Matrix metalloproteinases (MMPs) are required for re-epithelialization of cutaneous wounds

Abstract Matrix metalloproteinases (MMPs) are neutral zinc-dependent endopeptidases with substrate specificity for most extracellular matrix molecules. MMPs participate in physiological and pathological biological processes. In wound repair, several MMPs are upregulated in migrating epithelium although the biological significance of their presence is unknown. To elucidate the role of MMPs in epithelial migration of cutaneous wounds, a broad-spectrum synthetic MMP inhibitor (GM 6001, 10 mg/ml) was applied topically to partial-thickness wounds in domestic pigs to inhibit endogenous MMPs. The concentration of solubilized GM 6001 in wound fluid obtained from treated porcine wounds was 0.06 mg/ml (150 μM) as determined by high-performance liquid chromatography. Zymographic analysis showed that GM 6001 solubilized at this concentration abolished almost completely all enzymatic activity present in wound fluid. Epithelial coverage, assessed morphometrically, after 66 h of treatment was significantly decreased (P = 0.002) in GM 6001-treated wounds (50.0 ± 29.6%, mean ± SD, n = 6) compared with wounds treated with the vehicle (a hydrogel) alone (87.4 ± 10.6%, n = 6). Topical GM 6001 did not influence the degree of dermal inflammatory cell infiltrate or the in vivo incorporation of 5-bromo-2′-deoxyuridine (as a measure of epithelial proliferation in the wounds) indicating that the reduced re-epithelialization with GM 6001 was not due to interference with the inflammatory response or epithelial proliferation. Our results suggest that MMPs are directly involved and necessary in epithelial resurfacing of moist skin wounds. Received: 19 April 1999 / Received after revision: 16 August 1999 / Accepted: 19 August 1999     

Intracellular protein degradation in Leishmania tropica promastigotes

A labile class of proteins in the range of M_r = 30 000–60 000 which turn over rapidly have been demonstrated in Leishmania tropica promastigotes. The rate of protein degradation is increased by exhaustion of nutrients or inhibition of energy metabolism. Proteolysis is reduced when the parasites are provided with a readily utilizable carbon and energy source such as glucose, glutamate or proline. The breakdown of proteins in L. tropica is dependent on continuous protein synthesis probably for protease synthesis. It is suggested that the relatively high rate of intracellular protein degradation is an auxiliary means for generating carbon and energy sources during nutritional stress.     

Purification and characterization of an aminopeptidase from Plasmodium falciparum

A soluble aminopeptidase from Plasmodium falciparum was purified by high performance liquid chromatography. The enzyme has a molecular weight of 100 000 and pI 6.8. Activity can be monitored conveniently with L-alanine-p-nitroanilide or L-leucine-p-nitroanilide at 405 nm or with L-leucine-7-amido-4-methylcoumarin in a fluorescence assay. The enzyme is inhibited by bestatin and phosphoramidone but not by leupeptin, chymostatin, antipain or pepstatin. pH-rate studies indicated the presence of a group on the free enzyme, pKa = 6.6, which must be in the conjugate base form for activity. The aminopeptidase has an essential sulfhydryl group at the active site which is rapidly modified by Hg2+ or Zn2+, is slowly modified by p-hydroxymercuribenzoate, but is not accessible to iodoacetamide or N-ethylmaleimide. The aminopeptidase is inhibited noncompetitively by chloroquine, mefloquine and quinacrine (Ki = 410, 280 and 20 microM, respectively) but is not inhibited by quinine or primaquine. Hemin does not inhibit. Complexation of hemin with quinacrine prevents inhibition by quinacrine.     

Neutralization of proteases from Bothrops snake venoms by the aqueous extract from Casearia sylvestris (Flacourtiaceae).

Aqueous extract from Casearia sylvestris leaves, a typical plant from Brazilian open pastures, was able to neutralize the hemorrhagic activity caused by Bothrops asper, Bothrops jararacussu, Bothrops moojeni, Bothrops neuwiedi and Bothrops pirajai venoms. It also neutralized two hemorrhagic metalloproteinases from Bothrops asper venom. Proteolytic activity on casein induced by bothropic venoms and by isolated proteases, including Bn2 metalloproteinase from B. neuwiedi venom, was also inhibited by the C. sylvestris extract in different levels. The -fibrinogen chain was partially protected against degradation caused by B. jararacussu venom, when this venom was incubated with C. sylvestris extract. We also observed that this extract partially increased the time of plasma coagulation caused by B. jararacussu, B. moojeni and B. neuwiedi venoms. C. sylvestris extract did not induce proteolysis in any substrate assayed.     

Comparative tissue distribution of the processing enzymes “prohormone thiol protease”, and prohormone convertases 1 and 2, in human PTHrP-producing cell lines and mammalian neuroendocine tissues

Peptide hormones are generated by proteolytic processing of their respective protein precursors by several prohormone processing proteases. The peptide hormone PTHrP is widely expressed in normal and malignant tissues, where proPTHrP undergoes proteolytic processing to generate PTHrp peptides with distinct biological actions. In this study, the tissue distribution of the prohormone processing enzymes PTP, PC1, and PC2 were compared by immunohistochemistry in human PTHrP-producing cancer cell lines, and in mammalian neuroedocrine and other tissues from rat and bovine that contain peptide hormones. PTP, PC1, and PC2 were prominently expressed in PTHrP-expressing human cancer cell lines originating from tumors of the breast, lung prostate, as well as lymphoma. These processing enzymes also showed significant expression in normal mammalian neuroendocrine tissues from bovine and rat, including pituitary, hypothalamus, adrenal medulla, pancreas, and oter tissue. Most neuroendocrine tissues contained prominent levels of at least two of the three processing enzymes examined, and all tissues contained at least one of these three enzymes. Differential expression of processing enzyme proteins was also demonstrated by Western blots. The differential expression of PTP, PC1 and PC2 observed in certain cancer and normal neuroendocrine cell types postulates selective roles for these processing enzymes in different tissues for generating biologically active peptide hormones. Theses results support the importnce of these processing enzymes in their hypothesized roles in prohormone processing.     

Proteolytic activity regarding Sarconesiopsis magellanica (Diptera: Calliphoridae) larval excretions and secretions

Sarconesiopsis magellanica (Diptera: Calliphoridae) is a medically important necrophagous fly which is used for establishing the post-mortem interval. Diptera maggots release proteolytic enzymes contained in larval excretion and secretion (ES) products playing a key role in digestion. Special interest in proteolytic enzymes has also been aroused regarding understanding their role in wound healing since they degrade necrotic tissue during larval therapy. This study was thus aimed at identifying and characterising S. magellanica proteolytic enzyme ES products for the first time. These products were obtained from first-, second- and third-instar larvae taken from a previously-established colony. ES proteins were separated by SDS-PAGE and their proteolytic activity was characterised by zymograms and inhibition assays involving BAPNA (Nα-benzoyl-dl-Arg-p-nitroanilide) and SAPNA substrates, using synthetic inhibitors. The protein profile ranged from ∼69 kDa to ∼23 kDa; several of them coincided with the Lucilia sericata ES protein profile. Serine-protease hydrolysis activity (measured by zymogram) was confirmed when a ∼25 kDa band disappeared upon ES incubation with PMSF inhibitor at pH 7.8. Analysis of larval ES proteolytic activity on BAPNA and SAPNA substrates (determined by using TLCK and TPCK specific inhibitors) suggested a greater amount of trypsin-like protease. These results support the need for further experiments aimed at validating S. magellanica use in larval therapy.     

Protein quality control and degradation in cardiomyocytes

The heart is constantly under stress and cardiomyocytes face enormous challenges to correctly fold nascent polypeptides and keep mature proteins from denaturing. To meet the challenge, cardiomyocytes have developed multi-layered protein quality control (PQC) mechanisms which are carried out primarily by chaperones and ubiquitin–proteasome system mediated proteolysis. Autophagy may also participate in PQC in cardiomyocytes, especially under pathological conditions. Cardiac PQC often becomes inadequate in heart disease, which may play an important role in the development of congestive heart failure.     

Lectin activity of axenic and xenic Giardia lamblia trophozoites

The membrane bound lectin of Giardia lamblia is though to play an important role in the selective colonization of the proximal small intestine by the parasite. The lectin obtained from xenic lysetes of G.lamblia gave a titre of more than 1:2048 in the hemagglutination test with rabbit erythrocytes, whereas the axenic lysates required pre-tretment with proteases (trypsin and pronase) in order to obtain a titre of 1:32. Similar behavior was exhibited by enterocytes of the mouse small intestine when tested for agglutination. The acativation of xenic trophofzoites by the intestinal proteases and the probable role of bacterial enzymes has been discussed.