzyme-linked immunosorbent assay. Two-way and one-way ANOVA and Tukey test were used to analyze relationships among variables. Results. Healthy subjects had significantly higher salivary melatonin level (5.29±0.50 pg/mL) compared to patients with gingivitis (4.35±0.30 pg/mL) (P<0.001). The difference between salivary melatonin level in patients with gingivitis and periodontitis was significant (P<0.001). Level of melatonin in patients with generalized severe chronic periodontitis (3.39±0.10 pg/mL) was significantly lower than that in other groups (P<0.01). Conclusions. This study determined that salivary melatonin level in patients with periodontal diseases is lower than that in healthy subjects. Consequently we conclude that there is a negative correlation between melatonin level and the severity of disease, suggesting that melatonin might have a protective role against periodontal diseases, although further research is required to validate this hypothesis.

Evitar (l-Alanyl-l-Glutamine) Regulates Key Signaling Molecules in the Pathogenesis of Postoperative Tissue Fibrosis

2018 ◽  
Vol 26 (6) ◽  
pp. 724-733 ◽  
Author(s):  
Lynne M. Robertson ◽  
Nicole M. Fletcher ◽  
Michael P. Diamond ◽  
Ghassan M. Saed
Keyword(s):  
Cellular Response ◽  
Type I Collagen ◽  
Primary Cultures ◽  
Hypoxia Inducible Factor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Type I ◽  
Dependent Manner ◽  
Tissue Fibrosis ◽  
Hypoxia Inducible Factor 1Α ◽  
Episodic Hypoxia

Aims:Hypoxia and the resulting oxidative stress play a major role in postoperative tissue fibrosis. The objective of this study was to determine the effect of l-alanyl-l-glutamine (Ala-Gln) on key markers of postoperative tissue fibrosis: hypoxia-inducible factor (HIF) 1α and type I collagen.Methods:Primary cultures of human normal peritoneal fibroblasts (NPF) established from normal peritoneal tissue were treated with increasing doses of Ala-Gln (0, 1, 2, or 10 mM) with hypoxia ([2% O2] 0-48 hours; continuous hypoxia) or after hypoxia (0.5, 1, 2, 4 hours) and restoration of normoxia (episodic hypoxia) with immediate treatment with Ala-Gln. Hypoxia-inducible factor 1α and type 1 collagen levels were determined by enzyme-linked immunosorbent assay. Data were analyzed with 1-way analysis of variance followed by Tukey tests with Bonferroni correction.Results:Hypoxia-inducible factor 1α and type I collagen levels increased in untreated controls by 3- to 4-fold in response to continuous and episodic hypoxia in human NPF. Under continuous hypoxia, HIF-1α and type I collagen levels were suppressed by Ala-Gln in a dose-dependent manner. l-alanyl-l-glutamine treatment after episodic hypoxia also suppressed HIF-1α and type I collagen levels for up to 24 hours for all doses and up to 48 hours at the highest dose, regardless of exposure time to hypoxia.Conclusions:l-alanyl-l-glutamine significantly suppressed hypoxia-induced levels of key tissue fibrosis (adhesion) phenotype markers under conditions of continuous as well as episodic hypoxia in vitro. This effect of glutamine on molecular events involved in the cellular response to insult or injury suggests potential therapeutic value for glutamine in the prevention of postoperative tissue fibrosis.

scholarly journals Binding of Brucella protein, Bp26, to select extracellular matrix molecules

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Yasmin ElTahir ◽  
Amna Al-Araimi ◽  
Remya R. Nair ◽  
Kaija J. Autio ◽  
Hongmin Tu ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Type I Collagen ◽  
Host Cells ◽  
Mouse Serum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Target Cells ◽  
Type I ◽  
Dependent Manner ◽  
Biolayer Interferometry

Abstract Background Brucella is a facultative intracellular pathogen responsible for zoonotic disease brucellosis. Little is known about the molecular basis of Brucella adherence to host cells. In the present study, the possible role of Bp26 protein as an adhesin was explored. The ability of Brucella protein Bp26 to bind to extracellular matrix (ECM) proteins was determined by enzyme-linked immunosorbent assay (ELISA) and biolayer interferometry (BLI). Results ELISA experiments showed that Bp26 bound in a dose-dependent manner to both immobilized type I collagen and vitronectin. Bp26 bound weakly to soluble fibronectin but did not bind to immobilized fibronectin. No binding to laminin was detected. Biolayer interferometry showed high binding affinity of Bp26 to immobilized type I collagen and no binding to fibronectin or laminin. Mapping of Bp26 antigenic epitopes by biotinylated overlapping peptides spanning the entire sequence of Bp26 using anti Bp26 mouse serum led to the identification of five linear epitopes. Collagen and vitronectin bound to peptides from several regions of Bp26, with many of the binding sites for the ligands overlapping. The strongest binding for anti-Bp26 mouse serum, collagen and vitronectin was to the peptides at the C-terminus of Bp26. Fibronectin did not bind to any of the peptides, although it bound to the whole Bp26 protein. Conclusions Our results highlight the possible role of Bp26 protein in the adhesion process of Brucella to host cells through ECM components. This study revealed that Bp26 binds to both immobilized and soluble type I collagen and vitronectin. It also binds to soluble but not immobilized fibronectin. However, Bp26 does not bind to laminin. These are novel findings that offer insight into understanding the interplay between Brucella and host target cells, which may aid in future identification of a new target for diagnosis and/or vaccine development and prevention of brucellosis.

Immunoassay for quantifying type I collagen degradation products in urine evaluated

1994 ◽  
Vol 40 (11) ◽  
pp. 2022-2025 ◽  
Author(s):  
M Bonde ◽  
P Qvist ◽  
C Fledelius ◽  
B J Riis ◽  
C Christiansen
Keyword(s):  
Type I Collagen ◽  
Degradation Products ◽  
Mean Value ◽  
Collagen Degradation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Bone Resorption Marker ◽  
Type I ◽  
Analytical Recovery ◽  
Collagen Degradation Products ◽  
Better Than

Abstract An enzyme-linked immunosorbent assay (ELISA) for measuring type I collagen degradation products in urine &lt; 3 h was evaluated. The measuring range was 0.5-10.5 mg/L with a detection limit of 0.2 mg/L. Within-run and total CVs were 5.3% and 6.6%, respectively. Analytical recovery averaged 100%. The mean (+/- SD) concentrations in urine samples from a healthy premenopausal population (n = 102) were 250 +/- 110 mg/mol creatinine (Cr). A group of healthy postmenopausal women (n = 410) gave a mean value of 416 +/- 189 mg/mol Cr. Values obtained in the ELISA correlated well (r = 0.83) to HPLC values for the established bone resorption marker deoxypyridinoline (n = 214), slightly better than the correlation to hydroxyproline measurements (r = 0.78, n = 421). We conclude that the assay described here presents a useful tool for further elucidating the importance of type I collagen degradation products in urine.

scholarly journals Regulation of type I collagen mRNA in lung fibroblasts by cystine availability

1998 ◽  
Vol 331 (2) ◽  
pp. 417-422 ◽  
Author(s):  
David C. RISHIKOF ◽  
Ping-Ping KUANG ◽  
Christine POLIKS ◽  
Ronald H. GOLDSTEIN
Keyword(s):  
Amino Acids ◽  
Steady State ◽  
Amino Acid ◽  
Type I Collagen ◽  
State Level ◽  
Lung Fibroblasts ◽  
Type I ◽  
Mrna Levels ◽  
Collagen Mrna ◽  
Amino Acid Availability

The steady-state level of α1(I) collagen mRNA is regulated by amino acid availability in human lung fibroblasts. Depletion of amino acids decreases α1(I) collagen mRNA levels and repletion of amino acids induces rapid re-expression of α1(I) mRNA. In these studies, we examined the requirements for individual amino acids on the regulation of α1(I) collagen mRNA. We found that re-expression of α1(I) collagen mRNA was critically dependent on cystine but not on other amino acids. However, the addition of cystine alone did not result in re-expression of α1(I) collagen mRNA. Following amino acid depletion, the addition of cystine with selective amino acids increased α1(I) collagen mRNA levels. The combination of glutamine and cystine increased α1(I) collagen mRNA levels 6.3-fold. Methionine or a branch-chain amino acid (leucine, isoleucine or valine) also acted in combination with cystine to increase α1(I) collagen mRNA expression, whereas other amino acids were not effective. The prolonged absence of cystine lowered steady-state levels of α1(I) collagen mRNA through a mechanism involving decreases in both the rate of gene transcription as assessed by nuclear run-on experiments and mRNA stability as assessed by half-life determination in the presence of actinomycin D. The effect of cystine was not mediated via alterations in the level of glutathione, the major redox buffer in cells, as determined by the addition of buthionine sulphoximine, an inhibitor of γ-glutamylcysteine synthetase. These data suggest that cystine directly affects the regulation of α1(I) collagen mRNA.

‘Small’-proteoglycan: collagen interactions: Keratan sulphate proteoglycan associates with rabbit corneal collagen fibrils at the ‘a’ and ‘c’ bands

1985 ◽  
Vol 5 (9) ◽  
pp. 765-774 ◽  
Author(s):  
J. E. Scott ◽  
M. Haigh
Keyword(s):  
Binding Sites ◽  
Type I Collagen ◽  
Specific Binding ◽  
Corneal Stroma ◽  
Collagen Fibrils ◽  
Type I ◽  
Keratan Sulphate ◽  
Protein Rich ◽  
Small Proteoglycan ◽  
Corneal Collagen

l. Proteoglycans (PGs) in rabbit corneal stroma and mouse sclera have been stained for electron microscopy with Cupromeronic blue in a critical electrolyte concentration (CEC) mode, with and without prior digestion of the tissue by keratanase or chondroitinase ABC to remove the keratan sulphate (KS) or chondroitin-dermatan sulphates (CS or DS) respectively.2. Two classes of PGs, located orthogonally to the corneal collagen fibrils at either the ‘step’ (band ‘a’ or ‘c’) or gap zone (band ‘d’ or ‘e’) are shown to be KS-PGs or DS-PGs respectively. Four separate and specific PG binding sites on Type I collagen fibrils have thus been identified.3. Rabbit corneal KS and DS PGs each contain two kinds of PG (Gregory JD, Coster L & Damle SP (1982) J. Biol. Chem.257, 6965–6970). We propose that each ‘small’ protein-rich PG is associated with a specific binding site on the collagen fibril.

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