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Biomedical subjects

I Daudi

Publications and source records attributed to I Daudi.

4 recordsLinked to original sources
PubMed1991

Proteolysis of gelatin-bound fibronectin by activated leukocytes: a role for leukocyte elastase.

Fragmentation of subendothelial matrix-bound fibronectin by proteases released from stimulated leukocytes has been implicated in lung vascular injury. We studied the degradation of fibronectin bound to denatured collagen by inflammatory polymorphonuclear leukocytes (PMNL). Tissue culture wells coated with denatured collagen (gelatin) were pretreated with 125I rat plasma fibronectin to allow for fibronectin binding prior to the addition of rat inflammatory PMNL. The release of both intact and fragmented fibronectin from the 125I-labelled artificial matrix was quantified following the addition of PMNL stimulated by the phagocytosis of opsonized zymosan as well as leukocyte elastase. Stimulated PMNL released three times more radiolabelled fibronectin from the denatured collagen surface during a 4 h incubation as compared with unstimulated PMNL. This pattern of 125I-fibronectin release could also be elicited by the addition of purified leukocyte elastase alone, in the absence of PMNL. The release of radiolabelled fibronectin by stimulated PMNL was blocked in a dose-dependent manner by the addition of both methoxysuccinyl-alanine-alanine-valine chloromethyl ketone (AAPVCK), a leukocyte elastase specific inhibitor as well as phenylmethylsulfonylfluoride (PMSF), a non-specific serine protease inhibitor. Western blot analysis coupled with autoradiography confirmed the presence of fibronectin fragments in the medium after addition of PMNL or leukocyte elastase. The large molecular weight fragments (60-200 kD) were not labelled, but the smaller molecular weight fragments (less than 45 kD), derived from the artificial matrix, were labelled. Thus, fibronectin complexed with denatured collagen is susceptible to proteolytic degradation by stimulated inflammatory PMNL. Such a process may have a role in the pathogenesis of acute vascular injury following microvascular margination of activated blood leukocytes.

Amino Acid Chloromethyl Ketones↗
PubMed1991

Leukocyte elastase-independent proteolysis of gelatin-bound fibronectin by inflammatory macrophages.

Fragmentation of lung matrix fibronectin by proteases released from activated phagocytic cells has been implicated in lung vascular injury. We examined whether denatured collagen (gelatin)-bound fibronectin can be degraded by peritoneal exudate mononuclear phagocytes harvested from rats 96 h after intraperitoneal casein injection. Microtiter plates were pretreated with gelatin and then supplemented with purified 125I rat plasma fibronectin, which readily bound to the gelatin. Stimulated inflammatory exudate cells were added and proteolysis of the bound fibronectin was studied by the release of [125I]fibronectin fragments into the media. Following 2 h of incubation, peritoneal exudate mononuclear macrophages stimulated with opsonized zymosan released three times more radiolabeled fibronectin into the medium as compared to background controls, and 1.5 times more radiolabeled fibronectin as compared to cells not stimulated with zymosan. Western blot analysis and autoradiography confirmed the presence of fragments of fibronectin in the culture medium. Some of these fragments were clearly derived from the radiolabeled matrix, but others that were not labeled were potentially released directly from the added stimulated macrophages. The release of radiolabeled fibronectin was inhibited by N-p-tosyl-L-lysine chloromethyl ketone (TLCK), a trypsin specific inhibitor, but not by methoxysuccinyl-alanine-alanine-proline-valine-chloromethyl-ketone (AAPVCK), a leukocyte elastase-specific inhibitor. These results suggest that fibronectin bound to denatured collagen is susceptible to leukocyte elastase-independent enzymatic degradation by stimulated inflammatory exudate mononuclear phagocytic cells. Such proteolysis may mimic a pathological process associated with lung vascular injury during the sequestration of activated macrophages in the lung microcirculation and interstitium.

Animals↗
PubMed1989

Fibronectin fragments in lung lymph after thrombin-induced lung vascular injury.

Fibronectin is an adhesive glycoprotein found in plasma and lymph as well as between lung endothelial cells and their collagenous basement membranes. Fibronectin is highly sensitive to proteolytic cleavage. We determined if fragments of fibronectin appear in lung lymph in association with increased lung protein clearance after thrombin-induced intravascular coagulation. Thrombin was infused intravenously, (80 units/kg for 30 minutes) into sheep (n = 8) surgically prepared with chronic lung lymph fistulas. Plasma and lymph fibronectin was assayed by electroimmunoassay. Fibronectin fragments were detected using Western blot analysis. After thrombin infusion, lymph flow increased 650% above baseline within 1-2 hours in association with a 35% decline in lymph-to-plasma total protein concentration ratio. This was followed by a second phase (3.5-6 hours) of normalized lymph-to-plasma ratios coupled with sustained elevation of lymph flow. Lung protein clearance remained elevated (p less than 0.10) for 5.5 hours. Plasma fibronectin levels declined slightly over 1-5 hours (zero time = 597 +/- 64 micrograms/ml; 1.5 hours = 478 +/- 59 micrograms/ml) and then increased significantly (p less than 0.05) over 24-48 hours (760 +/- 85 micrograms/ml). The amount of low molecular weight fibronectin fragments in lung lymph increased over the 1.5-6 hours post-thrombin and then declined over 12-48 hours. Thus after thrombin infusion, fragments of fibronectin were usually detected in increased amounts of lung lymph in association with an elevation of lung protein clearance.

Animals↗