PubMed · 14950814
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A DELAUNAY, M GUILLAUMIE. 1952. [Collagenases].. https://pubmed.ncbi.nlm.nih.gov/14950814/
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We aimed to investigate the presence and activation status of matrix metalloproteinase (MMP)-2 and MMP-9 in vitreous samples from proliferative diabetic retinopathy (PDR) patients. Paired vitreous and serum samples were obtained from patients undergoing vitrectomy for the treatment of rhegmatogenous retinal detachment (RD) (65 patients) and PDR (67 patients). PDR patients were diagnosed for the presence of hemorrhage and/or patent new vessels. Quantitative assays were performed for vitreous protein content, MMP-2, MMP-9, tissue inhibitor of metalloproteinases-1 (TIMP-1) and hemoglobin. Qualitative evaluation of the MMP-2 and MMP-9 activation status was performed by zymography. Vitreous samples contained proMMP-2 but levels were unselectively related to total protein content. ProMMP-9 and activated MMP-9 levels were significantly increased in PDR patients (p<0.001 for both comparisons). In addition, TIMP-1 levels were significantly increased in PDR patients (p=0.004) and functionally inhibited activation of MMP-9 in vitreous samples. None of the parameters significantly differed between PDR patients with patent new vessels and those with inactive disease. However, activated MMP-9 levels in vitreous samples of PDR patients with hemorrhage (75.7+/-106.3 scanning units per 2 microl) were significantly higher than those in PDR patients without hemorrhage (7.1+/-16.2 scanning units per 2 microl) (p<0.001) and strongly correlated with hemoglobin levels (r=0.7525; p<0.001). Activated MMP-9 was not detected in paired serum samples. We conclude that activated MMP-9 might be involved in hemorrhagic transformation in patients with PDR.
Streptomyces strain 3B constitutively secreted collagenolytic enzymes during the post-exponential growth phase. Purification is described here leading to two collagenases (I and II) with specific activity of 3350 and 3600 U/mg, respectively, the highest activity obtained as yet for any streptomycete collagenase. Analysis of the purified enzymes by the method of zymography revealed that both I and II were homogeneous, with molar mass 116 and 97 kDa, respectively. Both collagenases were identical in their pH (7.5) and temperature optimum (37 degrees C). The inhibition profile of the enzymes by EDTA and 1,10-phenanthroline confirmed these enzymes to be metalloproteinases. By testing the activity with insoluble collagen, acid soluble collagen, gelatin, casein, elastin and Pz-PLGPR it was established that I and II are very specific for insoluble collagen and gelatin, showing a high activity toward acid soluble collagen and Pz-PLGPR. However, collagenases I and II failed to hydrolyze casein and elastin; they belong to true collagenases and resemble the clostridial enzymes.
BACKGROUND: The purpose of this study is to evaluate the effectiveness of a standardized mixture of purified enzymes (Liberase), for the isolation of human ovarian follicles. METHODS: This is an experimental prospective study. Ovarian biopsies were obtained from eight young women undergoing laparoscopy for benign gynaecological disease. Follicles were isolated by Liberase or collagenase enzymatic digestion. Follicle quality was assessed by evaluating their general morphology and viability after fluorescent staining, and the ultrastructure by electron microscopy. RESULTS: The number of fully isolated follicles recovered from the Liberase-treated group was lower than from the collagenase group (156 versus 263) despite equal-sized biopsies being taken. A high proportion of follicles (98.6%, 70/71) were viable after Liberase isolation and most follicles were of good morphology with a complete granulosa cell layer (70.4%, 31/44). Ultrastructural studies indicated that Liberase-isolated follicles showed signs of atresia only occasionally and that the oolemma-follicular cell interface was well preserved. CONCLUSIONS: Liberase treatment allows the isolation of highly viable follicles from human ovarian tissue, with an unaltered morphology and ultrastructure. This purified endotoxin-free enzyme preparation is a promising alternative to impure collagenase preparations for the reproducible isolation of intact primordial and primary follicles for culture and grafting purposes.