Cysteine proteinase activities in the fish pathogenPhilasterides dicentrarchi(Ciliophora: Scuticociliatida)

Parasitology ◽  
2004 ◽  
Vol 128 (5) ◽  
pp. 541-548 ◽  
Author(s):  
A. PARAMÁ ◽  
R. IGLESIAS ◽  
M. F. ÁLVAREZ ◽  
J. LEIRO ◽  
F. M. UBEIRA ◽  
...  
Keyword(s):  
Crude Extract ◽  
Type I Collagen ◽  
Cysteine Proteinase ◽  
Cysteine Proteases ◽  
Cysteine Protease Inhibitor ◽  
Type I ◽  
Molecular Weights ◽  
Reducing Conditions ◽  
Sds Page

This study investigated protease activities in a crude extract andin vitroexcretion/secretion (E/S) products ofPhilasterides dicentrarchi, a ciliate fish parasite causing economically significant losses in aquaculture. Gelatin/SDS–PAGE analysis (pH 4, reducing conditions) detected 7 bands with gelatinolytic activity (approximate molecular weights 30–63 kDa) in the crude extract. The banding pattern observed in analysis of E/S products was practically identical, except for 1 low-molecular-weight band detected in the crude extract but not in the E/S products. In assays with synthetic peptidep-nitroanilide substrates, the crude extract hydrolysed substrates characteristic of cysteine proteases, namely Z-Arg-Arg pNA, Bz-Phe-Val-Arg pNA and Z-Phe-Arg pNA. These activities were strongly inhibited by the cysteine protease inhibitor E-64 and by Ac-Leu-Val-Lys aldehyde, a potent inhibitor of cysteine proteases of the cathepsin B protease subfamily. The proteases present in the crude extract degraded both type-I collagen and haemoglobinin vitro, consistent with roles in tissue invasion and nutrition respectively. Again, E-64 completely (collagen) or markedly (haemoglobin) inhibited this degradation. Finally, the histolytic activity of the ciliate in turbot fibroblast monolayers was strongly reduced in the presence of E-64, confirming the importance of secreted cysteine proteinases in the biology ofPhilasterides dicentrarchi.

scholarly journals Relative rates of biosynthesis of collagen type I, type V and type VI in calf cornea

1991 ◽  
Vol 274 (2) ◽  
pp. 615-617 ◽  
Author(s):  
P Kern ◽  
M Menasche ◽  
L Robert
Keyword(s):  
Type I Collagen ◽  
Collagen Type ◽  
Corneal Stroma ◽  
Collagen Type I ◽  
Type I ◽  
Type Vi Collagen ◽  
Sds Page ◽  
Proline Incorporation ◽  
Type V

The biosynthesis of type I, type V and type VI collagens was studied by incubation of calf corneas in vitro with [3H]proline as a marker. Pepsin-solubilized collagen types were isolated by salt fractionation and quantified by SDS/PAGE. Expressed as proportions of the total hydroxyproline solubilized, corneal stroma comprised 75% type I, 8% type V and 17% type VI collagen. The rates of [3H]proline incorporation, linear up to 24 h for each collagen type, were highest for type VI collagen and lowest for type I collagen. From pulse-chase experiments, the calculated apparent half-lives for types I, V and VI collagens were 36 h, 10 h and 6 h respectively.

Bioactive Glass Templates for the Synthesis of Bone-Like Tissue In Vitro

1993 ◽  
Vol 331 ◽  
Author(s):  
A. Ei-Ghannam ◽  
P. Ducheyne ◽  
I. M. Shapiro
Keyword(s):  
Bioactive Glass ◽  
Porous Glass ◽  
Type I Collagen ◽  
Neonatal Rat ◽  
Bone Lesions ◽  
Type I ◽  
Sds Page ◽  
Biochemical Analyses ◽  
Ca:P Ratio

AbstractPorous glass templates of pore size 75–200 14m and 20–30% porosity were synthesized. The glass disks were conditioned in a modified tris buffer (SBF) for 48 hrs and then treated with tissue culture medium (TCM) for 1 hr at 37°C. Other porous glass templates were treated with either SBF or TCM. The conditioned glass disks were seeded with 106 neonatal rat calvaria osteoblasts and maintained in culture for 2, 5 or 7 days. It was found that the bioactive glass was rapidly invaded by cells which colonized the porous template. Morphological and biochemical analyses suggested that a bone-like tissue was formed inside templates conditioned with both SBF and TCM. By 7 days, the cells exhibited high alkaline phosphatase activity and synthesized osteocalcin. SDS-PAGE indicated the presence of type I collagen. SEM-EDAX analysis showed the formation of a bone-like tissue throughout the entire sample, while inspection of the Ca:P ratio indicated that the cells synthesized a mineral phase. Indeed, the FTIR spectra of the mineral confirmed that it was a biological hydroxyapatite. The rapid formation of the bone-like material within the template in vitro suggests that this template may be valuable clinically for the repair of bone lesions.

scholarly journals Cleavage of native type I collagen by human neutrophil elastase

1998 ◽  
Vol 330 (2) ◽  
pp. 897-902 ◽  
Author(s):  
Wa'el KAFIENAH ◽  
J. David BUTTLE ◽  
David BURNETT ◽  
P. Anthony HOLLANDER
Keyword(s):  
Neutrophil Elastase ◽  
Human Neutrophil ◽  
Type I Collagen ◽  
Cysteine Proteinase ◽  
Collagenolytic Activity ◽  
Human Neutrophil Elastase ◽  
Type I ◽  
Sds Page ◽  
Dose Dependent ◽  
Connective Tissue Pathology

The ability of purified human neutrophil elastase (EC 3.4.21.37) to cleave native type I collagen has been investigated. Soluble human, bovine or rat type I collagen was incubated with neutrophil elastase for 16 h at 25 °C before catalysis was stopped with 3,4-dichloroisocoumarin. Analysis by SDS/PAGE of the collagen digests revealed 3/4-length fragments similar in size to those produced by interstitial collagenase. The collagenolytic activity was dose dependent and was not due to a contaminating metalloproteinase or cysteine proteinase, as it was not inhibited by 1,10-phenanthroline, EDTA or L-trans-epoxysuccinyl-leucylamido-(4-guanidino)butane. The identity of the cleavage products was confirmed using a new antibody that recognizes the unwound α2(I)-chain. This detected the 3/4-length fragment of type I collagen following neutrophil elastase cleavage. In addition to cleaving soluble collagen, neutrophil elastase also cleaved reconstituted, radiolabelled type I collagen fibrils, at a rate of 16 μg/min per nmol. These results indicate that neutrophil elastase can cleave native type I collagen in the helix, an activity that might contribute to its roles in connective-tissue pathology.

scholarly journals Estrogen Reduces the Depth of Resorption Pits by Disturbing the Organic Bone Matrix Degradation Activity of Mature Osteoclasts

Endocrinology ◽  
2001 ◽  
Vol 142 (12) ◽  
pp. 5371-5378 ◽  
Author(s):  
Vilhelmiina Parikka ◽  
Petri Lehenkari ◽  
Mirja-Liisa Sassi ◽  
Jussi Halleen ◽  
Juha Risteli ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Type I Collagen ◽  
Bone Matrix ◽  
Organic Matrix ◽  
Cysteine Proteases ◽  
Bone Collagen ◽  
Type I ◽  
Matrix Degradation ◽  
Carboxyl Terminal

Abstract Decreased E2 levels after menopause cause bone loss through increased penetrative resorption. The reversal effect of E2 substitution therapy is well documented in vivo, although the detailed mechanism of action is not fully understood. To study the effects of E2 on bone resorption, we developed a novel in vitro bone resorption assay in which degradation of inorganic and organic matrix could be measured separately. E2 treatment significantly decreased the depth of resorption pits, although the area resorbed was not changed. Electron microscopy further revealed that the resorption pits were filled with nondegraded collagen, suggesting that E2 disturbed the organic matrix degradation. Two major groups of proteinases, matrix metalloproteinases (MMPs) and cysteine proteinases, have been suggested to participate in organic matrix degradation by osteoclasts. We show here that MMP-9 released a cross-linked carboxyl-terminal telopeptide of type I collagen from bone collagen, and cathepsin K released another C-terminal fragment, the C-terminal cross-linked peptide of type I collagen. E2 significantly inhibited the release of the C-terminal cross-linked peptide of type I collagen into the culture medium without affecting the release of cross-linked carboxyl-terminal telopeptide of type I collagen in osteoclast cultures. These results suggest that organic matrix degradation is initiated by MMPs and continued by cysteine proteases; the latter event is regulated by E2.

Collagen fibrillogenesis in vitro: interaction of types I and V collagen

1986 ◽  
Vol 44 ◽  
pp. 210-211
Author(s):  
Arthur J. Wasserman ◽  
Kathy C. Kloos ◽  
David E. Birk
Keyword(s):  
Type I Collagen ◽  
Corneal Stroma ◽  
Minor Component ◽  
Homogeneous Distribution ◽  
Type I ◽  
Type V Collagen ◽  
Fibril Morphology ◽  
Type V ◽  
In Vitro Interaction

Type I collagen is the predominant collagen in the cornea with type V collagen being a quantitatively minor component. However, the content of type V collagen (10-20%) in the cornea is high when compared to other tissues containing predominantly type I collagen. The corneal stroma has a homogeneous distribution of these two collagens, however, immunochemical localization of type V collagen requires the disruption of type I collagen structure. This indicates that these collagens may be arranged as heterpolymeric fibrils. This arrangement may be responsible for the control of fibril diameter necessary for corneal transparency. The purpose of this work is to study the in vitro assembly of collagen type V and to determine whether the interactions of these collagens influence fibril morphology.

scholarly journals Restoration of Osteogenesis by CRISPR/Cas9 Genome Editing of the Mutated COL1A1 Gene in Osteogenesis Imperfecta

2021 ◽  
Vol 10 (14) ◽  
pp. 3141
Author(s):  
Hyerin Jung ◽  
Yeri Alice Rim ◽  
Narae Park ◽  
Yoojun Nam ◽  
Ji Hyeon Ju
Keyword(s):  
Osteogenesis Imperfecta ◽  
Type I Collagen ◽  
Disease Modeling ◽  
Bone Fragility ◽  
Osteogenic Potential ◽  
Type I ◽  
Gene Correction ◽  
Col1a1 Gene ◽  
Collagen Formation

Osteogenesis imperfecta (OI) is a genetic disease characterized by bone fragility and repeated fractures. The bone fragility associated with OI is caused by a defect in collagen formation due to mutation of COL1A1 or COL1A2. Current strategies for treating OI are not curative. In this study, we generated induced pluripotent stem cells (iPSCs) from OI patient-derived blood cells harboring a mutation in the COL1A1 gene. Osteoblast (OB) differentiated from OI-iPSCs showed abnormally decreased levels of type I collagen and osteogenic differentiation ability. Gene correction of the COL1A1 gene using CRISPR/Cas9 recovered the decreased type I collagen expression in OBs differentiated from OI-iPSCs. The osteogenic potential of OI-iPSCs was also recovered by the gene correction. This study suggests a new possibility of treatment and in vitro disease modeling using patient-derived iPSCs and gene editing with CRISPR/Cas9.

scholarly journals Effects of chitosan-collagen dressing on wound healing in vitro and in vivo assays

2021 ◽  
Vol 19 ◽  
pp. 228080002198969
Author(s):  
Min-Xia Zhang ◽  
Wan-Yi Zhao ◽  
Qing-Qing Fang ◽  
Xiao-Feng Wang ◽  
Chun-Ye Chen ◽  
...  
Keyword(s):  
Wound Healing ◽  
Wound Dressing ◽  
Type I Collagen ◽  
Inhibition Zone ◽  
Negative Control ◽  
Type I ◽  
Nih3t3 Cells ◽  
Post Operation

The present study was designed to fabricate a new chitosan-collagen sponge (CCS) for potential wound dressing applications. CCS was fabricated by a 3.0% chitosan mixture with a 1.0% type I collagen (7:3(w/w)) through freeze-drying. Then the dressing was prepared to evaluate its properties through a series of tests. The new-made dressing demonstrated its safety toward NIH3T3 cells. Furthermore, the CCS showed the significant surround inhibition zone than empty controls inoculated by E. coli and S. aureus. Moreover, the moisture rates of CCS were increased more rapidly than the collagen and blank sponge groups. The results revealed that the CCS had the characteristics of nontoxicity, biocompatibility, good antibacterial activity, and water retention. We used a full-thickness excisional wound healing model to evaluate the in vivo efficacy of the new dressing. The results showed remarkable healing at 14th day post-operation compared with injuries treated with collagen only as a negative control in addition to chitosan only. Our results suggest that the chitosan-collagen wound dressing were identified as a new promising candidate for further wound application.

Development of Multilayered Chlorogenate-Peptide Based Biocomposite Scaffolds for Potential Applications in Ligament Tissue Engineering - An In Vitro Study

2017 ◽  
Vol 34 ◽  
pp. 37-56 ◽  
Author(s):  
Harrison T. Pajovich ◽  
Alexandra M. Brown ◽  
Andrew M. Smith ◽  
Sara K. Hurley ◽  
Jessica R. Dorilio ◽  
...  
Keyword(s):  
Tissue Regeneration ◽  
Type I Collagen ◽  
Phase Changes ◽  
Peptide Sequence ◽  
Proteoglycan Synthesis ◽  
Type I ◽  
Average Roughness ◽  
Potential Applications ◽  
Self Assembled

In this work, for the first time, chlorogenic acid, a natural phytochemical, was conjugated to a lactoferrin derived antimicrobial peptide sequence RRWQWRMKKLG to develop a self-assembled template. To mimic the components of extracellular matrix, we then incorporated Type I Collagen, followed by a sequence of aggrecan peptide (ATEGQVRVNSIYQDKVSL) onto the self-assembled templates for potential applications in ligament tissue regeneration. Mechanical properties and surface roughness were studied and the scaffolds displayed a Young’s Modulus of 169 MP and an average roughness of 72 nm respectively. Thermal phase changes were studied by DSC analysis. Results showed short endothermic peaks due to water loss and an exothermic peak due to crystallization of the scaffold caused by rearrangement of the components. Biodegradability studies indicated a percent weight loss of 27.5 % over a period of 37 days. Furthermore, the scaffolds were found to adhere to fibroblasts, the main cellular component of ligament tissue. The scaffolds promoted cell proliferation and displayed actin stress fibers indicative of cell motility and attachment. Collagen and proteoglycan synthesis were also promoted, demonstrating increased expression and deposition of collagen and proteoglycans. Additionally, the scaffolds exhibited antimicrobial activity against Staphylococcus epidermis bacteria, which is beneficial for minimizing biofilm formation if potentially used as implants. Thus, we have developed a novel biocomposite that may open new avenues to enhance ligament tissue regeneration.

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