Quantitative Zymography: Detection of Picogram Quantities of Gelatinases

Kleiner, David E.; Stetler‐Stevenson, William G.

doi:10.1006/abio.1994.1186

Cited by 785 publications

(444 citation statements)

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“…The level of proteinase activity in this study was measured using gelatin zymography and broad-spectrum MMP substrate hydrolysis. Zymography separates the proforms and active forms of MMPs but the technique will not distinguish between free MMPs and those complexed with their natural inhibitors, the TIMPs (Kleiner and Stetler Stevenson, 1994). Zymography, therefore, yields no absolute values on the levels of active and latent MMPs in vivo, but rather a representation of the levels of the active and latent forms in both free and complexed form in the various tissues studied.…”

Section: Discussionmentioning

confidence: 99%

Tissue levels of active matrix metalloproteinase-2 and -9 in colorectal cancer

Waas

Lomme

DeGroot³

et al. 2002

Br J Cancer

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The bioactivity of matrix metalloproteinases was studied in tissues from colorectal cancer patients by means of both quantitative gelatin zymography and a fluorometric activity assay. Next to paired samples of tumour tissue and distant normal mucosa (n=73), transitional tissue was analysed from a limited (n=33) number of patients. Broad-spectrum matrix metalloproteinase activity and both the active and latent forms of the gelatinases matrix metalloproteinase-2 and -9 were higher in tumour than in normal mucosa. The ratio's between active and latent forms of matrix metalloproteinase-2 and -9 were highest in tumour tissue and normal mucosa, respectively. Matrix metalloproteinase-2 levels, both active and latent forms, correlated inversely with stage of disease, the tumours without synchronous distant metastases containing significantly (P=0.005) more active matrix metalloproteinase-2 than the others. At much lower levels of activity, the same trend was observed in distant normal mucosa. The level of latent form of matrix metalloproteinase-9 in tumour depended on tumour location. Neither the active form of matrix metalloproteinase-9 nor broad-spectrum matrix metalloproteinase activity in tumour tissue did correlate with any of the clinicopathological parameters investigated. The results demonstrate explicit differences between the activity of matrix metalloproteinase-2 and -9, indicating different roles for both gelatinases in tumour progression. Such data are necessary in order to develop rational anti-cancer therapies based on inhibition of specific matrix metalloproteinases.

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Section: Discussionmentioning

confidence: 99%

Tissue levels of active matrix metalloproteinase-2 and -9 in colorectal cancer

Waas

Lomme

DeGroot³

et al. 2002

Br J Cancer

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“…Gelatinases were the first substances to be studied extensively in the brain because of the ease of detection by gelatin-substrate zymography; gelatin, which is embedded in the electrophoretic gel, is dissolved by the gelatinases, leaving a white region in the Coomassie blue-stained gel. The amount of enzyme can be quantified by zymography or enzyme-linked immunosorbent assay (ELISA) (Kleiner and Stetler-Stevenson, 1994).…”

Section: Studies Of Mmps In Brain Cell Culturesmentioning

confidence: 99%

Matrix metalloproteinases in neuroinflammation

Rosenberg

2002

Glia

783

667

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Matrix metalloproteinases (MMPs) are a gene family of neutral proteases that are important in normal development, wound healing, and a wide variety of pathological processes, including the spread of metastatic cancer cells, arthritic destruction of joints, atherosclerosis, and neuroinflammation. In the central nervous system (CNS), MMPs have been shown to degrade components of the basal lamina, leading to disruption of the blood-brain barrier (BBB), and to contribute to the neuroinflammatory response in many neurological diseases. Brain cells express both constitutive and inducible MMPs in response to cellular stress. MMPs are tightly regulated to avoid unwanted proteolysis. Secreted as inactive enzymes, the MMPs require activation by other proteases and free radicals. The MMPs are part of a larger class of metalloproteinases (MPs), which includes the recently discovered ADAMs (a disintegrin and metalloproteinase domain) and ADAMTS (a disintegrin and metalloproteinase thrombospondin) families. MPs have complex roles at the cell surface and within the extracellular matrix. At the cell surface, they act as sheddases, releasing growth factors, death receptors, and death-inducing ligands, making them important in cell survival and death. Tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors that regulate the activity of the MMPs. Synthetic inhibitors have been developed for the treatment of arthritis and cancer. These hydroxymate-based compounds have been shown to reduce injury in experimental allergic encephalomyelitis (EAE), experimental allergic neuritis (EAN), cerebral ischemia, intracerebral hemorrhage, and viral and bacterial infections. MPs have both beneficial and detrimental roles; understanding their expression in various CNS insults will allow for the use of MMP inhibitors in the treatment of neurological disorders. GLIA 39: 279 -291, 2002.

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“…41,42 This assay allows the identification of the pro forms of MMP-2 and MMP-9. 43 Briefly, plasma samples were subjected to electrophoresis on 7% SDS-PAGE co-polymerized with gelatin (1%) as the substrate. After electrophoresis was complete, the gel was incubated for 1 h at room temperature in a 2% Triton X-100 solution and incubated at 37 1C for 16 h in Tris-HCl buffer, pH 7.4, containing 10 mmol l À1 CaCl 2 .…”

Section: Sds-polyacrylamide Gel Electrophoresis (Page) Gelatin Zymogrmentioning

confidence: 99%

A genetic polymorphism of matrix metalloproteinase 9 (MMP-9) affects the changes in circulating MMP-9 levels induced by highly active antiretroviral therapy in HIV patients

et al. 2009

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We examined whether two functional polymorphisms (g.À1562C4T and g.À90(CA)14-24) in the matrix metalloproteinase (MMP)-9 gene or MMP-9 haplotypes affect the circulating levels of pro-MMP-9 and pro-MMP-9/TIMP-1 (tissue inhibitor of metalloproteinase-1) ratios in AIDS patients, and modulate alterations in these biomarkers after highly active antiretroviral therapy (HAART). We studied 82 patients commencing HAART. Higher pro-MMP-9 concentrations and pro-MMP-9/TIMP-1 ratios were found in CT/TT patients compared with CC patients. HAART decreased pro-MMP-9 levels and pro-MMP-9/TIMP-1 ratios in CT/TT patients, it did not modify pro-MMP-9 levels and it increased pro-MMP-9/TIMP-1 ratios in CC patients. The g.À90(CA)14-24 polymorphism, however, produced no significant effects. Moreover, we found no significant differences in HAARTinduced changes in plasma pro-MMP-9, TIMP-1 and pro-MMP-9/TIMP-1 ratios when different MMP-9 haplotypes were compared. These findings suggest that the g.À1562C4T polymorphism affects pro-MMP-9 levels in patients with AIDS and modulates the alterations in pro-MMP-9 levels caused by HAART, thus possibly affecting the risk of cardiovascular complications.

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Quantitative Zymography: Detection of Picogram Quantities of Gelatinases

Cited by 785 publications

References 0 publications

Tissue levels of active matrix metalloproteinase-2 and -9 in colorectal cancer

Tissue levels of active matrix metalloproteinase-2 and -9 in colorectal cancer

Matrix metalloproteinases in neuroinflammation

A genetic polymorphism of matrix metalloproteinase 9 (MMP-9) affects the changes in circulating MMP-9 levels induced by highly active antiretroviral therapy in HIV patients

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