scholarly journals Human neutrophil collagenase cleaves α1-antitrypsin

1990 ◽  
Vol 270 (3) ◽  
pp. 809-814 ◽  
Author(s):  
J Michaelis ◽  
M C Vissers ◽  
C C Winterbourn
Keyword(s):  
Type I Collagen ◽  
Serine Proteinase ◽  
Type I ◽  
Reaction Products ◽  
Western Blots ◽  
Sds Page ◽  
Neutrophil Collagenase ◽  
High Concentrations ◽  
Alpha 1 Antitrypsin ◽  
Neutrophil Gelatinase

Inactivation of the plasma serine-proteinase inhibitor alpha 1-antitrypsin (alpha 1-AT) by neutrophil metalloproteinases has been reported [Vissers, George, Bathurst, Brennan & Winterbourn (1987) Fed. Proc. Fed. Am. Soc. Exp. Biol. 46, 1390a; (1988) J. Clin. Invest. 82, 706-711; Desrochers & Weiss (1988) J. Clin. Invest. 81, 1646-1650]. To identify the enzyme responsible, supernatant from neutrophils stimulated with phorbol 12-myristate 13-acetate was subjected to preparative SDS/PAGE, both with and without activation of latent metalloproteinases with HgCl2. The lanes were subsequently sliced into pieces, the slices incubated with equimolar amounts of type I collagen and alpha 1-AT in the presence of HgCl2, and the reaction products separated by SDS/PAGE. With the latent supernatant, the characteristic collagen-cleavage products and cleaved alpha 1-AT were present in the same slices, corresponding to an Mr of 80,000-85,000. On treatment with HgCl2 both degradative activities underwent the same molecular-mass shift to a position corresponding to Mr 60,000-65,000. Western blots of neutrophil supernatants, using a polyclonal antibody to purified collagenase, showed Mr values of 83,000 for the latent enzyme and 63,000 for the HgCl2-activated enzyme. Neutrophil collagenase was purified to homogeneity and shown also to exist in a second latent form with Mr 70,000. When activated to the Mr-63,000 form by HgCl2 and incubated with equimolar amounts of collagen and alpha 1-AT, collagenase cleaved alpha 1-AT at almost twice the rate at which collagen was cleaved. alpha 1-AT cleavage was inhibited by 1,10-phenanthroline and by high concentrations of collagen. That the purified collagenase did not contain a contaminant proteinase such as stromelysin was indicated by inability of the preparation to cleave casein. Taken together these results lead us to conclude that neutrophil collagenase is capable of degrading alpha 1-AT. Neutrophil gelatinase also cleaved alpha 1-AT, but cleavage was slow when compared with its activity against gelatin.

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.

PLATELETS OF A PATIENT LACKING GLYCOPROTEINS lib AND Ilia AGGREGATE TO HIGH CONCENTRATIONS OF THROMBIN OR COLLAGEN

1987 ◽  
Author(s):  
J L McGregor ◽  
L McGregor ◽  
M Hans ◽  
A Sayegh ◽  
M C Trzeeiak ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Polyclonal Antibodies ◽  
Two Dimensional ◽  
Binding Studies ◽  
Western Blots ◽  
Glycoprotein Complex ◽  
Sds Page ◽  
Bleeding Episodes ◽  
High Concentrations ◽  
Induced Aggregation

The aim of this study was to investigate the platelets of a patient having bleeding episodes that began in infancy. The patient’s platelets in citrated-PRP did not aggregate when stimulated with ADP (5 and 10 uM), collagen (2.5 ug/ml), or sodium arachidonate (1 uM). However, washed patient platelets, in the presence of 2mM calcium, aggregated and secreted when stimulated with high concentrations of thrombin (0.36, 0.72 and lU/ml) or collagen (2, 4, 10 ug/ml). Monoclonal antibodies (Mab) LYP18 (directed against the IIb-IIIa glycoprotein complex) and LYP8 (anti-thrombospondin) inhibited thrombin and collagen induced aggregation of control but not the patient platelets. Patient thrombin -stimulated platelets did not bind 125I-labelled fibrinogen (40 to 320 ug/ml). Moreover, stimulating the washed patient's platelets with ADP (10-100 uM), in the presence of fibrinogen (2mg/ml), did not result in aggregation. Binding studies using Mab 125I-LYP2 (directed against the IIb-IIIa glycoprotein complex) showed the absence of the complex on the patient's platelets. The absence of the IIb-IIIa complex on the patient's platelets was also observed using crossed immunoelectro -phoresis and Mab 125I-LYP2 or 125I-LYP18. Individual glycoproteins (lib or Ilia) were not detected on silver stained two-dimensional (non-reduced/reduced) SDS-PAGE. Moreover, Western blots of |he patients platelets used in combination with anti-PLA or anti-LEK polyclonal antibodies failed to detect the presence of these two glycoproteins. These results indicate that this patient has Glanzmann's thrombasthenia or a variant of this disease. Moreover, this study shows that platelets lacking the IIb-IIIa glycoprotein complex can aggregate in responseto collagen or thrombin in the presence of physiological concentrations of calcium.

Susceptibility of Type I Collagen Containing Mutated α1(1) Chains to Cleavage by Human Neutrophil Collagenase

Matrix ◽  
1993 ◽  
Vol 13 (3) ◽  
pp. 181-186 ◽  
Author(s):  
Karen A. Hasty ◽  
Hong Wu ◽  
Michael Byrne ◽  
Mary B. Goldring ◽  
Jerome M. Seyer ◽  
...  
Keyword(s):  
Human Neutrophil ◽  
Type I Collagen ◽  
Type I ◽  
Neutrophil Collagenase ◽  
Human Neutrophil Collagenase

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 Inhibition of hydroxyapatite formation in collagen gels by chondroitin sulphate

1985 ◽  
Vol 228 (2) ◽  
pp. 463-469 ◽  
Author(s):  
G K Hunter ◽  
B L Allen ◽  
M D Grynpas ◽  
P T Cheng
Keyword(s):  
Type I Collagen ◽  
Chondroitin Sulphate ◽  
Collagen Gel ◽  
Calcium Pyrophosphate ◽  
Molar Ratio ◽  
Crystal Formation ◽  
Type I ◽  
Collagen Gels ◽  
High Concentrations ◽  
Hydroxyapatite Formation

Crystal growth in native collagen gels has been used to determine the role of extracellular matrix macromolecules in biological calcification phenomena. In this system, type I collagen gels containing sodium phosphate and buffered at pH 7.4 are overlayed with a solution containing CaCl2. Crystals form in the collagen gel adjacent to the gel-solution interface. Conditions were determined which permit the growth of crystals of hydroxyapatite [Ca10(PO4)6(OH)2]. At a Ca/P molar ratio of 2:1, the minimum concentrations of calcium and phosphate necessary for precipitation of hydroxyapatite are 10 mM and 5 mM, respectively. Under these conditions, precipitation is initiated at 18-24h, and is maximal between 24h and 6 days. Addition of high concentrations of chondroitin 4-sulphate inhibits the formation of hydroxyapatite in collagen gels; initiation of precipitation is delayed, and the final (equilibrium) amount of precipitation is decreased. Inhibition of hydroxyapatite formation requires concentrations of chondroitin sulphate higher than those required to inhibit calcium pyrophosphate crystal formation.

scholarly journals Human platelet glycoprotein IIIb binds to thrombospondin fragments bearing the C-terminal region, and/or the type I repeats (CSVTCG motif), but not to the N-terminal heparin-binding region

1992 ◽  
Vol 284 (1) ◽  
pp. 231-236 ◽  
Author(s):  
B Catimel ◽  
L Leung ◽  
H el Ghissasi ◽  
N Mercier ◽  
J McGregor
Keyword(s):  
Plasmodium Falciparum ◽  
Sequence Similarity ◽  
Terminal Region ◽  
Platelet Glycoprotein ◽  
Affinity Column ◽  
Type I ◽  
Heparin Binding ◽  
Western Blots ◽  
Reducing Conditions ◽  
Sds Page

Major blood membrane platelet glycoprotein IIIb (GPIIIb), also termed GPIV or CD365, has been identified as a receptor for thrombospondin (TSP), collagen and Plasmodium falciparum-infected erythrocytes. The aim of the present study was to identify region(s) of TSP involved in binding of GPIIIb. Proteolytic fragments of TSP (M(r) 140 kDa, 120-18 kDa and 27 kDa on SDS/PAGE under reducing conditions) were purified by f.p.l.c. and identified by N-terminal gas-phase sequencing, e.l.i.s.a. and Western blots using monoclonal antibodies directed against defined domains of TSP. The 140 kDa and 120-18 kDa fragments (C-terminal region), but not the 27 kDa fragment (N-terminal region), were shown to bind to GPIIIb by using e.l.i.s.a. and affinity-chromatography systems. TSP binding to a GPIIIb-affinity column was Ca(2+)-dependent and reduced by 45% in the presence of EDTA. Moreover, TSP was only partially eluted with EDTA from a Ca(2+)-equilibrated GPIIIb column. A fragment of 68 kDa, obtained by further digestion of the 140 kDa fragment, bound to the GPIIIb-Sepharose affinity column. This fragment, or stalk-like region, bears the TSP type I repeats that show sequence similarity to regions on properdin, Plasmodium falciparum proteins and antistasin. Peptides (CSVTCG or SVTCGGGV) representing these repeats bound isolated GPIIIb in a Ca(2+)-independent way, but did not completely inhibit the GPIIIb and TSP interaction. These studies indicate that GPIIIb binds to the TSP via the C-terminal region and/or the CSVTCG motif, but not to the N-terminal region. Interaction between GPIIIb and the TSP C-terminal region or the CSVTCG motif is respectively Ca(2+)-dependent and -independent.

Extraction and Partial Characterization of Pepsin-Soluble Collagens from the Skin of Amiurus Nebulosus

2011 ◽  
Vol 236-238 ◽  
pp. 2926-2934 ◽  
Author(s):  
Li Li Chen ◽  
Li Zhao ◽  
Hua Liu ◽  
Run Feng Wu
Keyword(s):  
Type I Collagen ◽  
Type I ◽  
Alternative Source ◽  
Soluble Collagen ◽  
Acid Soluble Collagen ◽  
Sds Page ◽  
Skin Collagen ◽  
Maximal Yield ◽  
Pepsin Soluble Collagen

Pepsin-soluble collagen (PSC) was successfully extracted from the skin of Amiurus nebulosus. The skin of Amiurus nebulosus was immersed in 0.3 mol/L acetic acid (1: 20, m: V) for 6 h at 37°C, while pepsin was added, at a level of 5000U/g dosage of defatted skin. The maximal yield of the collagen was 97.44%, which was higher than that of acid-soluble collagen (ASC) at 62.05%. Some properties of pepsin-soluble collagens from the skin of Amiurus nebulosus were characterized. Amino acid composition and SDS-PAGE suggested that the collagen might be classified as type I collagen. Moreover, FTIR investigations showed the existence of helical arrangements in PSC of Amiurus nebulosus skin of collagen. There is a possibility to use Amiurus nebulosus skin collagen as an alternative source of collagen for industrial purposes and subsequently it may maximize the economical value of the fish.

scholarly journals Uremic Toxins and Ciprofloxacin Affect Human Tenocytes In Vitro

2020 ◽  
Vol 21 (12) ◽  
pp. 4241
Author(s):  
Erman Popowski ◽  
Benjamin Kohl ◽  
Tobias Schneider ◽  
Joachim Jankowski ◽  
Gundula Schulze-Tanzil
Keyword(s):  
Type I Collagen ◽  
Uremic Toxins ◽  
Type I ◽  
Mrna Levels ◽  
Protein Levels ◽  
Cell Matrix ◽  
High Concentrations ◽  
High Doses ◽  
Very High

Tendinopathy is a rare but serious complication of quinolone therapy. Risk factors associated with quinolone-induced tendon disorders include chronic kidney disease accompanied by the accumulation of uremic toxins. Hence, the present study explored the effects of the representative uremic toxins phenylacetic acid (PAA) and quinolinic acid (QA), both alone and in combination with ciprofloxacin (CPX), on human tenocytes in vitro. Tenocytes incubated with uremic toxins +/- CPX were investigated for metabolic activity, vitality, expression of the dominant extracellular tendon matrix (ECM) protein type I collagen, cell-matrix receptor β1-integrin, proinflammatory interleukin (IL)-1β, and the ECM-degrading enzyme matrix metalloproteinase (MMP)-1. CPX, when administered at high concentrations (100 mM), suppressed tenocyte metabolism after 8 h exposure and at therapeutic concentrations after 72 h exposure. PAA reduced tenocyte metabolism only after 72 h exposure to very high doses and when combined with CPX. QA, when administered alone, led to scarcely any cytotoxic effect. Combinations of CPX with PAA or QA did not cause greater cytotoxicity than incubation with CPX alone. Gene expression of the pro-inflammatory cytokine IL-1β was reduced by CPX but up-regulated by PAA and QA. Protein levels of type I collagen decreased in response to high CPX doses, whereas PAA and QA did not affect its synthesis significantly. MMP-1 mRNA levels were increased by CPX. This effect became more pronounced in the form of a synergism following exposure to a combination of CPX and PAA. CPX was more tenotoxic than the uremic toxins PAA and QA, which showed only distinct suppressive effects.

Stability of the Magnesium Carbonate Apatite/Anionic Collagen Scaffolds: Effect of the Cross-Link Concentration

2011 ◽  
Vol 493-494 ◽  
pp. 844-848 ◽  
Author(s):  
Marcia S. Sader ◽  
Gutemberg Alves ◽  
Racquel Z. LeGeros ◽  
Gloria Dulce de Almeida Soares
Keyword(s):  
Bone Tissue ◽  
Culture Medium ◽  
Type I Collagen ◽  
Type I ◽  
Carbonate Apatite ◽  
Reaction Products ◽  
Collagen Scaffolds ◽  
Freeze Dried ◽  
The Cross

Natural bone constitutes of an inorganic phase (a biological nanoapatite) and an organic phase (mostly type I collagen). The challenge is to develop a material that can regenerate lost bone tissue with degradation and resorption kinetics compatible with the new bone formation. The aim of this study was to prepare self-organized magnesium and carbonate substituted apatite/collagen scaffolds, cross-linked with glutaraldehyde (GA). Bovine tendon was submitted to alkaline treatment resulting in a negatively charged collagen surface. The scaffolds were prepared by precipitation: simultaneous dropwise addition of solution containing calcium (Ca) and magnesium (Mg) ions and collagen into a buffered solution containing carbonate and phosphate ions in reaction vessel maintained at 37 °C, pH=8. The reaction products were cross-linked with 0.125 and 0.25% (v/v) glutaraldehyde (GA) solution and freeze-dried. The samples were characterized by Fourier-transformed infrared spectroscopy (FTIR). In vitro cytotoxicity (based on three parameters assays) and scaffolds degradation in culture medium and osteoblastic cells culture were performed in the cross-linked materials. No cytotoxic effects were observed. The cross-linked samples with the lower GA concentration showed a lower stability when placed in contact with culture medium. Human osteoblasts attached on the scaffolds surface cross-linked with 0.25% GA, forming a continuous layer after 14 days of incubation. These results showed potential application of the designed scaffolds for bone tissue engineering.

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