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Sample records for metalloproteases matricelles mmps

  1. Functional proteomic of Matrix Metallo-proteinases (MMP) dedicated to the detection of active forms of MMP in complex proteome; Proteomique fonctionnelle dediee aux Metalloproteases Matricelles (MMPs): developpement d'une methode extremement sensible permettant la detection des formes actives des MMPs dans des proteomes complexes

    Energy Technology Data Exchange (ETDEWEB)

    David, A

    2007-07-15

    The Matrix Metallo-proteinases (M.M.P.) represent a family of Zinc dependent extracellular proteinases able to cleave collectively all the proteins constituting the extracellular matrix. Currently, 23 human M.M.P. have been identified and are characterized by their sequence in amino-acids and their highly conserved 3 D structure. These enzymes are expressed constitutively during the tissue remodeling process. Their over-expression in various diseases tightly related to inflammatory processes (arthritis, emphysema, cancer) described M.M.P. as choice therapeutic targets. However, as the tissue remodeling implicates modification of cellular contacts, M.M.P. appear currently as proteins involved in signalling pathways. Recent works demonstrating that M.M.P. are able to cleave substrates, which are different than proteins constituting the extracellular matrix, reinforce this vision. In order to identify the individual role and the protein expression level of M.M.P. in pathological context, we developed a new technique of functional proteomics dedicated to the detection of active forms of M.M.P. in tumour samples. This technique relied on the development of a new photoaffinity probe, based on the structure of a potent phosphinic inhibitor of M.M.P., allowing targeting and isolating active forms of M.M.P. by photoaffinity labelling. Furthermore, as the new developed probe incorporated a radioactive element, photoaffinity labelling permitted to radiolabel the targeted proteins. This probe demonstrated in vitro its remarkable ability to covalently modify the h M.M.P.-12, with a singular cross-linking yield, determined at 42 %, displaying an extremely sensitive detection (2.5 fmoles of h M.M.P.-12). When added to complex proteome, the photoaffinity probe presents the same sensibility of detection for the h M.M.P.-12 (5 fmoles); importantly, in this case, h M.M.P.-12 represents only 0.001 % of the totality of the proteins present in the sample. Moreover, this technique allows us to identify active forms of gelatinases (M.M.P.-2 and -9) in an indirect manner by comparing the results of tumour samples treated with the photoaffinity probe and the results of tumour samples analysed in zymography. These studies indicate that the protein expression levels of active forms of M.M.P. are extremely low (fmoles) and do not permit any characterisation of those forms of M.M.P. by mass spectrometry, constituting a genuine challenge which can be approached by the development of new photoaffinity probes incorporating a biotin-group. The example of this class of proteins expressed in very low abundance under active form implicates to grant efforts to develop new strategies allowing the capture of the targeted proteins. (author)

  2. Physiology and pathophysiology of matrix metalloproteases

    NARCIS (Netherlands)

    Klein, T.; Bischoff, R.

    Matrix metalloproteases (MMPs) comprise a family of enzymes that cleave protein substrates based on a conserved mechanism involving activation of an active site-bound water molecule by a Zn(2+) ion. Although the catalytic domain of MMPs is structurally highly similar, there are many differences with

  3. Physiology and pathophysiology of matrix metalloproteases

    NARCIS (Netherlands)

    Klein, T; Bischoff, Rainer

    2010-01-01

    Matrix metalloproteases (MMPs) comprise a family of enzymes that cleave protein substrates based on a conserved mechanism involving activation of an active site-bound water molecule by a Zn(2+) ion. Although the catalytic domain of MMPs is structurally highly similar, there are many differences with

  4. Matrix Metalloproteases in Arterial Hypertension and their Trend after Antihypertensive Treatment

    OpenAIRE

    Hopps, E.; Lo Presti, R.; Caimi, G.

    2017-01-01

    Background/Aims: Arterial hypertension is characterized by vascular remodelling, atherosclerosis and cardiovascular complications. Matrix metalloproteases (MPPs) are endopeptidases produced by all the cells present in the vascular wall and are involved in the regulation of the extracellular matrix protein turnover. MMPs contribute to blood vessel formation, remodelling, angiogenesis; whereas an altered expression or activity of MMPs or their tissue inhibitors (TIMPs) results correlated with t...

  5. Development of an integrated system for activity-based profiling of matrix metallo-proteases

    NARCIS (Netherlands)

    Freije, Jan Robert

    2006-01-01

    Matrix metallo-proteases constitute a family of extracellular zinc-dependent endopeptidases that are involved in degradation of extracellular matrix (ECM) components and other bioactive non-ECM molecules. A plethora of studies have implicated important roles for MMPs in many diseases (including

  6. Matrix Metalloproteases in Arterial Hypertension and their Trend after Antihypertensive Treatment

    Directory of Open Access Journals (Sweden)

    Eugenia Hopps

    2017-06-01

    Full Text Available Background/Aims: Arterial hypertension is characterized by vascular remodelling, atherosclerosis and cardiovascular complications. Matrix metalloproteases (MPPs are endopeptidases produced by all the cells present in the vascular wall and are involved in the regulation of the extracellular matrix protein turnover. MMPs contribute to blood vessel formation, remodelling, angiogenesis; whereas an altered expression or activity of MMPs or their tissue inhibitors (TIMPs results correlated with the development and progression of cardiovascular complications. Methods: We examined the literature data regarding the role of MMPs in human hypertension, including their involvement in vascular remodelling, and the effects of some antihypertensive molecules on these MMP/TIMP profile. Results: The expression and the activity of some MMPs and TIMPs are impaired in human hypertension. An altered MMPs/TIMPs balance plays an important role in the vascular wall rearrangement, in response to hemodynamic changes which may induce myocardial hypertrophy and fibrosis leading to ventricular remodelling. Several studies have examined the effects of some antihypertensive molecules, such as ACE inhibitors, angiotensin receptor blockers, calcium-channel blockers, and aldosterone antagonists, on the MMPs/TIMPs profile by obtaining positive results. Conclusion: Considering the data taken into consideration, the authors believe that in clinical practice a strategic antihypertensive therapy directed to the MMPs profile, may be useful to decrease the risk of cardiovascular complications.

  7. Matrix Metalloproteases in Arterial Hypertension and their Trend after Antihypertensive Treatment.

    Science.gov (United States)

    Hopps, Eugenia; Lo Presti, Rosalia; Caimi, Gregorio

    2017-01-01

    Arterial hypertension is characterized by vascular remodelling, atherosclerosis and cardiovascular complications. Matrix metalloproteases (MPPs) are endopeptidases produced by all the cells present in the vascular wall and are involved in the regulation of the extracellular matrix protein turnover. MMPs contribute to blood vessel formation, remodelling, angiogenesis; whereas an altered expression or activity of MMPs or their tissue inhibitors (TIMPs) results correlated with the development and progression of cardiovascular complications. We examined the literature data regarding the role of MMPs in human hypertension, including their involvement in vascular remodelling, and the effects of some antihypertensive molecules on these MMP/TIMP profile. The expression and the activity of some MMPs and TIMPs are impaired in human hypertension. An altered MMPs/TIMPs balance plays an important role in the vascular wall rearrangement, in response to hemodynamic changes which may induce myocardial hypertrophy and fibrosis leading to ventricular remodelling. Several studies have examined the effects of some antihypertensive molecules, such as ACE inhibitors, angiotensin receptor blockers, calcium-channel blockers, and aldosterone antagonists, on the MMPs/TIMPs profile by obtaining positive results. Considering the data taken into consideration, the authors believe that in clinical practice a strategic antihypertensive therapy directed to the MMPs profile, may be useful to decrease the risk of cardiovascular complications. © 2017 The Author(s). Published by S. Karger AG, Basel.

  8. Therapeutic relevance of inhibitors of MMPs or of caspases in HD-induced injury in the ex vivo human skin model

    NARCIS (Netherlands)

    Mol, M.A.E.; Berg, R.M. van den; Chau, L.F.

    2004-01-01

    In order to prevent microvesication, direct and indirect inhibitors of matrix metalloproteases (MMPs) can be used to fully prevent HD-induced epidermal-dermal separation in organ-cultured human skin pieces. The MMP inhibitors show no effect on the massive epidermal cell damage caused by HD.

  9. COMPARATIVE ANALYSIS AND CLINICAL VALUE OF THE EXPRESSION OF METALLOPROTEASES AND THEIR INHIBITORS BY INTRATUMOR STROMAL MONONUCLEAR INFLAMMATORY CELLS AND THOSE AT THE INVASIVE FRONT OF BREAST CARCINOMAS

    OpenAIRE

    Gonzalez , Luis O; Gonzalez-Reyes , Salome; Marin , Laura; Gonzalez , Lucia; Gonzales , José-Manuel; Lamelas , M Luz; Merino , Antonio; Pidal , Ivan; Alvarez , Elena; Andicoechea , Alejandro; Del Casar , José M; Vizoso , Francisco J

    2010-01-01

    Abstract Aims: Matrix metalloproteases (MMPs) and their inhibitors (TIMPs) play an essential role in the degradation of stromal connective tissue and basement membrane components. The dynamic analysis of these components might help predict tumor agressiveness. Methods and Results: An immunohistochemical study was performed using tissue arrays and specific antibodies against MMPs -1, 2, 7, 9, 11, 13, 14, and TIMPs -1, 2 and 3. More than 5,000 determinations on cancer specimens ...

  10. Correlation of serum and urinary matrix metalloproteases/tissue inhibitors of metalloproteases with subclinical allograft fibrosis in renal transplantation.

    Science.gov (United States)

    Hirt-Minkowski, Patricia; Marti, Hans-Peter; Hönger, Gideon; Grandgirard, Denis; Leib, Stephen L; Amico, Patrizia; Schaub, Stefan

    2014-01-01

    Progressive interstitial fibrosis and tubular atrophy (IF/TA) is a leading cause of chronic allograft dysfunction. Increased extracellular matrix remodeling regulated by matrix metalloproteases (MMPs) and their inhibitors (TIMPs) has been implicated in the development of IF/TA. The aim of this study was to investigate whether urinary/serum MMPs/TIMPs correlate with subclinical IF/TA detected in surveillance biopsies within the first 6months post-transplant. We measured eight different MMPs/TIMPs simultaneously in urine and serum samples from patients classified as normal histology (n=15), IF/TA 1 (n=15) and IF/TA 2-3 (n=10). There was no difference in urinary MMPs/TIMPs among the three groups, and only 1/8 serum MMPs/TIMPs (i.e. MMP-1) was significantly elevated in biopsies with IF/TA 2-3 (p=0.01). In addition, urinary/serum MMPs/TIMPs were not different between surveillance biopsies demonstrating an early development of IF/TA (i.e. delta IF/TA≥1 compared to a previous biopsy obtained three months before; n=11) and stable grade of IF/TA (i.e. delta IF/TA=0; n=20). Next, we investigated whether urinary/serum MMP/TIMP levels are elevated during acute subclinical tubulitis in surveillance biopsies obtained within the first 6months post-transplant (n=25). Compared to biopsies with normal histology, serum MMPs/TIMPs were not different; however, all urinary MMP/TIMP levels were numerically higher during subclinical tubulitis (MMP-1, MMP-7, TIMP-1 with p≤0.04). We conclude that urinary/serum MMPs/TIMPs do hardly correlate with existing or early developing IF/TA in surveillance biopsies obtained within the first 6months post-transplant. This could be explained by the dynamic process of extracellular matrix remodeling, which seems to be active during acute tubulo-interstitial injury/inflammation, but not in quiescent IF/TA. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Metalloproteases and tendinopathy

    Science.gov (United States)

    Del Buono, Angelo; Oliva, Francesco; Osti, Leonardo; Maffulli, Nicola

    2013-01-01

    Summary Matrix metalloproteinases (MMP) are involved in the development of tendinopathy. These potent enzymes completely degrade all components of the connective tissue, modify the extracellular matrix (ECM), and mediate the development of painful tendinopathy. To control the local activity of activated proteinases, the same cells produce tissue inhibitors of metalloproteinases (TIMP). These latter bind to the enzyme and prevent degradation. The balance between the activities of MMPs and TIMPs regulates tendon remodeling, whereas an imbalance produces a collagen dis-regulation and disturbances in tendons. ADAMs (a disintegrin and metalloproteinase) are cell membrane-linked enzymes with proteolytic and cell signaling functions. ADAMTSs (ADAM with thrombospondin motifs) are secreted into the circulation and constitute a heterogenous family of proteases with both anabolic and catabolic functions. Further studies are needed to better define the mechanism of action, and whether these new strategies are safe and effective in larger models. PMID:23885345

  12. Developpement of a photoaffinity probe for the sensitive detection of matrix metallo-protease active forms from complex biological systems

    International Nuclear Information System (INIS)

    Nury, Catherine

    2012-01-01

    A new activity-based probe able to covalently modify the active site of proteases belonging to the matrix metallo-protease family (MMPs) has been developed in this thesis project. The probe was shown to behave as potent inhibitor of several MMPs, with nanomolar Ki values. This probe was also able to modify specifically only the free active site of MMPs, with particular high yields of cross-linking varying from 50 % to 11 %, depending of the MMPs tested. Using radioactivity as means of detection, this probe was able to detect active form of MMPs with a threshold of 1 femto-mole. Applied to the study of bronchoalveolar fluids (BAL) from mice exposed to nanoparticles by a lung aspiration protocol, this probe revealed the presence of the catalytic domain of MMP-12 under its active form, but not in control animals. When used to detect active form of MMPs from extracts obtained from human arteries of patient suffering from atherosclerosis, the probe was not able to detect such MMP active forms. Despite this negative result, the detection of active form of MMP in pathological fluid like BAL has never been reported before this work. Having validated this novel MMP activity-based probe, it will be possible to use it now for detecting MMPs from other pathological fluids or tissues extracts in which MMPs can be good markers of the pathology. (author) [fr

  13. Multifaceted role of matrix metalloproteinases (MMPs)

    OpenAIRE

    Singh, Divya; Srivastava, Sanjeev K.; Chaudhuri, Tapas K.; Upadhyay, Ghanshyam

    2015-01-01

    Matrix metalloproteinases (MMPs), a large family of calcium-dependent zinc-containing endopeptidases, are involved in the tissue remodeling and degradation of the extracellular matrix. MMPs are widely distributed in the brain and regulate various processes including microglial activation, inflammation, dopaminergic apoptosis, blood-brain barrier disruption, and modulation of ?-synuclein pathology. High expression of MMPs is well documented in various neurological disorders including Parkinson...

  14. Matrix metalloproteinases (MMPs) safeguard osteoblasts from apoptosis during transdifferentiation into osteocytes

    DEFF Research Database (Denmark)

    Karsdal, M A; Levin Andersen, Thomas; Bonewald, L

    2004-01-01

    of osteoblasts forced to transdifferentiate into osteocytes in 3D type I collagen gels were inhibited by more than 50% when exposed to 10 microM GM6001 and to Tissue Inhibitor of Metalloproteinase-2 (TIMP-2), a natural MT1-MMP inhibitor. This shows the importance of MMPs in safeguarding osteoblasts from......Osteoblasts undergo apoptosis or differentiate into either osteocytes or bone-lining cells after termination of bone matrix synthesis. In this study, we investigated the role of matrix metalloproteinases (MMPs) in differentiation of osteoblasts, bone formation, transdifferentiation into osteocytes......, and osteocyte apoptosis. This was accomplished by using calvarial sections from the MT1-MMP-deficient mouse and by culture of the mouse osteoblast cell line MC3T3-E1 and primary mouse calvarial osteoblasts. We found that a synthetic matrix metalloprotease inhibitor, GM6001, strongly inhibited bone formation...

  15. Helicobacter pylori and gastritis: the role of extracellular matrix metalloproteases, their inhibitors, and the disintegrins and metalloproteases--a systematic literature review.

    Science.gov (United States)

    Sampieri, Clara L

    2013-10-01

    Helicobacter pylori (H. pylori) is the etiologic agent of gastritis; it has been estimated that 50 % of the world's population could be infected by this bacteria. Gastritis may progress to chronic atrophic gastritis, a condition associated with the development of gastric cancer (GC). Several matrix metalloproteases (MMP) and tissue inhibitors of MMPs (TIMP) as well as disintegrins and metalloproteases (ADAM) have been reported as being involved in gastritis. Among other processes, these protein families participate in remodeling the extracellular matrix, cell signaling, immune response, angiogenesis, inflammation and epithelial mesenchymal transition. This systematic review analyzes the scientific evidence surrounding the relationship between members of the MMP, TIMP and ADAM families and infection by H. pylori in gastritis, considering both in vitro and in vivo studies. Given the potential clinical value of certain members of the MMP, TIMP and ADAM families as molecular markers in gastritis and the association of gastritis with GC, the need for further study is highlighted.

  16. Novel 19F Activatable Probe for the Detection of Matrix Metalloprotease-2 Activity by MRI/MRS

    OpenAIRE

    Yue, Xuyi; Wang, Zhe; Zhu, Lei; Wang, Yu; Qian, Chunqi; Ma, Ying; Kiesewetter, Dale O.; Niu, Gang; Chen, Xiaoyuan

    2014-01-01

    Matrix metalloproteases (MMPs) have been found to be highly expressed in a variety of malignant tumor tissues. Noninvasive visualization of MMP activity may play an important role in the diagnosis of MMP associated diseases. Here we report the design and synthesis of a set of fluorine-19 dendron-based magnetic resonance imaging (MRI) probes for real-time imaging of MMP-2 activity. The probes have the following features: (a) symmetrical fluorine atoms; (b) the number of fluorine atoms can be i...

  17. Immunohistochemical expression of matrix metalloprotease-2 and matrix metalloprotease-9 in the disks of patients with temporomandibular joint dysfunction.

    Science.gov (United States)

    Almeida, Luis Eduardo; Caporal, Karina; Ambros, Viviane; Azevedo, Marina; Noronha, Lucia; Leonardi, Rosy; Trevilatto, Paula C

    2015-01-01

    Matrix metalloproteases (MMPs) are tissue-remodeling enzymes that function during the remodeling process, such as in immune-inflammatory diseases. Metalloprotease-2 (MMP-2) and metalloprotease-9 (MMP-9) are gelatinases that degrade several types of extracellular matrix collagen. It is hypothesized that in temporomandibular joint (TMJ) dysfunction, MMP-2 and MMP-9 expression levels may be elevated. Therefore, the objective of this study is to determine the association of MMP-2 and MMP-9 expression with temporomandibular joint dysfunction using an immunohistochemical approach to evaluate the joint disk. A total of 45 human temporomandibular joint samples were collected, with 36 samples in the test group (patients with anterior disk displacement with reduction (n = 29) and without reduction (n = 7)) and nine samples in the control group. The immunostaining of the TMJ disks was statistically compared between the groups (P < 0.05). There was a statistically significant difference for the area of MMP-2 immunostaining between the control group and the displacement disks with reduction group (ADDwR) (P = 0.048) and between the groups with disk displacement and without reduction (ADDwoR) (P = 0.029). The expression of MMP-2 was significantly elevated in the ADDwoR group. No statistically significant difference was found between the variable area of MMP-9 expression in the disk with and without disk displacement, as determined by immunohistochemical analysis. However, there was an elevation of MMP-2 expression in the disks of patients with displacement and without reduction (more severe alteration). © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. Targeting a single function of the multifunctional matrix metalloprotease MT1-MMP

    DEFF Research Database (Denmark)

    Ingvarsen, Signe; Porse, Astrid; Erpicum, Charlotte

    2013-01-01

    the enzyme ability to activate proMMP-2 without interfering with the collagenolytic function or the general proteolytic activity of MT1-MMP. Using this antibody, we have shown that the MT1-MMP-catalyzed activation of proMMP-2 is involved in the outgrowth of cultured lymphatic endothelial cells in a collagen......The group of matrix metalloproteases (MMPs) is responsible for multiple processes of extracellular matrix remodeling in the healthy body but also for matrix and tissue destruction during cancer invasion and metastasis. The understanding of the contributions from each individual MMP, both in healthy...... and pathological events, has been complicated by the lack of specific inhibitors and the fact that some of the potent MMPs are multifunctional enzymes. These factors have also hampered the setup of therapeutic strategies targeting MMP activity. A tempting target is the membrane-associated MT1-MMP, which has well...

  19. Membrane-type matrix metalloproteases as diverse effectors of cancer progression.

    Science.gov (United States)

    Turunen, S Pauliina; Tatti-Bugaeva, Olga; Lehti, Kaisa

    2017-11-01

    Membrane-type matrix metalloproteases (MT-MMP) are pivotal regulators of cell invasion, growth and survival. Tethered to the cell membranes by a transmembrane domain or GPI-anchor, the six MT-MMPs can exert these functions via cell surface-associated extracellular matrix degradation or proteolytic protein processing, including shedding or release of signaling receptors, adhesion molecules, growth factors and other pericellular proteins. By interactions with signaling scaffold or cytoskeleton, the C-terminal cytoplasmic tail of the transmembrane MT-MMPs further extends their functionality to signaling or structural relay. MT-MMPs are differentially expressed in cancer. The most extensively studied MMP14/MT1-MMP is induced in various cancers along malignant transformation via pathways activated by mutations in tumor suppressors or proto-oncogenes and changes in tumor microenvironment including cellular heterogeneity, extracellular matrix composition, tissue oxygenation, and inflammation. Classically such induction involves transcriptional programs related to epithelial-to-mesenchymal transition. Besides inhibition by endogenous tissue inhibitors, MT-MMP activities are spatially and timely regulated at multiple levels by microtubular vesicular trafficking, dimerization/oligomerization, other interactions and localization in the actin-based invadosomes, in both tumor and the stroma. The functions of MT-MMPs are multifaceted within reciprocal cellular responses in the evolving tumor microenvironment, which poses the importance of these proteases beyond the central function as matrix scissors, and necessitates us to rethink MT-MMPs as dynamic signaling proteases of cancer. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. The timeline of metalloprotease events during oligofructose induced equine laminitis development.

    Science.gov (United States)

    Visser, M B; Pollitt, C C

    2012-01-01

    The role of matrix metalloproteases (MMPs) and the timeline of proteolysis during laminitis development are incompletely understood. To determine the temporal progression of selected MMPs and protease regulators during laminitis development. Five clinically normal Standardbred horses received, via nasogastric intubation, an oligofructose (OF) bolus (10 g/kg bwt). Laminitis induction proceeded for 48 h followed by euthanasia. Lamellar biopsies were obtained prior to dosing and at intervals during the treatment period for analysis (12, 18, 24, 30 and 36 h and at 48 h following euthanasia). Tissue samples were analysed by real-time PCR, zymography and western blotting. Activation of proMMP-2 occurs either simultaneously or at least 12 h following lamellar basement membrane (BM) damage, while no activation of proMMP-9 is seen during OF laminitis induction. Aggrecanase gene expression increased initially at 12-18 h post OF dosing, similar to BM changes. Gene expression of TIMP-2, a MMP regulator, decreases during laminitis development. The MMP-2/MT1-MMP complex may not play a major role in initiating lamellar BM damage. Aggrecanase and TIMP-2 gene expression appear related to BM lamellar changes. MMPs, historically thought to cause laminitis, do not appear to play an initiating role in the lamellar lesion. Other host derived proteases and degradation of alternative lamellar matrix components need to be considered. © 2011 EVJ Ltd.

  1. Human ADAM 12 (meltrin alpha) is an active metalloprotease

    DEFF Research Database (Denmark)

    Loechel, F; Gilpin, B J; Engvall, E

    1998-01-01

    The ADAMs (a disintegrin and metalloprotease) are a family of multidomain proteins with structural homology to snake venom metalloproteases. We recently described the cloning and sequencing of human ADAM 12 (meltrin alpha). In this report we provide evidence that the metalloprotease domain of ADAM...

  2. Partial purification and characterization of metalloprotease of ...

    African Journals Online (AJOL)

    USER

    2013-07-31

    Jul 31, 2013 ... We partially purified extracellular metalloprotease of a halotolerant alkaliphilic bacterium isolated from coastal sediment of Goa, India, which was identified as Bacillus cereus strain CS1 based on biochemical characteristics and 16S rDNA sequence. Zymogram of partially purified enzyme clearly revealed ...

  3. Effects of exercise training on the matrix metalloprotease response to acute exercise.

    Science.gov (United States)

    Urso, Maria L; Pierce, Joseph R; Alemany, Joseph A; Harman, Everett A; Nindl, Bradley C

    2009-07-01

    Matrix metalloproteases (MMPs) in the circulation are thought to modulate the activation of growth factors, cytokines, and angiogenesis, facilitating physiological adaptations to exercise training. The purpose of this work was to characterize serum MMP-1, MMP-2, MMP-3, and MMP-9 concentrations pre- and post-eight weeks of exercise training. We tested the hypothesis that exercise training would influence serum MMP concentrations in response to an acute resistance exercise test (ARET). Participants were randomized into an 8-week training program (5 days per week) that emphasized callisthenic (CT, N = 8) or resistance (RT, N = 8) exercise. Serum MMP concentrations (MMP-1, -2, -3, -9) were assessed in men (N = 16) in response to an acute bout of high-intensity resistance exercise (six sets of 10-RM squats with 2-min inter-set rest periods) both before and after 8 weeks of training. Training resulted in a temporal shift in the peak MMP-1 concentration from post-ARET to mid-ARET in both groups. Post-training, MMP-9 concentrations were increased immediately after the ARET in the CT group as compared to pre-training ARET concentrations. RT did not alter MMP-3 and -9 concentrations. These data suggest that the mode of exercise training influences the MMP response to an acute bout of exercise, revealing a possible role of MMPs in initiating training-specific adaptations.

  4. Commissioning of the new AGS MMPS transformers

    International Nuclear Information System (INIS)

    Bajon, E.; Badea, V. S.; Bannon, M.; Bonati, R.; Marneris, I. M.; Porqueddu, R.; Roser, T.; Sandberg, J.; Savatteri, S.

    2009-01-01

    The Brookhaven AGS Main Magnet Power Supply (MMPS) is a thyristor control supply rated at 5500 Amps. +/-9000 Volts. The peak magnet power is 50 MWatts. The power supply is fed from a motor/generator manufactured by Siemens. During rectify and invert operation the P Bank power supplies are used. During the flattops the F Bank power supplies are used. The P Bank power supplies are fed from two 23 MVA transformers and the F Bank power supplies are fed from two 5.3 MYA transformers. The fundamental frequency of the F Bank power supplies is 1440 Hz, however the fundamental frequency of the P banks was 720 Hz. It was very important to reduce the ripple during rectify to improve polarized proton operations. For this reason and also because the original transformers were 45 years old we replaced these transformers with new ones and we made the fundamental frequency of both P and F banks 1440 Hz. This paper will highlight the major hurdles that were involved during the installation of the new transformers. It will present waveforms while running at different power levels up to 6MW full load. It will show the transition from the F-Bank power supplies to the P-Banks and also show the improvements in ripple made on the P-Bank power supplies

  5. Commissioning of the new AGS MMPS transformers

    Energy Technology Data Exchange (ETDEWEB)

    Bajon,E.; Badea, V. S.; Bannon, M.; Bonati, R.; Marneris, I. M.; Porqueddu, r.; Roser, T.; Sandberg, J.; Savatteri, S.

    2009-05-04

    The Brookhaven AGS Main Magnet Power Supply (MMPS) is a thyristor control supply rated at 5500 Amps. +/-9000 Volts. The peak magnet power is 50 MWatts. The power supply is fed from a motor/generator manufactured by Siemens. During rectify and invert operation the P Bank power supplies are used. During the flattops the F Bank power supplies are used. The P Bank power supplies are fed from two 23 MVA transformers and the F Bank power supplies are fed from two 5.3 MYA transformers. The fundamental frequency of the F Bank power supplies is 1440 Hz, however the fundamental frequency of the P banks was 720 Hz. It was very important to reduce the ripple during rectify to improve polarized proton operations. For this reason and also because the original transformers were 45 years old we replaced these transformers with new ones and we made the fundamental frequency of both P and F banks 1440 Hz. This paper will highlight the major hurdles that were involved during the installation of the new transformers. It will present waveforms while running at different power levels up to 6MW full load. It will show the transition from the F-Bank power supplies to the P-Banks and also show the improvements in ripple made on the P-Bank power supplies.

  6. Intracellular Cleavage of the Cx43 C-Terminal Domain by Matrix-Metalloproteases: A Novel Contributor to Inflammation?

    Directory of Open Access Journals (Sweden)

    Marijke De Bock

    2015-01-01

    Full Text Available The coordination of tissue function is mediated by gap junctions (GJs that enable direct cell-cell transfer of metabolic and electric signals. GJs are formed by connexin (Cx proteins of which Cx43 is most widespread in the human body. Beyond its role in direct intercellular communication, Cx43 also forms nonjunctional hemichannels (HCs in the plasma membrane that mediate the release of paracrine signaling molecules in the extracellular environment. Both HC and GJ channel function are regulated by protein-protein interactions and posttranslational modifications that predominantly take place in the C-terminal domain of Cx43. Matrix metalloproteases (MMPs are a major group of zinc-dependent proteases, known to regulate not only extracellular matrix remodeling, but also processing of intracellular proteins. Together with Cx43 channels, both GJs and HCs, MMPs contribute to acute inflammation and a small number of studies reports on an MMP-Cx43 link. Here, we build further on these reports and present a novel hypothesis that describes proteolytic cleavage of the Cx43 C-terminal domain by MMPs and explores possibilities of how such cleavage events may affect Cx43 channel function. Finally, we set out how aberrant channel function resulting from cleavage can contribute to the acute inflammatory response during tissue injury.

  7. Identification of metalloprotease gene families in sugarcane

    Directory of Open Access Journals (Sweden)

    O.H.P. Ramos

    2001-12-01

    Full Text Available Metalloproteases play a key role in many physiological processes in mammals such as cell migration, tissue remodeling and processing of growth factors. They have also been identified as important factors in the patho-physiology of a number of human diseases, including cancer and hypertension. Many bacterial pathogens rely on proteases in order to infect the host. Several classes of metalloproteases have been described in humans, bacteria, snake venoms and insects. However, the presence and characterization of plant metalloproteases have rarely been described in the literature. In our research, we searched the sugarcane expressed sequence tag (SUCEST DNA library in order to identify, by homology with sequences deposited in other databases, metalloprotease gene families expressed under different conditions. Protein sequences from Arabidopsis thaliana and Glycine max were used to search the SUCEST data bank. Conserved regions corresponding to different metalloprotease domains and sequence motifs were identified in the reads to characterize each group of enzymes. At least four classes of sugarcane metalloproteases have been identified, i.e. matrix metalloproteases, zincins, inverzincins, and ATP-dependent metalloproteases. Each enzyme class was analyzed for its expression in different conditions and tissues.Metaloproteases exercem papéis importantes em muitos processos fisiológicos em mamíferos tais como migração celular, remodelamento tecidual e processamento de fatores de crescimento. Estas enzimas estão envolvidas também na pato-fisiologia de um grande número de doenças humanas como hipertensão e câncer. Muitas bactérias patogênicas dependem de proteases para infectar o hospedeiro. Diversas classes de metaloproteases foram descritas em seres humanos, bactérias, venenos de serpentes e insetos. No entanto, a presença e a caracterização de metaloproteases em plantas estão pouco descritas na literatura. Neste trabalho, foi

  8. Metalloprotease Dependent Release of Placenta Derived Fractalkine

    Directory of Open Access Journals (Sweden)

    Monika Siwetz

    2014-01-01

    Full Text Available The chemokine fractalkine is considered as unique since it exists both as membrane-bound adhesion molecule and as shed soluble chemoattractant. Here the hypothesis was tested whether placental fractalkine can be shed and released into the maternal circulation. Immunohistochemical staining of human first trimester and term placenta sections localized fractalkine at the apical microvillous plasma membrane of the syncytiotrophoblast. Gene expression analysis revealed abundant upregulation in placental fractalkine at term, compared to first trimester. Fractalkine expression and release were detected in the trophoblast cell line BeWo, in primary term trophoblasts and placental explants. Incubation of BeWo cells and placental explants with metalloprotease inhibitor Batimastat inhibited the release of soluble fractalkine and at the same time increased the membrane-bound form. These results demonstrate that human placenta is a source for fractalkine, which is expressed in the syncytiotrophoblast and can be released into the maternal circulation by constitutive metalloprotease dependent shedding. Increased expression and release of placental fractalkine may contribute to low grade systemic inflammatory responses in third trimester of normal pregnancy. Aberrant placental metalloprotease activity may not only affect the release of placenta derived fractalkine but may at the same time affect the abundance of the membrane-bound form of the chemokine.

  9. Polymorphism +17 C/G in Matrix Metalloprotease MMP8 decreases lung cancer risk

    Directory of Open Access Journals (Sweden)

    Tardón Adonina

    2008-12-01

    Full Text Available Abstract Background Matrix metalloproteases (MMPs constitute a family of enzymes capable of degrading different components of the extracellular matrix and are implicated in the invasion of tumor cells through the basement membrane. Polymorphisms in MMP genes may result in changes in the expression of MMPs being associated with the development and progression of cancer. We have investigated the association between three polymorphisms (-1607 1G/2G, +17 C/G and -77 A/G in the human collagenases MMP1, MMP8 and MMP13 and the risk of development or progression of lung cancer. Methods A hospital-based case-control study was designed including 501 lung cancer patients and 510 controls matched. Genotypes were determined by PCR-RFLP. Results were analyzed using unconditional logistic regression, Cox's proportional hazard regression, and the Kaplan-Meier method. Results The MMP1 and MMP13 promoter polymorphisms were not associated with lung cancer risk, while the C/G polymorphism in MMP8 was associated with a statistically significant decreased risk of developing lung cancer (ORadj = 0.65; 95%CI = 0.45–0.93. The Kaplan-Meier analysis showed that the polymorphisms in MMP1, MMP8 and MMP13 not seem to modify the overall survival. Multivariate analysis revealed that MMP1, MMP8 and MMP13 polymorphisms are not independent prognostic factors for overall survival. Conclusion This study suggests that the polymorphism in MMP8 is associated with a decreased lung cancer risk, which can be used as a prognostic marker in lung cancer.

  10. Human ADAM 12 (meltrin alpha) is an active metalloprotease

    DEFF Research Database (Denmark)

    Loechel, F; Gilpin, B J; Engvall, E

    1998-01-01

    The ADAMs (a disintegrin and metalloprotease) are a family of multidomain proteins with structural homology to snake venom metalloproteases. We recently described the cloning and sequencing of human ADAM 12 (meltrin alpha). In this report we provide evidence that the metalloprotease domain of ADAM...... 12 is catalytically active. We used the trapping mechanism of alpha2-macroglobulin to assay for protease activity of wild-type and mutant ADAM 12 proteins produced in a COS cell transfection system. We found that ADAM 12 is synthesized as a zymogen, with the prodomain maintaining the metalloprotease...... in a latent form, probably by means of a cysteine switch. The zymogen could be activated chemically by alkylation with N-ethylmaleimide. Cleavage of the prodomain at a site for a furin-like endopeptidase resulted in an ADAM 12 protein with proteolytic activity. The protease activity was sensitive...

  11. MicroRNAs in the Regulation of MMPs and Metastasis.

    Science.gov (United States)

    Abba, Mohammed; Patil, Nitin; Allgayer, Heike

    2014-03-25

    MicroRNAs are integral molecules in the regulation of numerous physiological cellular processes including cellular differentiation, proliferation, metabolism and apoptosis. Their function transcends normal physiology and extends into several pathological entities including cancer. The matrix metalloproteinases play pivotal roles, not only in tissue remodeling, but also in several physiological and pathological processes, including those supporting cancer progression. Additionally, the contribution of active MMPs in metastatic spread and the establishment of secondary metastasis, via the targeting of several substrates, are also well established. This review focuses on the important miRNAs that have been found to impact cancer progression and metastasis through direct and indirect interactions with the matrix metalloproteinases.

  12. The mitochondrial inner membrane AAA metalloprotease family in metazoans.

    Science.gov (United States)

    Juhola, M K; Shah, Z H; Grivell, L A; Jacobs, H T

    2000-09-15

    Three metalloproteases belonging to the AAA superfamily (Yme1p, Afg3p and Rca1p) are involved in protein turnover and respiratory chain complex assembly in the yeast inner mitochondrial membrane. Analysis of the completed genome sequences of Caenorhabditis elegans and Drosophila melanogaster indicates that this gene family typically comprises 3-4 members in metazoans. Phylogenetic analysis reveals three main branches represented, respectively, by Saccharomyces cerevisiae YME1, human SPG7 (paraplegin) and S. cerevisiae AFG3 and RCA1. mt-AAA metalloproteases are weak candidates for several previously studied Drosophila mutants. A full elucidation of the cellular and physiological roles of mt-AAA metalloproteases in metazoans will require the creation of targeted mutations.

  13. SIMULATIONS OF THE AGS MMPS STORING ENERGY IN CAPACITOR BANKS

    International Nuclear Information System (INIS)

    MARNERIS, I.; BADEA, V.S.; BONATI, R.; ROSER, T.; SANDBERG, J.

    2007-01-01

    The Brookhaven AGS Main Magnet Power Supply (MMPS) is a thyristor control supply rated at 5500 Amps, +/-9000 Volts. The peak magnet power is 50 MWatts. The power supply is fed from a motor/generator manufactured by Siemens. The generator is 3 phase 7500 Volts rated at 50 MVA. The peak power requirements come from the stored energy in the rotor of the motor/generator. The motor generator is about 45 years old, made by Siemens and it is not clear if companies will be manufacturing similar machines in the future. We are therefore investigating different ways of storing energy for future AGS MMPS operations. This paper will present simulations of a power supply where energy is stored in capacitor banks. Two dc to dc converters will be presented along with the control system of the power section. The switching elements will be IGCT's made by ABB. The simulation program used is called PSIM version 6.1. The average power from the local power authority into the power supply will be kept constant during the pulsing of the magnets at +/-50 MW. The reactive power will also be kept constant below 1.5 MVAR. Waveforms will be presented

  14. SIMULATIONS OF THE AGS MMPS STORING ENERGY IN CAPACITOR BANKS

    Energy Technology Data Exchange (ETDEWEB)

    MARNERIS,I.; BADEA, V.S.; BONATI, R.; ROSER, T.; SANDBERG, J.

    2007-06-25

    The Brookhaven AGS Main Magnet Power Supply (MMPS) is a thyristor control supply rated at 5500 Amps, +/-9000 Volts. The peak magnet power is 50 MWatts. The power supply is fed from a motor/generator manufactured by Siemens. The generator is 3 phase 7500 Volts rated at 50 MVA. The peak power requirements come from the stored energy in the rotor of the motor/generator. The motor generator is about 45 years old, made by Siemens and it is not clear if companies will be manufacturing similar machines in the future. We are therefore investigating different ways of storing energy for future AGS MMPS operations. This paper will present simulations of a power supply where energy is stored in capacitor banks. Two dc to dc converters will be presented along with the control system of the power section. The switching elements will be IGCT's made by ABB. The simulation program used is called PSIM version 6.1. The average power from the local power authority into the power supply will be kept constant during the pulsing of the magnets at +/-50 MW. The reactive power will also be kept constant below 1.5 MVAR. Waveforms will be presented.

  15. Reduction of the disintegrin and metalloprotease ADAM12 in preeclampsia

    DEFF Research Database (Denmark)

    Laigaard, Jennie; Sørensen, Tina; Placing, Sophie

    2005-01-01

    OBJECTIVES: The secreted form of ADAM12 is a metalloprotease that may be involved in placental and fetal growth. We examined whether the concentration of ADAM12 in first-trimester maternal serum could be used as a marker for preeclampsia. METHODS: We developed a semiautomated, time-resolved, immu...

  16. MicroRNAs in the Regulation of MMPs and Metastasis

    Directory of Open Access Journals (Sweden)

    Mohammed Abba

    2014-03-01

    Full Text Available MicroRNAs are integral molecules in the regulation of numerous physiological cellular processes including cellular differentiation, proliferation, metabolism and apoptosis. Their function transcends normal physiology and extends into several pathological entities including cancer. The matrix metalloproteinases play pivotal roles, not only in tissue remodeling, but also in several physiological and pathological processes, including those supporting cancer progression. Additionally, the contribution of active MMPs in metastatic spread and the establishment of secondary metastasis, via the targeting of several substrates, are also well established. This review focuses on the important miRNAs that have been found to impact cancer progression and metastasis through direct and indirect interactions with the matrix metalloproteinases.

  17. Essential domains of a disintegrin and metalloprotease with thrombospondin type 1 repeats-13 metalloprotease required for modulation of arterial thrombosis

    NARCIS (Netherlands)

    Xiao, Juan; Jin, Sheng-Yu; Xue, Jing; Sorvillo, Nicoletta; Voorberg, Jan; Zheng, X. Long

    2011-01-01

    A disintegrin and metalloprotease with thrombospondin type 1 repeats-13 (ADAMTS13) inhibits platelet aggregation and arterial thrombosis by cleavage of von Willebrand factor. However, the structural components of ADAMTS13 required for inhibition of arterial thrombosis are not fully defined. Using

  18. Matrix metalloproteinases (MMPs) in the endometrium of bitches.

    Science.gov (United States)

    Chu Py, Po-yin; Salamonsen, L A; Lee, C S; Wright, P J

    2002-03-01

    The relationships between activities of matrix metalloproteinases (MMPs) in the canine uterus and the occurrence of degeneration of the luminal epithelium, cystic endometrial hyperplasia, pyometra and uterine remodelling post partum were determined. Mature bitches (n = 10) were ovariectomized, treated with hormones (oestradiol benzoate, progestagen) and investigated at stages simulating pro-oestrus (n = 2), oestrus (n = 2), dioestrus (n = 2), and mid- (n = 2) and late (n = 2) anoestrus (3 and 9 weeks, respectively, after cessation of treatment with progestagen). Untreated bitches (n = 1 per group) served as controls (Expt 1). An additional 10 ovariectomized bitches, at the end of treatment-induced simulated dioestrus, were treated each day for a further 3 weeks either with the same dose (standard dose, n = 3), a decreased dose (n = 3) or an increased dose (n = 3) of progestagen, or no treatment (withdrawal dose, n = 1). These bitches were then investigated (Expt 2). A suture was placed in the lumen of one uterine horn of another five bitches at ovariectomy. Three of these bitches were treated to induce simulated dioestrus and two bitches served as untreated controls. In the hormone-treated bitches, the suture resulted in cystic endometrial hyperplasia in one bitch and in cystic endometrial hyperplasia with pyometra in two bitches. The control bitches showed no cystic endometrial hyperplasia or pyometra (Expt 3). Four intact bitches were studied at 2 (n = 1), 3 (n = 2) and 11 (n = 1) weeks post partum. Uterine tissues were also collected from two bitches with naturally occurring cystic endometrial hyperplasia with pyometra (Expt 4). All uteri were examined histologically and the activities of MMP-2, -7 and -9 (latent and active forms) were assessed using zymography of extracts of endometrium. In Expts 1 and 2, marked degeneration of the luminal epithelium in six of 25 bitches (simulated mid-anoestrus, withdrawal dose and decreased dose groups) was not associated

  19. Carbodiimide Inactivation of MMPs and Effect on Dentin Bonding

    Science.gov (United States)

    Mazzoni, A.; Apolonio, F.M.; Saboia, V.P.A.; Santi, S.; Angeloni, V.; Checchi, V.; Curci, R.; Di Lenarda, R.; Tay, F.R.; Pashley, D.H.; Breschi, L.

    2014-01-01

    The use of protein cross-linking agents during bonding procedures has been recently proposed to improve bond durability. This study aimed to use zymography and in situ zymography techniques to evaluate the ability of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) cross-linker to inhibit matrix metalloproteinase (MMP) activity. The hypotheses tested were that: (1) bonding procedures increase dentin gelatinolytic activity and (2) EDC pre-treatment prevents this enzymatic activity. The zymographic assay was performed on protein extracts obtained from dentin powder treated with Optibond FL or Scotchbond 1XT with or without 0.3M EDC pre-treatment. For in situ zymography, adhesive/dentin interfaces were created with the same adhesives applied to acid-etched dentin slabs pre-treated or not with EDC conditioner. Zymograms revealed increased expression of dentin endogenous MMP-2 and -9 after adhesive application, while the use of EDC as a primer inactivated dentin gelatinases. Results of in situ zymograpy showed that hybrid layers of tested adhesives exhibited intense collagenolytic activity, while almost no fluorescence signal was detected when specimens were pre-treated with EDC. The correlative analysis used in this study demonstrated that EDC could contribute to inactivate endogenous dentin MMPs within the hybrid layer created by etch-and-rinse adhesives. PMID:24334409

  20. MMPs regulate both development and immunity in the tribolium model insect.

    Directory of Open Access Journals (Sweden)

    Eileen Knorr

    Full Text Available BACKGROUND: Matrix metalloproteinases (MMPs are evolutionarily conserved and multifunctional effector molecules in development and homeostasis. In spite of previous, intensive investigation in vitro and in cell culture, their pleiotrophic functions in vivo are still not well understood. METHODOLOGY/PRINCIPAL FINDINGS: We show that the genetically amenable beetle Tribolium castaneum represents a feasible model organism to explore MMP functions in vivo. We silenced expression of three insect-type Tribolium MMP paralogs and their physiological inhibitors, TIMP and RECK, by dsRNA-mediated genetic interference (RNAi. Knock-down of MMP-1 arrested development during pupal morphogenesis giving phenotypes with altered antennae, compound eyes, wings, legs, and head. Parental RNAi-mediated knock-down of MMP-1 or MMP-2 resulted in larvae with non-lethal tracheal defects and with abnormal intestines, respectively, implicating additional roles of MMPs during beetle embryogenesis. This is different to findings from the fruit fly Drosophila melanogaster, in which MMPs have a negligible role in embryogenesis. Confirming pleiotrophic roles of MMPs our results also revealed that MMPs are required for proper insect innate immunity because systemic knock-down of Tribolium MMP-1 resulted in significantly higher susceptibility to the entomopathogenic fungus Beauveria bassiana. Moreover, mRNA levels of MMP-1, TIMP, and RECK, and also MMP enzymatic activity were significantly elevated in immune-competent hemocytes upon stimulation. To confirm collagenolytic activity of Tribolium MMP-1 we produced and purified recombinant enzyme and determined a similar collagen IV degrading activity as observed for the most related human MMP, MMP-19. CONCLUSIONS/SIGNIFICANCE: This is the first study, to our knowledge, investigating the in vivo role of virtually all insect MMP paralogs along with their inhibitors TIMP and RECK in both insect development and immunity. Our results from the

  1. Structure of Gentlyase, the neutral metalloprotease of Paenibacillus polymyxa

    Science.gov (United States)

    Ruf, Armin; Stihle, Martine; Benz, Jörg; Schmidt, Manfred; Sobek, Harald

    2013-01-01

    Gentlyase is a bacterial extracellular metalloprotease that is widely applied in cell culture and for tissue dissociation and that belongs to the family of thermolysin-like proteases. The structure of thermolysin has been known since 1972 and that of Bacillus cereus neutral protease since 1992. However, the structure determination of other Bacillus neutral proteases has been hindered by their tendency to cannibalistic autolysis. High calcium conditions that allow the concentration and crystallization of the active Gentlyase metalloprotease without autoproteolysis were identified using thermal fluorescent shift assays. X-ray structures of the protease were solved in the absence and in the presence of the inhibitor phosphoramidon at 1.59 and 1.76 Å resolution, respectively. No domain movement was observed upon inhibitor binding, although such movement is thought to be a general feature of the thermolysin-like protease family. Further analysis of the structure shows that the observed calcium dependency of Gentlyase stability may arise from a partly degenerated calcium site Ca1–2 and a deletion near site Ca3. PMID:23275160

  2. An exocellular thermolysin-like metalloprotease produced by Vibrio fluvialis: purification, characterization, and gene cloning.

    Science.gov (United States)

    Miyoshi, Shin-ichi; Sonoda, Yuka; Wakiyama, Hiroko; Rahman, Md Monzur; Tomochika, Ken-ichi; Shinoda, Sumio; Yamamoto, Shigeo; Tobe, Kazuo

    2002-09-01

    An exocellular metalloprotease produced by Vibrio fluvialis, an enteropathogenic vibrio, was purified and characterized. The metalloprotease (V. fluvialis protease [VFP]) was found to have very similar characteristics to V. vulnificus protease, including a molecular mass of 45kDa, sensitivity to chelating agents or competitive inhibitors for thermolysin-like metalloproteases, and the substrate specificity. The structural gene for VFP was also cloned, and its nucleotide sequence was determined. The deduced amino acid sequence confirmed that VFP was a member of the thermolysin family. VFP, like V. vulnificus protease, showed the haemagglutinating, permeability-enhancing and haemorrhagic activities in addition to the proteolytic activity toward oligopeptide, casein or elastin.

  3. Cloning and expression of ADAM related metalloproteases in Equine Laminitis

    Science.gov (United States)

    Coyne, Michael J.; Cousin, Hélène; Loftus, John P.; Johnson, Philip J.; Belknap, James K.; Gradil, Carlos M.; Black, Samuel J.; Alfandari, Dominique

    2010-01-01

    Equine laminitis is a debilitating disease affecting the digital laminae that suspends the distal phalanx within the hoof. While the clinical progression of the disease has been well documented, the molecular events associated with its pathogenesis remain largely unknown. We have investigated the expression of genes coding for proteins containing a Disintegrin and Metalloprotease domain (ADAM), as well as genes encoding the natural inhibitors of these enzymes (Tissue Inhibitor of MetalloProtease; TIMP) in horses with naturally acquired (acute, chronic and aggravated chronic cases collected in clinic) or experimentally-induced (black walnut extract and starch gruel models) laminitis using real time quantitative RT-PCR. Changes in expression of these enzymes and regulators may underlie the pathologic remodeling of lamellar tissue in laminitis. Genes encoding ADAMs involved in inflammation (ADAM-10 and ADAM-17), as well as those implicated in arthritis (ADAMTS-1, ADAMTS-4 and ADAMTS-5) were cloned, and the sequences used to generate specific oligonucleotide primers for the RT-qPCR experiments. Our results show that genes encoding ADAM-10 and 17 were not induced in most laminitic animals whereas ADAMTS-4 gene expression was strongly upregulated in practically all cases of experimentally induced and naturally acquired laminitis. The expression of MMP-9 and ADAMTS-5 was also increased in many of the laminitic horses. In addition, TIMP-2 gene expression was decreased in most laminitic horses, whereas expression of genes encoding other TIMPs, namely TIMP-1 and TIMP-3 was randomly increased or decreased in the various models. We conclude that elevated expression of lamellar ADAMTS-4 is a common feature of laminitis consistent with a central role of the gene product in the pathophysiology of laminitis. PMID:19131116

  4. Activation and proteolytic activity of the Treponema pallidum metalloprotease, pallilysin.

    Science.gov (United States)

    Houston, Simon; Hof, Rebecca; Honeyman, Lisa; Hassler, Julia; Cameron, Caroline E

    2012-01-01

    Treponema pallidum is a highly invasive pathogen that undergoes rapid dissemination to establish widespread infection. Previous investigations identified the T. pallidum adhesin, pallilysin, as an HEXXH-containing metalloprotease that undergoes autocatalytic cleavage and degrades laminin and fibrinogen. In the current study we characterized pallilysin's active site, activation requirements, cellular location, and fibrin clot degradation capacity through both in vitro assays and heterologous treponemal expression and degradation studies. Site-directed mutagenesis showed the pallilysin HEXXH motif comprises at least part of the active site, as introduction of three independent mutations (AEXXH [H¹⁹⁸A], HAXXH [E¹⁹⁹A], and HEXXA [H²⁰²A]) abolished pallilysin-mediated fibrinogenolysis but did not adversely affect host component binding. Attainment of full pallilysin proteolytic activity was dependent upon autocatalytic cleavage of an N-terminal pro-domain, a process which could not occur in the HEXXH mutants. Pallilysin was shown to possess a thrombin cleavage site within its N-terminal pro-domain, and in vitro studies confirmed cleavage of pallilysin with thrombin generates a truncated pallilysin fragment that has enhanced proteolytic activity, suggesting pallilysin can also exploit the host coagulation process to facilitate protease activation. Opsonophagocytosis assays performed with viable T. pallidum demonstrated pallilysin is a target of opsonic antibodies, consistent with a host component-interacting, surface-exposed cellular location. Wild-type pallilysin, but not the HEXXA mutant, degraded fibrin clots, and similarly heterologous expression of pallilysin in the non-invasive spirochete Treponema phagedenis facilitated fibrin clot degradation. Collectively these results identify pallilysin as a surface-exposed metalloprotease within T. pallidum that possesses an HEXXH active site motif and requires autocatalytic or host-mediated cleavage of a pro

  5. Activation and Proteolytic Activity of the Treponema pallidum Metalloprotease, Pallilysin

    Science.gov (United States)

    Houston, Simon; Hof, Rebecca; Honeyman, Lisa; Hassler, Julia; Cameron, Caroline E.

    2012-01-01

    Treponema pallidum is a highly invasive pathogen that undergoes rapid dissemination to establish widespread infection. Previous investigations identified the T. pallidum adhesin, pallilysin, as an HEXXH-containing metalloprotease that undergoes autocatalytic cleavage and degrades laminin and fibrinogen. In the current study we characterized pallilysin's active site, activation requirements, cellular location, and fibrin clot degradation capacity through both in vitro assays and heterologous treponemal expression and degradation studies. Site-directed mutagenesis showed the pallilysin HEXXH motif comprises at least part of the active site, as introduction of three independent mutations (AEXXH [H198A], HAXXH [E199A], and HEXXA [H202A]) abolished pallilysin-mediated fibrinogenolysis but did not adversely affect host component binding. Attainment of full pallilysin proteolytic activity was dependent upon autocatalytic cleavage of an N-terminal pro-domain, a process which could not occur in the HEXXH mutants. Pallilysin was shown to possess a thrombin cleavage site within its N-terminal pro-domain, and in vitro studies confirmed cleavage of pallilysin with thrombin generates a truncated pallilysin fragment that has enhanced proteolytic activity, suggesting pallilysin can also exploit the host coagulation process to facilitate protease activation. Opsonophagocytosis assays performed with viable T. pallidum demonstrated pallilysin is a target of opsonic antibodies, consistent with a host component-interacting, surface-exposed cellular location. Wild-type pallilysin, but not the HEXXA mutant, degraded fibrin clots, and similarly heterologous expression of pallilysin in the non-invasive spirochete Treponema phagedenis facilitated fibrin clot degradation. Collectively these results identify pallilysin as a surface-exposed metalloprotease within T. pallidum that possesses an HEXXH active site motif and requires autocatalytic or host-mediated cleavage of a pro-domain to attain

  6. A novel member of the thermolysin family, cloning and biochemical characterization of metalloprotease from Staphylococcus pseudintermedius

    NARCIS (Netherlands)

    Wladyka, Benedykt; Bista, Michal; Sabat, Artur J.; Bonar, Emilia; Grzeszczuk, Sabina; Hryniewicz, Waleria; Dubin, Adam

    2008-01-01

    Thermolysins constitute a family of secreted bacterial metalloproteases expressed, among others, by several pathogens. Strains of Staphylococcus pseudintermedius isolated from diseased dogs and judged as protease-positive, by skim milk agar plate culture, were investigated for protease content. No

  7. A scrutiny of matrix metalloproteinases in osteoclasts: evidence for heterogeneity and for the presence of MMPs synthesized by other cells

    DEFF Research Database (Denmark)

    Andersen, Thomas L; del Carmen Ovejero, Maria; Kirkegaard, Tove

    2004-01-01

    osteoclasts appears insensitive to MMP inhibitors. Our study shows that the confusion about osteoclastic MMPs in the literature reflects the remarkable ability of osteoclasts to adapt to their environment, as required by the structural or functional diversity of bone tissue. Our observations provide basic...... information needed for understanding the emerging role of MMPs in controlling cell signaling and bone resorption....

  8. Expression of the Cydia pomonella granulovirus matrix metalloprotease enhances Autographa californica multiple nucleopolyhedrovirus virulence and can partially substitute for viral cathepsin.

    Science.gov (United States)

    Ishimwe, Egide; Hodgson, Jeffrey J; Passarelli, A Lorena

    2015-07-01

    The Cydia pomonella granulovirus open reading frame 46 (CpGV-ORF46) contains predicted domains found in matrix metalloproteases (MMPs), a family of zinc-dependent endopeptidases that degrade extracellular matrix proteins. We showed that CpGV-MMP was active in vitro. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) expressing CpGV-ORF46 replicated similarly to a control virus lacking CpGV-ORF46 in cultured cells. The effects of AcMNPV expressing CpGV-MMP on virus infection in cultured cells and Trichoplusia ni larvae in the presence or absence of other viral degradative enzymes, cathepsin and chitinase, were evaluated. In the absence of cathepsin and chitinase or cathepsin alone, larval time of death was significantly delayed. This delay was compensated by the expression of CpGV-MMP. CpGV-MMP was also able to promote larvae melanization in the absence of cathepsin and chitinase. In addition, CpGV-MMP partially substituted for cathepsin in larvae liquefaction when chitinase, which is usually retained in the endoplasmic reticulum, was engineered to be secreted. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Novel Apigenin Based Small Molecule that Targets Snake Venom Metalloproteases

    Science.gov (United States)

    Anusha, Sebastian; Hemshekhar, Mahadevappa; Chandra Nayaka, Siddaiah; Kemparaju, Kempaiah; Basappa; Girish, Kesturu S.; Rangappa, Kanchugarakoppal S.

    2014-01-01

    The classical antivenom therapy has appreciably reduced snakebite mortality rate and thus is the only savior drug available. Unfortunately, it considerably fails to shield the viper bite complications like hemorrhage, local tissue degradation and necrosis responsible for severe morbidity. Moreover, the therapy is also tagged with limitations including anaphylaxis, serum sickness and poor availability. Over the last decade, snake venom metalloproteases (SVMPs) are reported to be the primary component responsible for hemorrhage and tissue degradation at bitten site. Thus, antivenom inability to offset viper venom-induced local toxicity has been a basis for an insistent search for SVMP inhibitors. Here we report the inhibitory effect of compound 5d, an apigenin based molecule against SVMPs both in silico and in vivo. Several apigenin analogues are synthesized using multicomponent Ugi reactions. Among them, compound 5d effectively abrogated Echis carinatus (EC) venom-induced local hemorrhage, tissue necrosis and myotoxicity in a dose dependant fashion. The histopathological study further conferred effective inhibition of basement membrane degradation, and accumulation of inflammatory leucocytes at the site of EC venom inoculation. The compound also protected EC venom-induced fibrin and fibrinogen degradation. The molecular docking of compound 5d and bothropasin demonstrated the direct interaction of hydroxyl group of compound with Glu146 present in hydrophobic pocket of active site and does not chelate Zn2+. Hence, it is concluded that compound 5d could be a potent agent in viper bite management. PMID:25184206

  10. Differential Matrix Metalloprotease (MMP Expression Profiles Found in Aged Gingiva.

    Directory of Open Access Journals (Sweden)

    Suhee Kim

    Full Text Available The periodontium undergoes age-related cellular and clinical changes, but the involved genes are not yet known. Here, we investigated age-related genetic changes in gingiva at the transcriptomic level. Genes that were differentially expressed between young and old human gingiva were identified by RNA sequencing and verified by real-time PCR. A total of 1939 mRNA transcripts showed significantly differential expression between young and old gingival tissues. Matrix metalloprotease (MMP regulation was the top pathway involved in gingival aging. MMP3, MMP9, MMP12, and MMP13 were upregulated in old gingival tissues, concomitantly with interleukin-1 beta (IL1B expression. In vitro experiments using human gingival fibroblasts (hGFs showed that MMP12 was upregulated in old hGFs compared to young hGFs. Moreover, the MMP3, MMP9 and IL1B levels were more highly stimulated by infection with the oral bacterium, Fusobacterium nucleatum, in old hGFs compared to young hGFs. Collectively, these findings suggest that, in gingiva, the upregulation of MMP12 may be a molecular hallmark of natural aging, while the upregulations of MMP3, MMM9, and IL1B may indicate externally (e.g., infection-induced aging. These findings contribute to our understanding of the molecular targets involved in gingival aging.

  11. Correlations Between MMPs and TIMPs Levels in Aqueous Humor from High Myopia and Cataract Patients.

    Science.gov (United States)

    Jia, Yan; Hu, Dan-Ning; Sun, Jing; Zhou, Jibo

    2017-04-01

    To study the relationships between matrix metalloproteinases (MMP)-2, MMP-3, and tissue inhibitors of matrix metalloproteinases (TIMP)-1, TIMP-2, and TIMP-3 aqueous humor levels in patients with high myopia or cataract. MMPs and TIMPs protein levels in 65 aqueous humor samples collected from patients with high myopia or cataract during cataract or clear lens extraction surgery were measured with the Luminex xMAP Technology. The relationship between MMPs and TIMPs levels was analyzed with Spearman's correlation test. MMP-2 levels, but not MMP-3 levels, were increased in the aqueous humor from high-myopia patients. Levels of TIMP-1, -2, and -3 were positively and very significantly correlated with the MMP-2 levels (TIMP-1: r=0.626, p 0.05; TIMP-3: r = 0.127, p > 0.05) and TIMP-1 levels only marginally correlated with MMP-3 levels (r = 0.278, 0.01myopia.

  12. MMPs/TIMPs and inflammatory signalling de-regulation in human incisional hernia tissues

    Science.gov (United States)

    Guillen-Marti, Jordi; Diaz, Ramon; Quiles, Maria T; Lopez-Cano, Manuel; Vilallonga, Ramon; Huguet, Pere; Ramon-y-Cajal, Santiago; Sanchez-Niubo, Albert; Reventós, Jaume; Armengol, Manel; Arbos, Maria A

    2009-01-01

    Background: Incisional hernia is a common and important complication of laparotomies. Epidemiological studies allude to an underlying biological cause, at least in a subset of population. Interest has mainly focused on abnormal collagen metabolism. However, the role played by other determinants of extracellular matrix (ECM) composition is unknown. To date, there are few laboratory studies investigating the importance of biological factors contributing to incisional hernia development. We performed a descriptive tissue-based analysis to elucidate the possible relevance of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in association with local cytokine induction in human incisional hernia tissues. The expression profiles of MMPs, TIMPs and pro-inflammatory cytokine signalling were investigated in aponeurosis and skeletal muscle specimens taken intraoperatively from incisional hernia (n= 10) and control (n= 10) patients. Semiquantitative RT-PCR, zymography and immunoblotting analyses were done. Incisional hernia samples displayed alterations in the microstructure and loss of ECM, as assessed by histological analyses. Moreover, incisional hernia tissues showed increased MMP/TIMP ratios and de-regulated inflammatory signalling (tumor necrosis factor [TNFA] and interleukin [IL]-6 tended to increase, whereas aponeurosis TNFA receptors decreased). The changes were tissue-specific and were detectable at the mRNA and/or protein level. Statistical analyses showed several associations between individual MMPs, TIMPs, interstitial collagens and inflammatory markers. The increment of MMPs in the absence of a counterbalance by TIMPs, together with an ongoing de-regulated inflammatory signalling, may contribute in inducing a functional defect of the ECM network by post-translational mechanisms, which may trigger abdominal wall tissue loss and eventual rupture. The notable TIMP3 protein down-regulation in incisional hernia fascia may be of pathophysiological

  13. MMPs and soluble ICAM-5 increase neuronal excitability within in vitro networks of hippocampal neurons.

    Directory of Open Access Journals (Sweden)

    Mark Niedringhaus

    Full Text Available Matrix metalloproteinases (MMPs are zinc-dependent endopeptidases that are released from neurons in an activity dependent manner. Published studies suggest their activity is important to varied forms of learning and memory. At least one MMP can stimulate an increase in the size of dendritic spines, structures which represent the post synaptic component for a large number of glutamatergic synapses. This change may be associated with increased synaptic glutamate receptor incorporation, and an increased amplitude and/or frequency of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA mini excitatory post-synaptic currents (EPSCs. An associated increase in the probability of action potential occurrence would be expected. While the mechanism(s by which MMPs may influence synaptic structure and function are not completely understood, MMP dependent shedding of specific cell adhesion molecules (CAMs could play an important role. CAMs are ideally positioned to be cleaved by synaptically released MMPs, and shed N terminal domains could potentially interact with previously unengaged integrins to stimulate dendritic actin polymerization with spine expansion. In the present study, we have used multielectrode arrays (MEAs to investigate MMP and soluble CAM dependent changes in neuronal activity recorded from hippocampal cultures. We have focused on intercellular adhesion molecule-5 (ICAM-5 in particular, as this CAM is expressed on glutamatergic dendrites and shed in an MMP dependent manner. We show that chemical long-term potentiation (cLTP evoked changes in recorded activity, and the dynamics of action potential bursts in particular, are altered by MMP inhibition. A blocking antibody to β(1 integrins has a similar effect. We also show that the ectodomain of ICAM-5 can stimulate β(1 integrin dependent increases in spike counts and burst number. These results support a growing body of literature suggesting that MMPs have important effects on neuronal

  14. MMPs regulate both development and immunity in the Tribolium model insect

    OpenAIRE

    Knorr, E.; Schmidtberg, H.; Vilcinskas, A.; Altincicek, B.

    2009-01-01

    BACKGROUND: Matrix metalloproteinases (MMPs) are evolutionarily conserved and multifunctional effector molecules in development and homeostasis. In spite of previous, intensive investigation in vitro and in cell culture, their pleiotrophic functions in vivo are still not well understood. METHODOLOGY/PRINCIPAL FINDINGS: We show that the genetically amenable beetle Tribolium castaneum represents a feasible model organism to explore MMP functions in vivo. We silenced expression of three insect-t...

  15. Metalloproteases in Trypanosoma rangeli-infected Rhodnius prolixus

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    D Feder

    1999-11-01

    Full Text Available Protease activities in the haemolymph and fat body in a bloodsucking insect, Rhodnius prolixus, infected with Trypanosoma rangeli, were investigated. After SDS-polyacrylamide gel electrophoresis containing gelatin as substrate, analysis of zymograms performed on samples of different tissues of controls and insects inoculated or orally infected with short or long epimastigotes of T. rangeli, demonstrated distinct patterns of protease activities: (i proteases were detected in the haemolymph of insects which were fed on, or inoculated with, short epimastigotes of T. rangeli (39 kDa and 33 kDa, respectively, but they were not observed in the fat body taken from these insects; (ii protease was also presented in the fat bodies derived from naive insects or controls inoculated with sterile phosphate-saline buffer (49 kDa, but it was not detected in the haemolymph of these insects; (iii no protease activity was observed in both haemolymph and fat bodies taken from insects inoculated with, or fed on, long epimastigotes of T. rangeli. Furthermore, in short epimastigotes of T. rangeli extracts, three bands of the protease activities with apparent molecular weights of 297, 198 and 95 kDa were detected while long epimastigotes preparation presented only two bands of protease activities with molecular weights of 297 and 198 kDa. The proteases from the insect infected with T. rangeli and controls belong to the class of either metalloproteases or metal-activated enzymes since they are inhibited by 1,10-phenanthroline. The significance of these proteases in the insects infected with short epimastigotes of T. rangeli is discussed in relation to the success of the establishment of infection of these parasites in its vector, R. prolixus.

  16. Impact of Leishmania Metalloprotease GP63 on Macrophage Signalling

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    Amandine eIsnard

    2012-05-01

    Full Text Available Several Leishmania surface molecules are known to be important virulence factors. For instance, LPG is recognized as one of the key virulence factor for Leishmania. Interestingly, recent findings permit to believe that the Leishmania GP63 could be also a critical one. GP63 is a metalloprotease found in all Leishmania species under different forms going from membrane-bound to extracellularly secreted ones. Even before parasite entries into the host macrophage, GP63 provides parasite resistance to the complement-mediated lysis and facilitate promastigote engulfment by macrophages. Additionally, it has been found that the degradation of proteins from the macrophage extracellular matrix by GP63 could confer protection to promastigotes, as well as amastigotes, during their initial interaction with the host cell. More recently, GP63 has been observed to rapidly enter within the host macrophage -in part via lipid raft microdomains- and to be pivotal for the subversion of host innate immune response. For instance, it has been found to be responsible for the activation of negative regulatory mechanisms involving activation of protein tyrosine phosphatases (PTPs; SHP-1, PTP1B and TCPTP that lead to the alteration of several key signalling pathways utilizing JAK and MAP kinases family members, as well as the pivotal IRAK-1 kinase for toll like-dependent signalling. In addition, it has been recently reported that inactivation of some transcription factors such as AP-1 occurs directly in the nuclear environment of the infected cells, and to involve the cleavage and degradation of c-jun and c-fos family members by GP63. Altogether, this signalling inactivation under the mediation of GP63 concurs to inhibit important antimicrobial actions usually under the regulation of the innate immune response, and therefore favouring the survival and propagation of the parasite once into its host intracellular environment.

  17. Tissue Inhibitor of Metalloprotease-2 (TIMP-2): Bioprocess Development, Physicochemical, Biochemical, and Biological Characterization of Highly Expressed Recombinant Protein.

    Science.gov (United States)

    Chowdhury, Anandã; Brinson, Robert; Wei, Beiyang; Stetler-Stevenson, William G

    2017-12-12

    Tissue inhibitor of metalloprotease-2 (TIMP-2) is a secreted 21 kDa multifunctional protein first described as an endogenous inhibitor of matrix metalloproteinases (MMPs) that prevents breakdown of the extracellular matrix often observed in chronic diseases. TIMP-2 diminishes the level of growth factor-mediated cell proliferation in vitro, as well as neoangiogenesis and tumor growth in vivo independent of its MMP inhibitory activity. These physiological properties make TIMP-2 an excellent candidate for further preclinical development as a biologic therapy of cancer. Here we present a straightforward bioprocessing methodology that yields >35 mg/L recombinant human TIMP-2 6XHis-tagged protein (rhTIMP-2) from suspension cultures of HEK-293-F cells. Enhanced rhTIMP-2-6XHis yields were achieved by optimization of both TIMP-2 cDNA codon sequence and cell culture conditions. Using a two-step chromatographic process, we achieved >95% purity with minimal processing losses. Purified rhTIMP-2-6XHis was free of mouse antigen contamination. Circular dichroism spectroscopy indicated a well-folded rhTIMP-2-6XHis that is highly stable and refractory to pH changes. Two-dimensional heteronuclear single-quantum coherence nuclear magnetic resonance of full length rhTIMP-2-6XHis also indicated a monodisperse, well-folded protein preparation. Purified rhTIMP-2-6XHis inhibited MMP-2 enzymatic activity in a dose-dependent fashion with an IC 50 of ∼1.4 nM. Pretreatment of A549 lung cancer and JygMC(A) triple-negative breast cancer cells with rhTIMP-2-6XHis in low-nanomolar amounts inhibited EGF-induced proliferation to basal (unstimulated) levels. This study therefore not only offers a robust bioprocess methodology for rhTIMP-2 production but also characterizes critical physicochemical and biological attributes that are useful for monitoring quality control of the production process.

  18. Metaloproteinasas de la matriz extracelular (mmps en Odontología

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    Vanesa Pereira Prado

    Full Text Available Resumen La mayoría de los tejidos del organismo se encuentran en un constante proceso de renovación, modulado por una vasta cantidad de proteínas que inducen la síntesis y degradación tisular a lo largo del tiempo. Las metaloproteinasas de la matriz extracelular (MMPs son una familia de 25 endopeptidasas zinc dependientes encargadas de escindir los componentes inmediatos de la matriz extracelular (MEC. Ciertas metaloproteinasas de la matriz extracelular, especialmente MMP-2 y MMP-9, se ven implicadas en el desarrollo del germen dental así como con la proliferación e invasión de los tumores odontogénicos, siendo un factor a considerar sobre la posible agresividad tumoral y la base para el tratamiento de los mismos. A su vez, ambas MMPs actúan en la progresión de las lesiones cariosas así como en progresión de la periodontitis apical de pacientes con necrosis pulpar. Varios patógenos periodontales, promueven la secreción tanto de MMP-2 como MMP-9 favoreciendo la destrucción de los tejidos periodontales, íntimamente relacionadas con el avance de la enfermedad. En pacientes bajo tratamiento ortodóncico se encuentran altos niveles de estas MMPs, tanto en zonas de compresión como de tensión, relacionando los movimientos dentales con la renovación de los tejidos. Esta revisión analiza su expresión y utilidad relacionándolas con diferentes disciplinas en el área de las ciencias odontológicas, enfatizando su potencial como biomarcadores

  19. Hydrogen sulfide and sodium nitroprusside compete to activate/deactivate MMPs in bone tissue homogenates

    Directory of Open Access Journals (Sweden)

    Vacek TP

    2013-03-01

    Full Text Available Thomas P Vacek, Natia Qipshidze, Suresh C Tyagi Department of Physiology and Biophysics, University of Louisville School of Medicine, Louisville, KY, USA Background: Bone microvascular remodeling is the primary predictor of bone structure and function. Remodeling by its very nature implies synthesis and degradation of the extracellular matrix. Normally, 50% of total protein in the vessel wall is elastin. During remodeling, elastin is degraded by specialized matrix metalloproteinases (MMPs. Because the turnover of elastin is 1000-fold slower than that of collagen, most of the elastin is replaced by stiffer collagen. Stiffer vessels impose pressure on the aortic valve, causing regurgitation and increased pulse pressure. On the other hand, high MMP activity will cause vascular dilatation, leading to aneurysm. Therefore, balanced constitutive remodeling is necessary for adequate bone structure and function. Interestingly, collagen-degrading MMPs are involved in various pathological conditions, including osteoporosis, osteoarthritis, and cardiovascular disease. Sodium nitroprusside is a nitric oxide donor that could potentially alter MMP activity via vasodilation in vivo, but can also produce peroxynitrite, which activates MMPs by combining with superoxide. Moreover, hydrogen sulfide is a known antioxidant as well as a vasodilator, and is also speculated to contribute directly to MMP activity. We hypothesized that hydrogen sulfide reduced activity of MMP in ex vivo bone tissue homogenates and that sodium nitroprusside would increase MMP activity in vitro. Methods: We surgically removed the tibia and femur from anesthetized mice, and prepared bone tissue homogenates using a mortar and pestle, measured the protein concentration with a spectrophotometer, and detected MMP activity using gelatin gel zymography. Results: Our data showed increased MMP activity at a sodium nitroprusside concentration of 1 µM, and MMP activity increased exponentially. There

  20. Role of matrix metalloprotease-2 in oxidant activation of Ca ATPase ...

    Indian Academy of Sciences (India)

    Unknown

    Exposure of bovine pulmonary artery smooth muscle plasma membrane suspension with the oxidant H2O2. (1 mM) stimulated Ca2+ATPase activity. We sought to determine the role of matrix metalloprotease-2 (MMP-2) in stimulating Ca2+ATPase activity by H2O2 in the smooth muscle plasma membrane. The smooth ...

  1. Role of matrix metalloprotease-2 in oxidant activation of Ca 2 ...

    Indian Academy of Sciences (India)

    The protease activity and the Ca2+ATPase activity were prevented by the antioxidant, vitamin E, indicating that the effect produced by H2O2 was due to reactive oxidant species(es). Both basal and H2O2 stimulated MMP-2 activity and Ca2+ATPase activity were inhibited by the general inhibitors of matrix metalloproteases: ...

  2. Metalloprotease Peptide Inhibitors: A Semester-Long Organic Synthetic Research Project for the Introductory Laboratory Course

    Science.gov (United States)

    Pontrello, Jason K.

    2015-01-01

    A semester-long research project to synthesize unique compounds designed after published metalloprotease peptide inhibitors is presented. The research project encompasses a set of nine organic chemistry reactions traditionally taught in the second semester lab course, and the procedures are derived from scientific literature. The two principle…

  3. Imaging Matrix Metalloproteases in Spontaneous Colon Tumors: Validation by Correlation with Histopathology.

    Science.gov (United States)

    Hensley, Harvey; Cooper, Harry S; Chang, Wen-Chi L; Clapper, Margie L

    2017-01-01

    The use of fluorescent probes in conjunction with white-light colonoscopy is a promising strategy for improving the detection of precancerous colorectal lesions, in particular flat (sessile) lesions that do not protrude into the lumen of the colon. We describe a method for determining the sensitivity and specificity of an enzymatically activated near-infrared probe (MMPSense680) for the detection of colon lesions in a mouse model (APC +/Min-FCCC ) of spontaneous colorectal cancer. Fluorescence intensity correlates directly with the activity of matrix metalloproteinases (MMPs). Overexpression of MMPs is an early event in the development of colorectal lesions. Although the probe employed serves as a reporter of the activity of MMPs, our method can be applied to any fluorescent probe that targets an early molecular event in the development of colorectal tumors.

  4. Matrix metalloproteinases (MMPs) inhibitory effects of an octameric oligopeptide isolated from abalone Haliotis discus hannai.

    Science.gov (United States)

    Nguyen, Van-Tinh; Qian, Zhong-Ji; Ryu, Bomi; Kim, Kil-Nam; Kim, Daekyung; Kim, Young-Mog; Jeon, You-Jin; Park, Won Sun; Choi, Il-Whan; Kim, Geun Hyung; Je, Jae-Young; Jung, Won-Kyo

    2013-11-01

    Abalone (Haliotis discus hannai) is a marine gastropod, and an important fishery and food industrial resource that is massively maricultured in Asia, Africa, Australia and America. However, its health benefits have rarely been studied for nutraceutical and pharmaceutical application. In this study, the purified abalone oligopeptide (AOP) with anti-matrix metalloproteinases (anti-MMPs) effects was isolated from the digests of abalone intestine using recycle HPLC with a JAI W253 column and an OHpak SB-803 HQ column. The AOP was identified as Ala-Glu-Leu-Pro-Ser-Leu-Pro-Gly (MW=782.4 Da) with a de novo peptide sequencing technique using a tandem mass spectrometer. The AOP exhibited a specific inhibitory effect against MMP-2/-9 activity and attenuated protein expression of p50 and p65 in the human fibrosarcoma (HT1080) cells, dose-dependently. The results presented illustrate that the AOP could inhibit MMP-2/-9 expression in HT1080 cells via the nuclear factor-kappaB (NF-κB)-mediated pathway. This data suggest that the AOP from H. discus hannai intestine may possess therapeutic and preventive potential for the treatment of MMPs-related disorders such as angiogenesis and cardiovascular diseases. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. MT-MMPs in pre-eclamptic placenta: relationship to soluble endoglin production.

    Science.gov (United States)

    Kaitu'u-Lino, T J; Tuohey, L; Ye, L; Palmer, K; Skubisz, M; Tong, S

    2013-02-01

    Pre-eclampsia is a serious complication of pregnancy, characterized by severe endothelial dysfunction resulting in hypertension, proteinuria and maternal end-organ damage. Soluble endoglin is an anti-angiogenic factor released from placenta that has been linked to severe pre-eclampsia. We recently reported MMP-14 is capable of cleaving endoglin to release soluble endoglin from placenta, however inhibition studies only partially repressed production. To this end we have sought to identify other proteases that mediate endoglin shedding from placenta. MMP-14 is one of six-membrane-type (MT-) MMPs, a sub-family of the MMP superfamily, so named because they are membrane bound. MMP-15 is phylogenetically the closest MMP relative to MMP-14, however its inhibition has no effect on soluble endoglin production from placenta. Here we aimed to characterize the remaining four MT-MMPs (MMP-16, -17, -24 and -25) in severe early-onset pre-eclamptic placenta and assess their relative contribution to soluble endoglin production. Immunolocalisation studies revealed MMP-16, -24 and -25 were localized to the syncytiotrophoblast, the same site as endoglin, whilst MMP-17 was predominantly localized to fetal vessels and underlying stroma. MMP-17 protein was significantly (p placentas compared to gestationally matched pre-term controls, whilst MMP-25 mRNA was significantly (p placenta and indicates that MMP-14 is the only MT-MMP that contributes to soluble endoglin production in pre-eclampsia. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Amlodipine and Atorvastatin Improved Hypertensive Cardiac Remodeling through Regulation of MMPs/TIMPs in SHR Rats

    Directory of Open Access Journals (Sweden)

    Jingchao Lu

    2016-06-01

    Full Text Available Background: MMPs/TIMPs system is well known to play important roles in pressure overload-induced cardiac remodeling, and Amlodipine and Atorvastatin have been showed to exert favourable protective effects on cardiovascular disease, however, it is not clear whether Amlodipine and Atorvastatin can improve hypertensive cardiac remodeling and whether the MMPs/TIMPs system is involved. The present study aims to answer these questions. Methods: 36 weeks old male spontaneous hypertension (SHR rats were randomly divided into four groups: 1. SHR control group, 2. Amlodipine alone (10 mg/kg/d group, 3. Atorvastatin alone (10 mg/kg/d group, 4.Combination of Amlodipine and Atorvastatin (10 mg/kg/d for each group. Same gender, weight and age of Wistar-Kyoto (WKY rats with normal blood pressure were used as normal control. Drugs were administered by oral gavage over 12 weeks. The blood pressure and left ventricle mass index were measured. Enzyme activity of MMP-2 and MMP-9 was assessed with Gelatin zymography. MMP-2, MMP-9, TIMP-1 and TIMP-2 mRNA and protein expression was studied by RT-PCR and Western blot. Single factor ANOVA and LSD-t test were used in statistical analysis. Results: Treatment with Amlodipine alone or combination with atorvastatin significantly decreased blood pressure, left ventricle mass index in SHR rats (P Conclusion: Amlodipine and Atorvastatin could improve ventricular remodeling in SHR rats through intervention with the imbalance of MMP-2/TIMP-2 and MMP-9/TIMP-1 system.

  7. New Strategies for the Next Generation of Matrix-Metalloproteinase Inhibitors: Selectively Targeting Membrane-Anchored MMPs with Therapeutic Antibodies

    Directory of Open Access Journals (Sweden)

    Laetitia Devy

    2011-01-01

    Full Text Available MMP intervention strategies have met with limited clinical success due to severe toxicities. In particular, treatment with broad-spectrum MMP-inhibitors (MMPIs caused musculoskeletal pain and inflammation. Selectivity may be essential for realizing the clinical potential of MMPIs. Here we review discoveries pinpointing membrane-bound MMPs as mediators of mechanisms underlying cancer and inflammation and as possible therapeutic targets for prevention/treatment of these diseases. We discuss strategies to target these therapeutic proteases using highly selective inhibitory agents (i.e., human blocking antibodies against individual membrane-bound MMPs.

  8. Rosiglitazone attenuates the metalloprotease/anti-metalloprotease imbalance in emphysema induced by cigarette smoke: involvement of extracellular signal-regulated kinase and NFκB signaling

    Directory of Open Access Journals (Sweden)

    Hou G

    2015-04-01

    Full Text Available Gang Hou, Yan Yin, Dan Han, Qiu-yue Wang, Jian Kang Department of Respiratory Medicine, the First Hospital of China Medical University, Shenyang, People’s Republic of China Objective: We investigated how rosiglitazone attenuated cigarette smoke (CS-induced emphysema in a rat model. In particular, we focused on its possible effects on the imbalance between metalloprotease (MMP and anti-MMP activity, mitogen-activated protein kinase (MAPK phosphorylation, and nuclear factor kappa-light-chain-enhancer of activated B cell (NFκB signaling pathway over-activation.Methods: A total of 36 Wistar rats were divided into three groups (n=12 each: animals were exposed to CS for 12 weeks in the absence (the CS group or presence of 30 mg/kg rosiglitazone (the rosiglitazone-CS [RCS] group; a control group was treated with the rosiglitazone vehicle only, without any CS exposure. Histopathology of lung tissue in all groups was evaluated to grade severity of the disease. Expression levels of peroxisome proliferator-activated receptor γ (PPARγ, MMP2, and MMP9 in lung tissue were determined and compared using Western blotting and immunohistochemistry. Activation of MAPKs, NFκB, and the nuclear factor of kappa light polypeptide gene enhancer in B-cell inhibitor, alpha (IκBα phosphorylation in lung tissue was examined by Western blotting.Results: Emphysema-related pathology, based on inter-alveolar wall distance and alveolar density, was less severe in the RCS group than in the CS group. Compared with the CS group, levels of PPARγ were higher in the RCS group, and levels of MMP2 and MMP9 proteins were lower in the RCS rats. Levels of activated MAPKs and NFκB were also lower, while the IκBαphosphorylation was increased in the lung tissue of RCS rats.Conclusion: Our findings suggest that oral administration of rosiglitazone attenuates the metalloprotease activity induced by CS, and the underlying mechanism might involve the activation of signaling pathways

  9. Effect of collagen type IV, MMPs and TIMPs on remodeling of radiation pulmonary injury

    International Nuclear Information System (INIS)

    Diao Ruiying; Song Liangwen; Wang Shaoxia; Yin Jiye

    2007-01-01

    Objective: To explore the effect of collagen type IV, matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs(TIMPs) on early remodeling after radiation pulmonary injury. Methods: Right lungs of rats were irradiated by 60 Co γ-rays at a dose of 20 Gy to induce radiation pulmonary injury, and the lung specimens were taken at weeks 1, 2, 4 after irradiation. Quantitative analysis was performed on pulmonary collagen type IV, MMP-2, MMP-9, TIMP-2, TIMP-1 at the level of gene expression and protein synthesis using real-time PCR or immunohistochemistry. Results: Gene detection using real-time PCR: gene expression of collagen type IV increased at week 1 and decreased at week 2 after irradiation; MMP-2 reached peak at week 2 in which an opposed alteration trend was displayed; MMP-9 appeared a significant trend of elevation, then decrease and elevation again which was similar to those of collagen type IV; expression of TIMP-1 was lower, and there was no marked difference among all time points; TIMP-2 displayed a trend of slight elevation, then decrease and elevation again, which was opposed to MMP-2. Immunohistochemistry-image analysis: Pulmonary collagen type IV obviously increased at week 1, and began to decrease at week 2; MMP-2 decreased at week 2 and then increased; an opposed alteration trend to that of collagen type IV was displayed; alteration trend of MMP-9 was similar to that of collagen type IV but the extent was higher; gene expression of TIMP-1 slightly increased at 2 week and an opposed trend to of MMP-9 was displayed. Conclusions: Collagen type IV, MMP-2, MMP-9 and their tissue inhibitors were involved in ineffective remodeling in the early radiation pulmonary injury; MMP-2 and MMP-9 play an important role in degradation of collagen type IV; Disturbance of collagen type IV degradation might have relationship with the initiation of pulmonary fibrosis. (authors)

  10. Molecular detection and analysis of a novel metalloprotease gene of entomopathogenic Serratia marcescens strains in infected Galleria mellonella.

    Science.gov (United States)

    Tambong, J T; Xu, R; Sadiku, A; Chen, Q; Badiss, A; Yu, Q

    2014-04-01

    Serratia marcescens strains isolated from entomopathogenic nematodes (Rhabditis sp.) were examined for their pathogenicity and establishment in wax moth (Galleria mellonella) larvae. All the Serratia strains were potently pathogenic to G. mellonella larvae, leading to death within 48 h. The strains were shown to possess a metalloprotease gene encoding for a novel serralysin-like protein. Rapid establishment of the bacteria in infected larvae was confirmed by specific polymerase chain reaction (PCR) detection of a DNA fragment encoding for this protein. Detection of the viable Serratia strains in infected larvae was validated using the SYBR Green reverse transcriptase real-time PCR assay targeting the metalloprotease gene. Nucleotide sequences of the metalloprotease gene obtained in our study showed 72 single nucleotide polymorphisms (SNP) and 3 insertions compared with the metalloprotease gene of S. marcescens E-15. The metalloprotease gene had 60 synonymous and 8 nonsynonymous substitutions relative to the closest GenBank entry, S. marcescens E-15. A comparison of the amino acid composition of the new serralysin-like protein with that of the serralysin protein of S. marcescens E-15 revealed differences at 11 positions and a new aspartic acid residue. Analysis of the effect of protein variation suggests that a new aspartic acid residue resulting from nonsynonymous nucleotide mutations in the protein structure could have the most significant effect on its biological function. The new metalloprotease gene and (or) its product could have applications in plant agricultural biotechnology.

  11. ADAM 12, a disintegrin metalloprotease, interacts with insulin-like growth factor-binding protein-3

    DEFF Research Database (Denmark)

    Shi, Z; Xu, Wei; Loechel, F

    2000-01-01

    Insulin-like growth factor-binding protein (IGFBP)-3 binds the insulin-like growth factors with high affinity and modulates their actions. Proteolytic cleavage of IGFBP-3 may regulate insulin-like growth factor bioavailability. IGFBP-3 is extensively degraded in serum during pregnancy; however......, as yet the pregnancy-specific protease, or proteases, have not been identified. We utilized a yeast two-hybrid assay and a human placental cDNA library to investigate IGFBP-3-interacting proteins. A disintegrin and metalloprotease-12 (ADAM 12), a member of a family of metalloprotease disintegrins...... protein. To verify the interaction between ADAM 12 and IGFBP-3, an expression construct containing an ADAM 12-S cDNA was transfected into COS-1 cells. Co-precipitation was observed when conditioned medium was analyzed by immunoprecipitation with an antibody against either ADAM 12 or IGFBP-3 followed...

  12. Identifying common metalloprotease inhibitors by protein fold types using Fourier transform mass spectrometry.

    Science.gov (United States)

    Mitchell, Jennifer K; Pitcher, Desley; McArdle, Bernadette M; Alnefelt, Terese; Duffy, Sandra; Avery, Vicky; Quinn, Ronald J

    2007-12-01

    Fourteen natural products, known to inhibit other proteins of the Zincin-like fold class, were screened for inhibition of the Zincin-like fold metalloprotease thermolysin using mass spectrometry. Fourier Transform Mass Spectrometry was successful in identifying actinonin, a known inhibitor of astacin and stromelysin, to be an inhibitor of thermolysin. Molecular modelling studies have shown that specificity within the Zincin-like fold is determined by Protein Fold Topology.

  13. Comparison of the Internal Dynamics of Metalloproteases Provides New Insights on Their Function and Evolution.

    Directory of Open Access Journals (Sweden)

    Henrique F Carvalho

    Full Text Available Metalloproteases have evolved in a vast number of biological systems, being one of the most diverse types of proteases and presenting a wide range of folds and catalytic metal ions. Given the increasing understanding of protein internal dynamics and its role in enzyme function, we are interested in assessing how the structural heterogeneity of metalloproteases translates into their dynamics. Therefore, the dynamical profile of the clan MA type protein thermolysin, derived from an Elastic Network Model of protein structure, was evaluated against those obtained from a set of experimental structures and molecular dynamics simulation trajectories. A close correspondence was obtained between modes derived from the coarse-grained model and the subspace of functionally-relevant motions observed experimentally, the later being shown to be encoded in the internal dynamics of the protein. This prompted the use of dynamics-based comparison methods that employ such coarse-grained models in a representative set of clan members, allowing for its quantitative description in terms of structural and dynamical variability. Although members show structural similarity, they nonetheless present distinct dynamical profiles, with no apparent correlation between structural and dynamical relatedness. However, previously unnoticed dynamical similarity was found between the relevant members Carboxypeptidase Pfu, Leishmanolysin, and Botulinum Neurotoxin Type A, despite sharing no structural similarity. Inspection of the respective alignments shows that dynamical similarity has a functional basis, namely the need for maintaining proper intermolecular interactions with the respective substrates. These results suggest that distinct selective pressure mechanisms act on metalloproteases at structural and dynamical levels through the course of their evolution. This work shows how new insights on metalloprotease function and evolution can be assessed with comparison schemes that

  14. Modulation of the epithelial sodium channel (ENaC by bacterial metalloproteases and protease inhibitors.

    Directory of Open Access Journals (Sweden)

    Michael B Butterworth

    Full Text Available The serralysin family of metalloproteases is associated with the virulence of multiple gram-negative human pathogens, including Pseudomonas aeruginosa and Serratia marcescens. The serralysin proteases share highly conserved catalytic domains and show evolutionary similarity to the mammalian matrix metalloproteases. Our previous studies demonstrated that alkaline protease (AP from Pseudomonas aeruginosa is capable of activating the epithelial sodium channel (ENaC, leading to an increase in sodium absorption in airway epithelia. The serralysin proteases are often co-expressed with endogenous, intracellular or periplasmic inhibitors, which putatively protect the bacterium from unwanted or unregulated protease activities. To evaluate the potential use of these small protein inhibitors in regulating the serralysin induced activation of ENaC, proteases from Pseudomonas aeruginosa and Serratia marcescens were purified for characterization along with a high affinity inhibitor from Pseudomonas. Both proteases showed activity against in vitro substrates and could be blocked by near stoichiometric concentrations of the inhibitor. In addition, both proteases were capable of activating ENaC when added to the apical surfaces of multiple epithelial cells with similar slow activation kinetics. The high-affinity periplasmic inhibitor from Pseudomonas effectively blocked this activation. These data suggest that multiple metalloproteases are capable of activating ENaC. Further, the endogenous, periplasmic bacterial inhibitors may be useful for modulating the downstream effects of the serralysin virulence factors under physiological conditions.

  15. Activation of MMPs in Macrophages by Mycobacterium tuberculosis via the miR-223-BMAL1 Signaling Pathway.

    Science.gov (United States)

    Lou, Jun; Wang, Yongli; Zhang, Zhimin; Qiu, Weiqiang

    2017-12-01

    An interaction between Mycobacterium tuberculosis and macrophages constitutes an essential step in tuberculosis development, as macrophages exert both positive and negative effects on M. tuberculosis-triggered organ lesions. In this study, we focused on the regulation of the expression of matrix metalloproteinases (MMPs), which is responsible for lung matrix degradation and bacteria dissection, in macrophages following M. tuberculosis infection. Female BALB/c mice were intravenously injected with the M. tuberculosis strain H37Rv at 0 h zeitgeber time (ZT0) or 12 h zeitgeber time (ZT12). The expression and activity of MMP-1, -2, -3, and -9 in lungs and spleens were then evaluated. In vitro, peritoneal macrophages were harvested at ZT0 or at ZT12 and infected with 10 MOI M. tuberculosis. The expression of MMPs, microRNA-223 and BMAL1 was analyzed by qRT-PCR and/or Western blot. The binding of BMAL1 3'-UTR by miR-223 was confirmed by luciferase activity assay. Additionally, wild-type BMAL1 or NLS mut BMAL1 plasmids were transfected to evaluate the effect of BMAL1 on MMPs. The results showed a differential expression of MMPs in mice tissues infected at different times. M. tuberculosis infection caused enhanced MMP-1, -9, and miR-223 expression, with inhibited BMAL1 expression. MiR-223 modulated BMAL1 expression via the direct binding of BMAL1 3'-UTR. Furthermore, wild-type BMAL1 other than NLS mut BMAL1 attenuated MMPs expression in M. tuberculosis-infected macrophages. Overall, this study demonstrated a potential involvement of circadian rhythm in MMP activation by M. tuberculosis in macrophages. J. Cell. Biochem. 118: 4804-4812, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  16. A comparative study of effect of autograft compared with allograft anterior cruciate ligament reconstruction on expressions ofLOXsandMMPs.

    Science.gov (United States)

    Wang, Wei-Ming; Ma, Xiao-Jun; Huang, Shi-Bo; Ren, Liu-Bao; Liu, Yu-Peng

    2017-04-30

    The present study aimed to compare the effect of autograft or allograft anterior cruciate ligament (ACL) reconstruction on the expressions of lipoxygenases ( LOXs ) and matrix metalloproteinases ( MMPs ) in a New Zealand white rabbit model. New Zealand white rabbits were divided randomly into control, sham, autograft and allograft groups. At the 4th and 8th week after operation, biomechanical testing was performed to measure the primary length, cross-sectional area, maximum tensile load and stiffness of ACL, and HE staining was used to observe cell morphology and fibre alignment of ACL. At the 2nd, 4th and 8th week after operation, quantitative real-time PCR (qRT-PCR), Western blotting and immunohistochemistry were applied to detect LOXs and MMPs expressions, and expressions of adenomatous polyposis coli (APC)/Wnt signalling pathway-related proteins. At the 4th and 8th week after operation, the maximum tensile load and stiffness were higher in the autograft group than in the allograft group, and the values at the 8th week were higher than those at the 4th week after operation. The fibroblast proliferation in the allograft group was more significant than that in the autograft group. Compared with the control group, LOXs and MMPs expressions and the positive expression rates of LOXs and MMPs proteins were elevated, and the values in the allograft group were higher than those in the autograft group at all time points. At 8th week after operation, compared with the autograft group, Wnt expression was higher and APC expression was lower in the allograft group. Autograft and allograft ACL reconstruction can promote LOXs and MMPs expressions by activating the APC/Wnt signalling pathway. © 2017 The Author(s).

  17. Structure-Based Design and Synthesis of a New Phenylboronic-Modified Affinity Medium for Metalloprotease Purification

    Directory of Open Access Journals (Sweden)

    Shangyong Li

    2016-12-01

    Full Text Available Metalloproteases are emerging as useful agents in the treatment of many diseases including arthritis, cancer, cardiovascular diseases, and fibrosis. Studies that could shed light on the metalloprotease pharmaceutical applications require the pure enzyme. Here, we reported the structure-based design and synthesis of the affinity medium for the efficient purification of metalloprotease using the 4-aminophenylboronic acid (4-APBA as affinity ligand, which was coupled with Sepharose 6B via cyanuric chloride as spacer. The molecular docking analysis showed that the boron atom was interacting with the hydroxyl group of Ser176 residue, whereas the hydroxyl group of the boronic moiety is oriented toward Leu175 and His177 residues. In addition to the covalent bond between the boron atom and hydroxyl group of Ser176, the spacer between boronic acid derivatives and medium beads contributes to the formation of an enzyme-medium complex. With this synthesized medium, we developed and optimized a one-step purification procedure and applied it for the affinity purification of metalloproteases from three commercial enzyme products. The native metalloproteases were purified to high homogeneity with more than 95% purity. The novel purification method developed in this work provides new opportunities for scientific, industrial and pharmaceutical projects.

  18. Cloning and expression of ADAM-related metalloproteases in equine laminitis.

    Science.gov (United States)

    Coyne, Michael J; Cousin, Hélène; Loftus, John P; Johnson, Philip J; Belknap, James K; Gradil, Carlos M; Black, Samuel J; Alfandari, Dominique

    2009-06-15

    Equine laminitis is a debilitating disease affecting the digital laminae that suspend the distal phalanx within the hoof. While the clinical progression of the disease has been well documented, the molecular events associated with its pathogenesis remain largely unknown. Using real time quantitative PCR (RT-qPCR), we have investigated the expression of genes coding for proteins containing a Disintegrin and Metalloprotease domain (ADAM), as well as genes encoding the natural inhibitors of these enzymes (tissue inhibitor of metalloprotease; TIMP) in horses with naturally-acquired (acute, chronic and aggravated chronic clinical cases) or experimentally-induced (black walnut extract (BWE) and starch gruel models) laminitis. Changes in expression of these enzymes and regulators may underlie the pathologic remodeling of lamellar tissue in laminitis. Genes encoding ADAMs involved in inflammation (ADAM-10 and ADAM-17), as well as those implicated in arthritis (ADAMTS-1, ADAMTS-4 and ADAMTS-5) were cloned, and the sequences used to generate specific oligonucleotide primers for the RT-qPCR experiments. Our results show that genes encoding ADAM-10 and ADAM-17 were not induced in most laminitic animals, whereas ADAMTS-4 gene expression was strongly upregulated in nearly all horses with experimentally-induced and naturally-acquired laminitis. The expression of matrix metalloproteases (MMP)-9 and ADAMTS-5 was also increased in many of the laminitic horses. In addition, TIMP-2 gene expression was decreased in most laminitic horses, whereas expression of genes encoding other TIMPs, namely TIMP-1 and TIMP-3, was randomly increased or decreased in the various models. We conclude that increased expression of lamellar ADAMTS-4 is a common feature of laminitis consistent with a central role of the gene product in the pathophysiology of the disease.

  19. Cell-surface metalloprotease ADAM12 is internalized by a clathrin- and Grb2-dependent mechanism

    DEFF Research Database (Denmark)

    Hansen, Dorte Stautz; Leyme, Anthony; Grandal, Michael Vibo

    2012-01-01

    ADAM12 (A Disintegrin And Metalloprotease 12), a member of the ADAMs family of transmembrane proteins, is involved in ectodomain shedding, cell-adhesion and signaling, with important implications in cancer. Therefore, mechanisms that regulate the levels and activity of ADAM12 at the cell...... that regulates ADAM cell surface levels and show that ADAM12 internalization involves the clathrin-dependent pathway and Grb2.......-surface are possibly crucial in these contexts. We here investigated internalization and subsequent recycling or degradation of ADAM12 as a potentially important regulatory mechanism. Our results show that ADAM12 is constitutively internalized primarily via the clathrin-dependent pathway and is subsequently detected...

  20. Functional Analysis of a Breast Cancer-Associated Mutation in the Intracellular Domain of the Metalloprotease ADAM12

    DEFF Research Database (Denmark)

    Stautz, Dorte; Wewer, Ulla M; Kveiborg, Marie

    2012-01-01

    A recently identified breast cancer-associated mutation in the metalloprotease ADAM12 alters a potential dileucine trafficking signal, which could affect protein processing and cellular localization. ADAM12 belongs to the group of A Disintegrin And Metalloproteases (ADAMs), which are typically...... membrane-associated proteins involved in ectodomain shedding, cell-adhesion, and signaling. ADAM12 as well as several members of the ADAM family are over-expressed in various cancers, correlating with disease stage. Three breast cancer-associated somatic mutations were previously identified in ADAM12......, and two of these, one in the metalloprotease domain and another in the disintegrin domain, were investigated and found to result in protein misfolding, retention in the secretory pathway, and failure of zymogen maturation. The third mutation, p.L792F in the ADAM12 cytoplasmic tail, was not investigated...

  1. Time-Dependent Alterations of MMPs, TIMPs and Tendon Structure in Human Achilles Tendons after Acute Rupture.

    Science.gov (United States)

    Minkwitz, Susann; Schmock, Aysha; Kurtoglu, Alper; Tsitsilonis, Serafeim; Manegold, Sebastian; Wildemann, Britt; Klatte-Schulz, Franka

    2017-10-20

    A balance between matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) is required to maintain tendon homeostasis. Variation in this balance over time might impact on the success of tendon healing. This study aimed to analyze structural changes and the expression profile of MMPs and TIMPs in human Achilles tendons at different time-points after rupture. Biopsies from 37 patients with acute Achilles tendon rupture were taken at surgery and grouped according to time after rupture: early (2-4 days), middle (5-6 days), and late (≥7 days), and intact Achilles tendons served as control. The histological score increased from the early to the late time-point after rupture, indicating the progression towards a more degenerative status. In comparison to intact tendons, qRT-PCR analysis revealed a significantly increased expression of MMP-1, -2, -13, TIMP-1, COL1A1, and COL3A1 in ruptured tendons, whereas TIMP-3 decreased. Comparing the changes over time post rupture, the expression of MMP-9, -13, and COL1A1 significantly increased, whereas MMP-3 and -10 expression decreased. TIMP expression was not significantly altered over time. MMP staining by immunohistochemistry was positive in the ruptured tendons exemplarily analyzed from early and late time-points. The study demonstrates a pivotal contribution of all investigated MMPs and TIMP-1, but a minor role of TIMP-2, -3, and -4, in the early human tendon healing process.

  2. Relationship of PDCD5 expression with apoptosis, inflammatory factors and MMPs/TIMPs expression in degenerated intervertebral disc tissue

    Directory of Open Access Journals (Sweden)

    Xiao-Hua Gu

    2017-11-01

    Full Text Available Objective: To study the relationship of PDCD5 expression with apoptosis, inflammatory factors and MMPs/TIMPs expression in degenerated intervertebral disc tissue. Methods: Patients with lumbar disc herniation who were treated in the Seventh People's Hospital of Shanghai between March 2015 and February 2017 were selected as the LDH group and patients with violent thoracolumbar vertebral fracture were selected as the control group. The intervertebral disc tissue was collected to determine the mRNA expression of PDCD5 as well as the protein levels of apoptosis molecules, inflammatory factors and MMPs/TIMPs molecules. Results: PDCD5 mRNA expression in intervertebral disc tissue of LDH group was significantly higher than that of control group; Caspase-3, Caspase-8, Fas, Caspase-9, Bax, SDF-1, CXCR-4, TNF-α, PGE2, MMP1, MMP2, MMP8 and MMP9 protein levels in intervertebral disc tissue of LDH group were significantly higher than those of control group and positively correlated with PDCD5 mRNA expression while TIMP1 and TIMP2 protein levels were significantly lower than those of control group and negatively correlated with PDCD5 mRNA expression. Conclusion: The high expression of PDCD5 in degenerated intervertebral disc tissue can activate apoptosis and inflammatory response and cause MMPs/ TIMPs imbalance.

  3. SIMULATION RESULTS OF RUNNING THE AGS MMPS, BY STORING ENERGY IN CAPACITOR BANKS.

    Energy Technology Data Exchange (ETDEWEB)

    MARNERIS, I.

    2006-09-01

    The Brookhaven AGS is a strong focusing accelerator which is used to accelerate protons and various heavy ion species to equivalent maximum proton energy of 29 GeV. The AGS Main Magnet Power Supply (MMPS) is a thyristor control supply rated at 5500 Amps, +/-go00 Volts. The peak magnet power is 49.5 Mwatts. The power supply is fed from a motor/generator manufactured by Siemens. The motor is rated at 9 MW, input voltage 3 phase 13.8 KV 60 Hz. The generator is rated at 50 MVA its output voltage is 3 phase 7500 Volts. Thus the peak power requirements come from the stored energy in the rotor of the motor/generator. The rotor changes speed by about +/-2.5% of its nominal speed of 1200 Revolutions per Minute. The reason the power supply is powered by the Generator is that the local power company (LIPA) can not sustain power swings of +/- 50 MW in 0.5 sec if the power supply were to be interfaced directly with the AC lines. The Motor Generator is about 45 years old and Siemens is not manufacturing similar machines in the future. As a result we are looking at different ways of storing energy and being able to utilize it for our application. This paper will present simulations of a power supply where energy is stored in capacitor banks. The simulation program used is called PSIM Version 6.1. The control system of the power supply will also be presented. The average power from LIPA into the power supply will be kept constant during the pulsing of the magnets at +/-50 MW. The reactive power will also be kept constant below 1.5 MVAR. Waveforms will be presented.

  4. Tempol inhibits TGF-β and MMPs upregulation and prevents cardiac hypertensive changes.

    Science.gov (United States)

    Rizzi, Elen; Castro, Michele M; Ceron, Carla S; Neto-Neves, Evandro M; Prado, Cibele M; Rossi, Marcos A; Tanus-Santos, Jose E; Gerlach, Raquel F

    2013-04-30

    Increased oxidative stress upregulates matrix metalloproteinases (MMPs) and transforming grow factor (TGF-β), which are involved in hypertensive cardiac remodeling. We tested the hypothesis that tempol (an antioxidant) could prevent these alterations in two-kidney, one-clip (2K1C) hypertension. Sham-operated or hypertensive rats were treated with tempol (18 mg.kg(-1)day(-1) or vehicle) for 8 weeks. Systolic blood pressure was monitored weekly. At the end of the treatment, a catheter was inserted into the left carotid artery and into the left ventricle (LV) to assess arterial blood pressure and contractile function. Morphometry of the LV was carried out in hematoxylin/eosin sections and fibrosis was assessed in picrosirius red-stained sections. Cardiac TGF-β level was evaluated by immunofluorescence. Cardiac MMP-2 levels and activity were determined by gelatin zymography, in situ zymography, and immunofluorescence. Cardiac superoxide production was evaluated by dihydroethidium probe. Tempol treatment attenuated 2K1C-induced hypertension and reversed the contractile dysfunction in 2K1C rats. Cardiac hypertrophy was ameliorated by antioxidant treatment. Hypertensive rats showed increased cardiac MMP-2 levels, however tempol did not decrease MMP-2 levels. Increased TGF-β level, total gelatinolytic activity and oxidative stress were found in untreated 2K1C rats. Tempol treatment decreased oxidative stress, TGF-β levels, and gelatinolytic activity in 2K1C rats to control levels. Tempol blunted the increases in TGF-β, the proteolytic imbalance, and the morphological and functional alterations found in 2K1C-induced cardiac hypertrophy. These findings are consistent with the idea that antioxidants may help to prevent hypertension-induced cardiac hypertrophy. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  5. Secretome analysis to elucidate metalloprotease-dependent ectodomain shedding of glycoproteins during neuronal differentiation.

    Science.gov (United States)

    Tsumagari, Kazuya; Shirakabe, Kyoko; Ogura, Mayu; Sato, Fuminori; Ishihama, Yasushi; Sehara-Fujisawa, Atsuko

    2017-02-01

    Many membrane proteins are subjected to limited proteolyses at their juxtamembrane regions, processes referred to as ectodomain shedding. Shedding ectodomains of membrane-bound ligands results in activation of downstream signaling pathways, whereas shedding those of cell adhesion molecules causes loss of cell-cell contacts. Secreted proteomics (secretomics) using high-resolution mass spectrometry would be strong tools for both comprehensive identification and quantitative measurement of membrane proteins that undergo ectodomain shedding. In this study, to elucidate the ectodomain shedding events that occur during neuronal differentiation, we establish a strategy for quantitative secretomics of glycoproteins released from differentiating neuroblastoma cells into culture medium with or without GM6001, a broad-spectrum metalloprotease inhibitor. Considering that most of transmembrane and secreted proteins are N-glycosylated, we include a process of N-glycosylated peptides enrichment as well as isotope tagging in our secretomics workflow. Our results show that differentiating N1E-115 neurons secrete numerous glycosylated polypeptides in metalloprotease-dependent manners. They are derived from cell adhesion molecules such as NCAM1, CADM1, L1CAM, various transporters and receptor proteins. These results show the landscape of ectodomain shedding and other secretory events in differentiating neurons and/or during axon elongation, which should help elucidate the mechanism of neurogenesis and the pathogenesis of neurological disorders. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  6. Elucidating the Origin of Long Residence Time Binding for Inhibitors of the Metalloprotease Thermolysin.

    Science.gov (United States)

    Cramer, Jonathan; Krimmer, Stefan G; Fridh, Veronica; Wulsdorf, Tobias; Karlsson, Robert; Heine, Andreas; Klebe, Gerhard

    2017-01-20

    Kinetic parameters of protein-ligand interactions are progressively acknowledged as valuable information for rational drug discovery. However, a targeted optimization of binding kinetics is not easy to achieve, and further systematic studies are necessary to increase the understanding about molecular mechanisms involved. We determined association and dissociation rate constants for 17 inhibitors of the metalloprotease thermolysin by surface plasmon resonance spectroscopy and correlated kinetic data with high-resolution crystal structures in complex with the protein. From the structure-kinetics relationship, we conclude that the strength of interaction with Asn112 correlates with the rate-limiting step of dissociation. This residue is located at the beginning of a β-strand motif that lines the binding cleft and is commonly believed to align a substrate for catalysis. A reduced mobility of the Asn112 side chain owing to an enhanced engagement in charge-assisted hydrogen bonds prevents the conformational adjustment associated with ligand release and transformation of the enzyme to its open state. This hypothesis is supported by kinetic data of ZF P LA, a known pseudopeptidic inhibitor of thermolysin, which blocks the conformational transition of Asn112. Interference with this retrograde induced-fit mechanism results in variation of the residence time of thermolysin inhibitors by a factor of 74 000. The high conservation of this structural motif within the M4 and M13 metalloprotease families underpins the importance of this feature and has significant implications for drug discovery.

  7. Disseminated intravascular coagulation caused by moojenactivase, a procoagulant snake venom metalloprotease.

    Science.gov (United States)

    Sartim, Marco A; Cezarette, Gabriel N; Jacob-Ferreira, Anna L; Frantz, Fabiani G; Faccioli, Lucia H; Sampaio, Suely V

    2017-10-01

    Snake venom toxins that activate coagulation factors are key players in the process of venom-induced coagulopathy, and account for severe clinical manifestations. The present study applies a variety of biochemical, hematological, and histopathological approaches to broadly investigate the intravascular and systemic effects of moojenactivase (MooA), the first described PIIId subclass metalloprotease isolated from Bothrops sp. venom that activates coagulation factors. MooA induced consumption coagulopathy with high toxic potency, characterized by prolongation of prothrombin and activated partial thromboplastin time, consumption of fibrinogen and the plasma coagulation factors X and II, and thrombocytopenia. MooA promoted leukocytosis and expression of the proinflammatory cytokines interleukin-6 and tumor necrosis factor-α, accompanied by tissue factor-dependent procoagulant activity in peripheral blood mononuclear cells. This metalloprotease also caused intravascular hemolysis, elevated plasma levels of creatine kinase-MB, aspartate transaminase, and urea/creatinine, and induced morphopathological alterations in erythrocytes, heart, kidney, and lungs associated with thrombosis and hemorrhage. Diagnosis of MooA-induced disseminated intravascular coagulation represents an important approach to better understand the pathophysiology of Bothrops envenomation and develop novel therapeutic strategies targeting hemostatic disturbances. Copyright © 2017. Published by Elsevier B.V.

  8. Matrix Metalloprotease 2-Responsive Multifunctional Liposomal Nanocarrier for Enhanced Tumor Targeting

    Science.gov (United States)

    Zhu, Lin; Kate, Pooja; Torchilin, Vladimir P.

    2012-01-01

    A novel “smart” multifunctional drug delivery system was successfully developed to respond to the up-regulated matrix metalloprotease 2 (MMP2) in the tumor microenvironment and improve cancer cell-specific delivery of loaded drugs. The system represents a surface-functionalized liposomal nanocarrier, for which two functional polyethylene glycol (PEG)-lipid conjugates were synthesized and characterized. The functionalized liposome was further modified with the tumor cell-specific anti-nucleosome monoclonal antibody (mAb 2C5). In the resulting system, several drug delivery strategies were combined in the same nanocarrier in a simple way and coordinated in an optimal fashion. The functions of the nanocarrier include: i) the hydrophilic and flexible long PEG chains to prevent nanocarrier non-specific interactions and prolong its circulation time; ii) a nanoscale size of the system that allows for its passive tumor targeting via the enhanced permeability and retention (EPR) effect; iii) a mAb 2C5 to allow for the specific targeting of tumor cells; iv) a matrix metalloprotease 2-sensitive bond between PEG and lipid that undergoes cleavage in the tumor by the highly expressed extracellular MMP2 for the removal of PEG chains; v) The cell-penetrating peptide (TATp) triggering of the enhanced intracellular delivery of the system after long-chain PEG removal and exposure of the previously hidden surface-attached TATp. It is shown that such a design can enhance the targetability and internalization of nanocarriers in cancer cells. PMID:22409425

  9. Effect of simvastatin on MMPs and TIMPs in cigarette smoke-induced rat COPD model

    Directory of Open Access Journals (Sweden)

    Sun J

    2017-02-01

    Full Text Available Jiawei Sun,1,* Jie Bao,2,* Yanan Shi,3 Bin Zhang,4 Lindong Yuan,5 Junhong Li,1 Lihai Zhang,1 Mo Sun,6 Ling Zhang,2 Wuzhuang Sun1 1Department of Respiratory Medicine, First Hospital of Hebei Medical University, 2Department of Respiratory Medicine, Chest Hospital of Hebei Province, 3Maternal and Child Health Care Center of Hebei Province, 4Department of Emergency, First Hospital of Hebei Medical University, Shijiazhuang, 5Department of Respiratory Medicine, People’s Hospital of Liaocheng, Liaocheng, 6Hebei Medical University, Shijiazhuang, People’s Republic of China *These authors contributed equally to this work Background: Proteases may play an important role in the development of chronic obstructive pulmonary disease and emphysema in response to cigarette smoke exposure (CSE. The current study was designed to investigate the expression of matrix metalloproteinase (MMP-8, MMP-9, MMP-12, tissue inhibitor of MMP (TIMP-1, and TIMP-4 in rat lung tissues in response to CSE, and assessed the effect of simvastatin in regulating expression of MMPs and TIMPs.Methods: Thirty normal Sprague Dawley (SD rats were divided into control (n=10, CSE (n=10, and CSE plus simvastatin (n=10 groups. Animals were whole-body exposed to the cigarette smoke in the box for 1 hour each time, twice a day, 5 days a week for 16 weeks. Animals of CSE + simvastatin group were intra-gastrically administered simvastatin at a dose of 5 mg/kg/day followed by CSE. Bronchoalveolar lavage fluid was harvested for inflammatory cell count and lung tissues were stained for morphologic examination. Expression of mRNA and protein level of MMP-8, MMP-9, MMP-12, TIMP-1, and TIMP-4 was assessed by real-time reverse transcription polymerase chain reaction and immunohistochemistry, respectively.Results: CSE resulted in a significant increase of mean linear intercept (MLI: 34.6±2.0 µm and bronchial wall thickness and diameter (BWT/D, 0.250±0.062 compared to control (MLI: 24.0±1.7 µm, BWT

  10. The Matrix Metalloproteases and Endothelin-1 in Infection-Associated Preterm Birth

    Directory of Open Access Journals (Sweden)

    Nicole S. Olgun

    2010-01-01

    Full Text Available Preterm birth (PTB is clinically defined as any delivery which occurs before the completion of 37 weeks of gestation, and is currently the most important problem in obstetrics. In the United States, PTB accounts for 12-13% of all live births, and, with the exception of fetuses suffering from anomalies, is the primary cause of perinatal mortality. While the risk factors for PTB are numerous, the single most common cause is intrauterine infection. As there is currently no FDA-approved therapy for infection-associated PTB, understanding the pathogenesis of preterm labor (PTL and delivery should be given high priority. The matrix metalloproteinases (MMPs are a family of enzymes that have been implicated in normal parturition as well as infection-triggered rupture of membranes and preterm birth. Several lines of evidence also suggest a role for endothelin-1 (ET-1 in infection-associated preterm delivery. This paper focuses on the evidence that the MMPs and ET-1 act in the same molecular pathway in preterm birth.

  11. Generation of biologically active endostatin fragments from human collagen XVIII by distinct matrix metalloproteases

    International Nuclear Information System (INIS)

    Heljasvaara, Ritva; Nyberg, Pia; Luostarinen, Jani; Parikka, Mataleena; Heikkilae, Pia; Rehn, Marko; Sorsa, Timo; Salo, Tuula; Pihlajaniemi, Taina

    2005-01-01

    Endostatin, a potent inhibitor of endothelial cell proliferation, migration, angiogenesis and tumor growth, is proteolytically cleaved from the C-terminal noncollagenous NC1 domain of type XVIII collagen. We investigated the endostatin formation from human collagen XVIII by several MMPs in vitro. The generation of endostatin fragments differing in molecular size (24-30 kDa) and in N-terminal sequences was identified in the cases of MMP-3, -7, -9, -13 and -20. The cleavage sites were located in the protease-sensitive hinge region between the trimerization and endostatin domains of NC1. MMP-1, -2, -8 and -12 did not show any significant activity against the C-terminus of collagen XVIII. The anti-proliferative effect of the 20-kDa endostatin, three longer endostatin-containing fragments generated in vitro by distinct MMPs and the entire NC1 domain, on bFGF-stimulated human umbilical vein endothelial cells was established. The anti-migratory potential of some of these fragments was also studied. In addition, production of endostatin fragments between 24-30 kDa by human hepatoblastoma cells was shown to be due to MMP action on type XVIII collagen. Our results indicate that certain, especially cancer-related, MMP family members can generate biologically active endostatin-containing polypeptides from collagen XVIII and thus, by releasing endostatin fragments, may participate in the inhibition of endothelial cell proliferation, migration and angiogenesis

  12. JAMM: A Metalloprotease-like zinc site in the proteasome and signalosome

    Energy Technology Data Exchange (ETDEWEB)

    Ambroggio, X.I.; Rees, D.C.; Deshaies, R.J.

    2005-06-27

    The JAMM (JAB1/MPN/Mov34 metalloenzyme) motif in Rpn11 and Csn5 underlies isopeptidase activities intrinsic to the proteasome and signalosome, respectively. We show here that the archaebacterial protein AfJAMM possesses the key features of a zinc metalloprotease, yet with a distinct fold. The histidine and aspartic acid of the conserved EXnHS/THX7SXXD motif coordinate a zinc, whereas the glutamic acid hydrogen-bonds an aqua ligand. By analogy to the active site of thermolysin, we predict that the glutamic acid serves as an acid-base catalyst and the second serine stabilizes a tetrahedral intermediate. Mutagenesis of Csn5 confirms these residues are required for Nedd8 isopeptidase activity. The active site-like architecture specified by the JAMM motif motivates structure-based approaches to the study of JAMM domain proteins and the development of therapeutic proteasome and signalosome inhibitors.

  13. JAMM: a metalloprotease-like zinc site in the proteasome and signalosome.

    Directory of Open Access Journals (Sweden)

    Xavier I Ambroggio

    2004-01-01

    Full Text Available The JAMM (JAB1/MPN/Mov34 metalloenzyme motif in Rpn11 and Csn5 underlies isopeptidase activities intrinsic to the proteasome and signalosome, respectively. We show here that the archaebacterial protein AfJAMM possesses the key features of a zinc metalloprotease, yet with a distinct fold. The histidine and aspartic acid of the conserved EX(nHS/THX(7SXXD motif coordinate a zinc, whereas the glutamic acid hydrogen-bonds an aqua ligand. By analogy to the active site of thermolysin, we predict that the glutamic acid serves as an acid-base catalyst and the second serine stabilizes a tetrahedral intermediate. Mutagenesis of Csn5 confirms these residues are required for Nedd8 isopeptidase activity. The active site-like architecture specified by the JAMM motif motivates structure-based approaches to the study of JAMM domain proteins and the development of therapeutic proteasome and signalosome inhibitors.

  14. A silanediol inhibitor of the metalloprotease thermolysin: synthesis and comparison with a phosphinic acid inhibitor.

    Science.gov (United States)

    Kim, Jaeseung; Sieburth, Scott McN

    2004-04-30

    A silanediol inhibitor of the metalloprotease thermolysin was prepared for comparison to a known phosphinic acid inhibitor, providing the first comparison of these second-row element based transition-state analogues. Inhibition of thermolysin by the silanediol (K(i) = 41 nM) was comparable to that of the phosphinic acid (K(i) = 10 nM) even though the silanediol is uncharged and thereby lacks the intrinsic Coulombic attraction of the phosphinate anion to the active-site zinc cation. This silanediol protease inhibitor is the least sterically encumbered example prepared to date and, therefore, the most prone toward polymerization. Hydrolysis of a difluorosilane intermediate to the silanediol leads cleanly to a monomeric product.

  15. JAMM: A Metalloprotease-Like Zinc Site in the Proteasome and Signalosome

    Science.gov (United States)

    Ambroggio, Xavier I; Rees, Douglas C

    2004-01-01

    The JAMM (JAB1/MPN/Mov34 metalloenzyme) motif in Rpn11 and Csn5 underlies isopeptidase activities intrinsic to the proteasome and signalosome, respectively. We show here that the archaebacterial protein AfJAMM possesses the key features of a zinc metalloprotease, yet with a distinct fold. The histidine and aspartic acid of the conserved EXnHS/THX7SXXD motif coordinate a zinc, whereas the glutamic acid hydrogen-bonds an aqua ligand. By analogy to the active site of thermolysin, we predict that the glutamic acid serves as an acid-base catalyst and the second serine stabilizes a tetrahedral intermediate. Mutagenesis of Csn5 confirms these residues are required for Nedd8 isopeptidase activity. The active site-like architecture specified by the JAMM motif motivates structure-based approaches to the study of JAMM domain proteins and the development of therapeutic proteasome and signalosome inhibitors. PMID:14737182

  16. The Metalloprotease Mpl Supports Listeria monocytogenes Dissemination through Resolution of Membrane Protrusions into Vacuoles.

    Science.gov (United States)

    Alvarez, Diego E; Agaisse, Hervé

    2016-06-01

    Listeria monocytogenes is an intracellular pathogen that disseminates within the intestinal epithelium through acquisition of actin-based motility and formation of plasma membrane protrusions that project into adjacent cells. The resolution of membrane protrusions into vacuoles from which the pathogen escapes results in bacterial spread from cell to cell. This dissemination process relies on the mlp-actA-plcB operon, which encodes ActA, a bacterial nucleation-promoting factor that mediates actin-based motility, and PlcB, a phospholipase that mediates vacuole escape. Here we investigated the role of the metalloprotease Mpl in the dissemination process. In agreement with previous findings showing that Mpl is required for PlcB activation, infection of epithelial cells with the ΔplcB or Δmpl strains resulted in the formation of small infection foci. As expected, the ΔplcB strain displayed a strong defect in vacuole escape. However, the Δmpl strain showed an unexpected defect in the resolution of protrusions into vacuoles, in addition to the expected but mild defect in vacuole escape. The Δmpl strain displayed increased levels of ActA on the bacterial surface in protrusions. We mapped an Mpl-dependent processing site in ActA between amino acid residues 207 to 238. Similar to the Δmpl strain, the ΔactA207-238 strain displayed increased levels of ActA on the bacterial surface in protrusions. Although the ΔactA207-238 strain displayed wild-type actin-based motility, it formed small infection foci and failed to resolve protrusions into vacuoles. We propose that, in addition to its role in PlcB processing and vacuole escape, the metalloprotease Mpl is required for ActA processing and protrusion resolution. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  17. A novel synthetic quinolinone inhibitor presents proteolytic and hemorrhagic inhibitory activities against snake venom metalloproteases.

    Science.gov (United States)

    Baraldi, Patrícia T; Magro, Angelo J; Matioli, Fábio F; Marcussi, Silvana; Lemke, Ney; Calderon, Leonardo A; Stábeli, Rodrigo G; Soares, Andreimar M; Correa, Arlene G; Fontes, Marcos R M

    2016-02-01

    Metalloproteases play a fundamental role in snake venom envenomation inducing hemorrhagic, fibrigen(ogen)olytic and myotoxic effects in their victims. Several snake venoms, such as those from the Bothrops genus, present important local effects which are not efficiently neutralized by conventional serum therapy. Consequently, these accidents may result in permanent sequelae and disability, creating economic and social problems, especially in developing countries, leading the attention of the World Health Organization that considered ophidic envenomations a neglected tropical disease. Aiming to produce an efficient inhibitor against bothropic venoms, we synthesized different molecules classified as quinolinones - a group of low-toxic chemical compounds widely used as antibacterial and antimycobacterial drugs - and tested their inhibitory properties against hemorrhage caused by bothropic venoms. The results from this initial screening indicated the molecule 2-hydroxymethyl-6-methoxy-1,4-dihydro-4-quinolinone (Q8) was the most effective antihemorrhagic compound among all of the assayed synthetic quinolinones. Other in vitro and in vivo experiments showed this novel compound was able to inhibit significantly the hemorrhagic and/or proteolytic activities of bothropic crude venoms and isolated snake venom metalloproteases (SVMPs) even at lower concentrations. Docking and molecular dynamic simulations were also performed to get insights into the structural basis of Q8 inhibitory mechanism against proteolytic and hemorrhagic SVMPs. These structural studies demonstrated that Q8 may form a stable complex with SVMPs, impairing the access of substrates to the active sites of these toxins. Therefore, both experimental and structural data indicate that Q8 compound is an interesting candidate for antiophidic therapy, particularly for the treatment of the hemorrhagic and necrotic effects induced by bothropic venoms. Copyright © 2015 Elsevier B.V. and Société Française de

  18. Activation of the epidermal growth factor receptor (EGFR) by a novel metalloprotease pathway.

    LENUS (Irish Health Repository)

    Bergin, David A

    2008-11-14

    Neutrophil Elastase (NE) is a pro-inflammatory protease present at higher than normal levels in the lung during inflammatory disease. NE regulates IL-8 production from airway epithelial cells and can activate both EGFR and TLR4. TACE\\/ADAM17 has been reported to trans-activate EGFR in response to NE. Here, using 16HBE14o-human bronchial epithelial cells we demonstrate a new mechanism by which NE regulates both of these events. A high molecular weight soluble metalloprotease activity detectable only in supernatants from NE-treated cells by gelatin and casein zymography was confirmed to be meprin alpha by Western immunoblotting. In vitro studies demonstrated the ability of NE to activate meprin alpha, which in turn could release soluble TGFalpha and induce IL-8 production from 16HBE14o- cells. These effects were abrogated by actinonin, a specific meprin inhibitor. NE-induced IL-8 expression was also inhibited by meprin alpha siRNA. Immunoprecipitation studies detected EGFR\\/TLR4 complexes in NE-stimulated cells overexpressing these receptors. Confocal studies confirmed colocalization of EGFR and TLR4 in 16HBE14o- cells stimulated with meprin alpha. NFkappaB was also activated via MyD88 in these cells by meprin alpha. In bronchoalveolar lavage fluid from NE knock-out mice infected intra-tracheally with Pseudomonas aeruginosa meprin alpha was significantly decreased compared with control mice, and was significantly increased and correlated with NE activity, in bronchoalveolar lavage fluid from individuals with cystic fibrosis but not healthy controls. The data describe a previously unidentified lung metalloprotease meprin alpha, and its role in NE-induced EGFR and TLR4 activation and IL-8 production.

  19. Expression of matrix metalloproteinases (MMPs) in primary human breast cancer and breast cancer cell lines: New findings and review of the literature

    International Nuclear Information System (INIS)

    Köhrmann, Andrea; Kammerer, Ulrike; Kapp, Michaela; Dietl, Johannes; Anacker, Jelena

    2009-01-01

    Matrix metalloproteinases (MMPs) are a family of structural and functional related endopeptidases. They play a crucial role in tumor invasion and building of metastatic formations because of their ability to degrade extracellular matrix proteins. Under physiological conditions their activity is precisely regulated in order to prevent tissue disruption. This physiological balance seems to be disrupted in cancer making tumor cells capable of invading the tissue. In breast cancer different expression levels of several MMPs have been found. To fill the gap in our knowledge about MMP expression in breast cancer, we analyzed the expression of all known human MMPs in a panel of twenty-five tissue samples (five normal breast tissues, ten grade 2 (G2) and ten grade 3 (G3) breast cancer tissues). As we found different expression levels for several MMPs in normal breast and breast cancer tissue as well as depending on tumor grade, we additionally analyzed the expression of MMPs in four breast cancer cell lines (MCF-7, MDA-MB-468, BT 20, ZR 75/1) commonly used in research. The results could thus be used as model for further studies on human breast cancer. Expression analysis was performed on mRNA and protein level using semiquantitative RT-PCR, Western blot, immunohistochemistry and immunocytochemistry. In summary, we identified several MMPs (MMP-1, -2, -8, -9, -10, -11, -12, -13, -15, -19, -23, -24, -27 and -28) with a stronger expression in breast cancer tissue compared to normal breast tissue. Of those, expression of MMP-8, -10, -12 and -27 is related to tumor grade since it is higher in analyzed G3 compared to G2 tissue samples. In contrast, MMP-7 and MMP-27 mRNA showed a weaker expression in tumor samples compared to healthy tissue. In addition, we demonstrated that the four breast cancer cell lines examined, are constitutively expressing a wide variety of MMPs. Of those, MDA-MB-468 showed the strongest mRNA and protein expression for most of the MMPs analyzed. MMP-1, -2

  20. Potential of Pseudoshikonin I Isolated from Lithospermi Radix as Inhibitors of MMPs in IL-1β-Induced SW1353 Cells

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    Dae Young Lee

    2016-08-01

    Full Text Available Pseudoshikonin I, the new bioactive constituent of Lithospermi radix, was isolated from this methanol extract by employing reverse-phase medium-pressure liquid chromatography (MPLC using acetonitrile/water solvent system as eluents. The chemical structure was determined based on spectroscopic techniques, including 1D NMR (1H, 13C, DEPT, 2D NMR (gCOSY, gHMBC, gHMQC, and QTOF/MS data. In this study, we demonstrated the effect of pseudoshikonin I on matrix-metalloproteinase (MMPs activation and expression in interleukin (IL-1β-induced SW1353 chondrosarcoma cells. MMPs are considered important for the maintenance of the extracellular matrix. Following treatment with PS, active MMP-1, -2, -3, -9, -13 and TIMP-2 were quantified in the SW1353 cell culture supernatants using a commercially available ELISA kit. The mRNA expression of MMPs in SW1353 cells was measured by RT-PCR. Pseudoshikonin I treatment effectively protected the activation on all tested MMPs in a dose-dependent manner. TIMP-2 mRNA expression was significantly upregulated by pseudoshikonin I treatment. Overall, we elucidated the inhibitory effect of pseudoshikonin on MMPs, and we suggest its use as a potential novel anti-osteoarthritis agent.

  1. Cyclic mechanical strain-induced proliferation and migration of human airway smooth muscle cells: role of EMMPRIN and MMPs.

    Science.gov (United States)

    Hasaneen, Nadia A; Zucker, Stanley; Cao, Jian; Chiarelli, Christian; Panettieri, Reynold A; Foda, Hussein D

    2005-09-01

    Airway smooth muscle (ASM) proliferation and migration are major components of airway remodeling in asthma. Asthmatic airways are exposed to mechanical strain, which contributes to their remodeling. Matrix metalloproteinase (MMP) plays an important role in remodeling. In the present study, we examined if the mechanical strain of human ASM (HASM) cells contributes to their proliferation and migration and the role of MMPs in this process. HASM were exposed to mechanical strain using the FlexCell system. HASM cell proliferation, migration and MMP release, activation, and expression were assessed. Our results show that cyclic strain increased the proliferation and migration of HASM; cyclic strain increased release and activation of MMP-1, -2, and -3 and membrane type 1-MMP; MMP release was preceded by an increase in extracellular MMP inducer; Prinomastat [a MMP inhibitor (MMPI)] significantly decreased cyclic strain-induced proliferation and migration of HASM; and the strain-induced increase in the release of MMPs was accompanied by an increase in tenascin-C release. In conclusion, cyclic mechanical strain plays an important role in HASM cell proliferation and migration. This increase in proliferation and migration is through an increase in MMP release and activation. Pharmacological MMPIs should be considered in the pursuit of therapeutic options for airway remodeling in asthma.

  2. Value of preoperative serum LC3 and MMPs combined with TSH detection in diagnosis of papillary thyroid carcinoma

    Directory of Open Access Journals (Sweden)

    Yan-Fei Lu

    2016-03-01

    Full Text Available Objective: To study the value of preoperative serum LC3 and MMPs combined with TSH detection in diagnosis of papillary thyroid carcinoma and provide reference for clinical diagnosis and treatment. Methods: A total of 80 cases of patients with papillary thyroid carcinoma treated in our hospital from March 2010 to March 2014 were analyzed, and serum TSH, MMP2/9, TIMP1/2 and LC3 levels of patients before operation were detected by ELISA. Healthy subjects and patients with benign neoplasm of thyroid during the same period were taken as control. Results: Serum TSH, MMP2, MMP9 and LC3 levels in patients with papillary thyroid carcinoma significantly increased, TIMP1 and TIMP2 levels significantly decreased, and compared with healthy subjects and patients with benign neoplasm of thyroid, there were significant statistical differences; at the same time, above parameters in serum were not related to gender, but closely related to age, clinical stage and diameter of tumor as well as lymph node. Conclusion: Preoperative detection of serum LC3 and MMPs combined with TSH levels has important reference significance in diagnosis of papillary thyroid carcinoma.

  3. Identification of metalloprotease/disintegrins in Xenopus laevis testis with a potential role in fertilization.

    Science.gov (United States)

    Shilling, F M; Krätzschmar, J; Cai, H; Weskamp, G; Gayko, U; Leibow, J; Myles, D G; Nuccitelli, R; Blobel, C P

    1997-06-15

    Proteins containing a membrane-anchored metalloprotease domain, a disintegrin domain, and a cysteine-rich region (MDC proteins) are thought to play an important role in mammalian fertilization, as well as in somatic cell-cell interactions. We have identified PCR sequence tags encoding the disintegrin domain of five distinct MDC proteins from Xenopus laevis testis cDNA. Four of these sequence tags (xMDC9, xMDC11.1, xMDC11.2, and xMDC13) showed strong similarity to known mammalian MDC proteins, whereas the fifth (xMDC16) apparently represents a novel family member. Northern blot analysis revealed that the mRNA for xMDC16 was only expressed in testis, and not in heart, muscle, liver, ovaries, or eggs, whereas the mRNAs corresponding to the four other PCR products were expressed in testis and in some or all somatic tissues tested. The xMDC16 protein sequence, as predicted from the full-length cDNA, contains a metalloprotease domain with the active-site sequence HEXXH, a disintegrin domain, a cysteine-rich region, an EGF repeat, a transmembrane domain, and a short cytoplasmic tail. To study a potential role for these xMDC proteins in fertilization, peptides corresponding to the predicted integrin-binding domain of each protein were tested for their ability to inhibit X. laevis fertilization. Cyclic and linear xMDC16 peptides inhibited fertilization in a concentration-dependent manner, whereas xMDC16 peptides that were scrambled or had certain amino acid replacements in the predicted integrin-binding domain did not affect fertilization. Cyclic and linear xMDC9 peptides and linear xMDC13 peptides also inhibited fertilization similarly to xMDC16 peptides, whereas peptides corresponding to the predicted integrin-binding site of xMDC11.1 and xMDC11.2 did not. These results are discussed in the context of a model in which multiple MDC protein-receptor interactions are necessary for fertilization to occur.

  4. SlpE is a calcium-dependent cytotoxic metalloprotease associated with clinical isolates of Serratia marcescens.

    Science.gov (United States)

    Stella, Nicholas A; Callaghan, Jake D; Zhang, Liang; Brothers, Kimberly M; Kowalski, Regis P; Huang, Jean J; Thibodeau, Patrick H; Shanks, Robert M Q

    Serralysin-like proteases are found in a wide variety of bacteria. These metalloproteases are frequently implicated in virulence and are members of the widely conserved RTX-toxin family. We identified a serralysin-like protease in the genome of a clinical isolate of Serratia marcescens that is highly similar to the canonical serralysin protein, PrtS. This gene was named serralysin-like protease E, SlpE, and was found in the majority (67%) of tested clinical isolates, but was absent from most tested non-clinical isolates including the insect pathogen and reference S. marcescens strain Db11. Purified recombinant SlpE exhibited calcium-dependent protease activity similar to metalloproteases PrtS and SlpB. Induction of slpE in the low-protease-producing S. marcescens strain PIC3611 highly elevated extracellular protease activity, and extracellular secretion required the lipD type 1 secretion system gene. Transcription of slpE was highly reduced in an eepR transcription factor mutant. Mutation of the slpE gene in a highly proteolytic clinical isolate reduced its protease activity, and evidence suggests that SlpE confers cytotoxicity of S. marcescens to the A549 airway carcinoma cell line. Together, these data reveal SlpE to be an EepR-regulated cytotoxic metalloprotease associated with clinical isolates of an important opportunistic pathogen. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  5. Garlic Supplementation Ameliorates UV-Induced Photoaging in Hairless Mice by Regulating Antioxidative Activity and MMPs Expression

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    Hye Kyung Kim

    2016-01-01

    Full Text Available UV exposure is associated with oxidative stress and is the primary factor in skin photoaging. UV-induced reactive oxygen species (ROS cause the up-regulation of metalloproteinase (MMPs and the degradation of dermal collagen and elastic fibers. Garlic and its components have been reported to exert antioxidative effects. The present study investigated the protective effect of garlic on UV-induced photoaging and MMPs regulation in hairless mice. Garlic was supplemented in the diet, and Skh-1 hairless mice were exposed to UV irradiation five days/week for eight weeks. Mice were divided into four groups; Non-UV, UV-irradiated control, UV+1% garlic powder diet group, and UV+2% garlic powder diet group. Chronic UV irradiation induced rough wrinkling of the skin with hyperkeratosis, and administration of garlic diminished the coarse wrinkle formation. UV-induced dorsal skin and epidermal thickness were also ameliorated by garlic supplementation. ROS generation, skin and serum malondialdehyde levels were significantly increased by UV exposure and were ameliorated by garlic administration although the effects were not dose-dependent. Antioxidant enzymes such as superoxide dismutase and catalase activities in skin tissues were markedly reduced by UV irradiation and garlic treatment increased these enzyme activities. UV-induced MMP-1 and MMP-2 protein levels were suppressed by garlic administration. Furthermore, garlic supplementation prevented the UV-induced increase of MMP-1 mRNA expression and the UV-induced decrease of procollagen mRNA expression. These results suggest that garlic may be effective for preventing skin photoaging accelerated by UV irradiation through the antioxidative system and MMP regulation.

  6. Garlic Supplementation Ameliorates UV-Induced Photoaging in Hairless Mice by Regulating Antioxidative Activity and MMPs Expression.

    Science.gov (United States)

    Kim, Hye Kyung

    2016-01-08

    UV exposure is associated with oxidative stress and is the primary factor in skin photoaging. UV-induced reactive oxygen species (ROS) cause the up-regulation of metalloproteinase (MMPs) and the degradation of dermal collagen and elastic fibers. Garlic and its components have been reported to exert antioxidative effects. The present study investigated the protective effect of garlic on UV-induced photoaging and MMPs regulation in hairless mice. Garlic was supplemented in the diet, and Skh-1 hairless mice were exposed to UV irradiation five days/week for eight weeks. Mice were divided into four groups; Non-UV, UV-irradiated control, UV+1% garlic powder diet group, and UV+2% garlic powder diet group. Chronic UV irradiation induced rough wrinkling of the skin with hyperkeratosis, and administration of garlic diminished the coarse wrinkle formation. UV-induced dorsal skin and epidermal thickness were also ameliorated by garlic supplementation. ROS generation, skin and serum malondialdehyde levels were significantly increased by UV exposure and were ameliorated by garlic administration although the effects were not dose-dependent. Antioxidant enzymes such as superoxide dismutase and catalase activities in skin tissues were markedly reduced by UV irradiation and garlic treatment increased these enzyme activities. UV-induced MMP-1 and MMP-2 protein levels were suppressed by garlic administration. Furthermore, garlic supplementation prevented the UV-induced increase of MMP-1 mRNA expression and the UV-induced decrease of procollagen mRNA expression. These results suggest that garlic may be effective for preventing skin photoaging accelerated by UV irradiation through the antioxidative system and MMP regulation.

  7. The Metalloprotease Meprin β Is an Alternative β-Secretase of APP.

    Science.gov (United States)

    Becker-Pauly, Christoph; Pietrzik, Claus U

    2016-01-01

    The membrane bound metalloprotease meprin β is important for collagen fibril assembly in connective tissue formation and for the detachment of the intestinal mucus layer for proper barrier function. Recent proteomic studies revealed dozens of putative new substrates of meprin β, including the amyloid precursor protein (APP). It was shown that APP is cleaved by meprin β in distinct ways, either at the β-secretase site resulting in increased levels of Aβ peptides, or at the N-terminus releasing 11 kDa, and 20 kDa peptide fragments. The latter event was discussed to be rather neuroprotective, whereas the ectodomain shedding of APP by meprin β reminiscent to BACE-1 is in line with the amyloid hypothesis of Alzheimer's disease, promoting neurodegeneration. The N-terminal 11 kDa and 20 kDa peptide fragments represent physiological cleavage products, since they are found in human brains under different diseased or non-diseased states, whereas these fragments are completely missing in brains of meprin β knock-out animals. Meprin β is not only a sheddase of adhesion molecules, such as APP, but was additionally demonstrated to cleave within the prodomain of ADAM10. Activated ADAM10, the α-secretase of APP, is then able to shed meprin β from the cell surface thereby abolishing the β-secretase activity. All together meprin β seems to be a novel player in APP processing events, even influencing other enzymes involved in APP cleavage.

  8. Cloning, sequencing and expression of the gene encoding the extracellular metalloprotease of Aeromonas caviae.

    Science.gov (United States)

    Kawakami, K; Toma, C; Honma, Y

    2000-01-01

    A gene (apk) encoding the extracellular protease of Aeromonas caviae Ae6 has been cloned and sequenced. For cloning the gene, the DNA genomic library was screened using skim milk LB agar. One clone harboring plasmid pKK3 was selected for sequencing. Nucleotide sequencing of the 3.5 kb region of pKK3 revealed a single open reading frame (ORF) of 1,785 bp encoding 595 amino acids. The deduced polypeptide contained a putative 16-amino acid signal peptide followed by a large propeptide. The N-terminal amino acid sequence of purified recombinant protein (APK) was consistent with the DNA sequence. This result suggested a mature protein of 412 amino acids with a molecular mass of 44 kDa. However, the molecular mass of purified recombinant APK revealed 34 kDa by SDS-PAGE, suggesting that further processing at the C-terminal region took place. The 2 motifs of zinc binding sites deduced are highly conserved in the APK as well as in other zinc metalloproteases including Vibrio proteolyticus neutral protease, Emp V from Vibrio vulnificus, HA/P from Vibrio cholerae, and Pseudomonas aeruginosa elastase. Proteolytic activity was inhibited by EDTA, Zincov, 1,10-phenanthroline and tetraethylenepentamine while unaffected by the other inhibitors tested. The protease showed maximum activity at pH 7.0 and was inactivated by heating at 80 C for 15 min. These results together suggest that APK belongs to the thermolysin family of metalloendopeptidases.

  9. Vibrio cholerae hemagglutinin(HA)/protease: An extracellular metalloprotease with multiple pathogenic activities.

    Science.gov (United States)

    Benitez, Jorge A; Silva, Anisia J

    2016-06-01

    Vibrio cholerae of serogroup O1 and O139, the etiological agent of the diarrheal disease cholera, expresses the extracellular Zn-dependent metalloprotease hemagglutinin (HA)/protease also reported as vibriolysin. This enzyme is also produced by non-O1/O139 (non-cholera) strains that cause mild, sporadic illness (i.e. gastroenteritis, wound or ear infections). Orthologs of HA/protease are present in other members of the Vibrionaceae family pathogenic to humans and fish. HA/protease belongs to the M4 neutral peptidase family and displays significant amino acid sequence homology to Pseudomonas aeruginosa elastase (LasB) and Bacillus thermoproteolyticus thermolysin. It exhibits a broad range of potentially pathogenic activities in cell culture and animal models. These activities range from the covalent modification of other toxins, the degradation of the protective mucus barrier and disruption of intestinal tight junctions. Here we review (i) the structure and regulation of HA/protease expression, (ii) its interaction with other toxins and the intestinal mucosa and (iii) discuss the possible role(s) of HA/protease in the pathogenesis of cholera. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Role of a disintegrin and metalloprotease 10 in Staphylococcus aureus α-hemolysin–mediated cellular injury

    Science.gov (United States)

    Wilke, Georgia A.; Wardenburg, Juliane Bubeck

    2010-01-01

    Staphylococcus aureus α-hemolysin (Hla), a potent cytotoxin, plays an important role in the pathogenesis of staphylococcal diseases, including those caused by methicillin-resistant epidemic strains. Hla is secreted as a water-soluble monomer that undergoes a series of conformational changes to generate a heptameric, β-barrel structure in host membranes. Structural maturation of Hla depends on its interaction with a previously unknown proteinaceous receptor in the context of the cell membrane. It is reported here that a disintegrin and metalloprotease 10 (ADAM10) interacts with Hla and is required to initiate the sequence of events whereby the toxin is transformed into a cytolytic pore. Hla binding to the eukaryotic cell requires ADAM10 expression. Further, ADAM10 is required for Hla-mediated cytotoxicity, most notably when the toxin is present at low concentrations. These data thus implicate ADAM10 as the probable high-affinity toxin receptor. Upon Hla binding, ADAM10 relocalizes to caveolin 1-enriched lipid rafts that serve as a platform for the clustering of signaling molecules. It is demonstrated that the Hla–ADAM10 complex initiates intracellular signaling events that culminate in the disruption of focal adhesions. PMID:20624979

  11. Role of a disintegrin and metalloprotease 10 in Staphylococcus aureus alpha-hemolysin-mediated cellular injury.

    Science.gov (United States)

    Wilke, Georgia A; Bubeck Wardenburg, Juliane

    2010-07-27

    Staphylococcus aureus alpha-hemolysin (Hla), a potent cytotoxin, plays an important role in the pathogenesis of staphylococcal diseases, including those caused by methicillin-resistant epidemic strains. Hla is secreted as a water-soluble monomer that undergoes a series of conformational changes to generate a heptameric, beta-barrel structure in host membranes. Structural maturation of Hla depends on its interaction with a previously unknown proteinaceous receptor in the context of the cell membrane. It is reported here that a disintegrin and metalloprotease 10 (ADAM10) interacts with Hla and is required to initiate the sequence of events whereby the toxin is transformed into a cytolytic pore. Hla binding to the eukaryotic cell requires ADAM10 expression. Further, ADAM10 is required for Hla-mediated cytotoxicity, most notably when the toxin is present at low concentrations. These data thus implicate ADAM10 as the probable high-affinity toxin receptor. Upon Hla binding, ADAM10 relocalizes to caveolin 1-enriched lipid rafts that serve as a platform for the clustering of signaling molecules. It is demonstrated that the Hla-ADAM10 complex initiates intracellular signaling events that culminate in the disruption of focal adhesions.

  12. New Terpenoids from Chamaecyparis formosensis (Cupressaceae Leaves with Modulatory Activity on Matrix Metalloproteases 2 and 9

    Directory of Open Access Journals (Sweden)

    Meng-Lun Chang

    2018-03-01

    Full Text Available Chamaecyparis formosensis is Taiwan’s most representative tree, and has high economic value. To date, only a few active chemical constituents have been reported for C. formosensis. In this study, 37 secondary metabolites, including three new compounds (1–3, were extracted from the leaves of C. formosensis. The compounds isolated from the ethyl acetate layer were used at different concentrations to treat HT-1080 human fibrosarcoma cells and to evaluate their effects on matrix metalloprotease 2 (MMP-2 and 9 (MMP-9 expression. Based on extensive analysis of data from high-resolution mass spectrometry (HR-MS as well as nuclear magnetic resonance (NMR, infrared (IR, and ultraviolet (UV spectroscopy, the new compounds were identified as 11,12-dihydroxyisodaucenoic acid (1, 12-hydroxyisodaucenoic acid (2, and 1-oxo-2α,3β-dihydroxytotarol (3. Known compounds 4–37 were identified by comparing their spectroscopic data with data reported in the literature. Biological activity tests by gelatin zymographic analysis revealed that seven compounds, including new compound 2, have no cytotoxic effect on HT-1080 cells and were found to increase MMP-2 or MMP-9 expression by 1.25- to 1.59-fold at lower concentrations of 10–50 µM. These naturally derived regulatory compounds could potentially serve as a novel pharmaceutical basis for medical purposes.

  13. Development and validation of novel enzyme activity methods to assess inhibition of matrix metalloproteinases (MMPs) in human serum by antibodies against enzyme therapeutics.

    Science.gov (United States)

    Edkins, Thomas J; Alhadeff, Jack A; Kwok, Vincent; Kalensky, Charles; Rock, Marie T; Vidmar, Thomas J; Del Tito, Benjamin J

    2012-11-01

    This paper summarizes the development and validation of five enzyme activity methods to assess the specific inhibition of human endogenous matrix metalloproteinases MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin 1), MMP-8 (collagenase 2) and MMP-13 (collagenase 3) by anti-Collagenase Clostridium histolyticum (CCH) antibodies in human serum. These MMPs are of interest since antibodies against a therapeutic enzyme may cross-react with, and inactivate, the MMPs. The validated methods utilize spiked exogenous individual MMPs added to serum to determine if the serum inhibits MMP enzyme activity. Factors evaluated and optimized during development include pH, reaction time and temperature, inhibitor concentration for the positive control, and substrate and serum concentration. Characteristics established during validation for each MMP activity inhibition method included intra- and inter-assay precision and recovery, recovery in the pooled normal human serum samples, bench-top stability at room temperature and on wet ice, and assay cut-point determination. Precision results ranged from ~1 to 12% CV, recoveries of the activities of the exogenous MMPs ranged from ~84 to 90% and cut-point values ranged from 67 to 91%. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. Alternative Roles of STAT3 and MAPK Signaling Pathways in the MMPs Activation and Progression of Lung Injury Induced by Cigarette Smoke Exposure in ACE2 Knockout Mice.

    Science.gov (United States)

    Hung, Yi-Han; Hsieh, Wen-Yeh; Hsieh, Jih-Sheng; Liu, Fon-Chang; Tsai, Chin-Hung; Lu, Li-Che; Huang, Chen-Yi; Wu, Chien-Liang; Lin, Chih-Sheng

    2016-01-01

    Inflammation-mediated abnormalities in the renin-angiotensin system (RAS) and expression of matrix metalloproteinases (MMPs) are implicated in the pathogenesis of lung injury. Angiotensin converting enzyme II (ACE2), an angiotensin converting enzyme (ACE) homologue that displays antagonist effects on ACE/angiotensin II (Ang II) axis, could also play a protective role against lung diseases. However, the relationship between ACE2 and MMPs activation in lung injury is still largely unclear. The purpose of this study is to investigate whether MMPs activity could be affected by ACE2 and which ACE2 derived signaling pathways could be also involved via using a mouse model with lung injury induced by cigarette smoke (CS) exposure for 1 to 3 weeks. Wild-type (WT; C57BL/6) and ACE2 KO mice (ACE2(-/-)) were utilized to study CS-induced lung injury. Increases in the resting respiratory rate (RRR), pulmonary immunokines, leukocyte infiltration and bronchial hyperplasia were observed in the CS-exposed mice. Compared to WT mice, more serious physiopathological changes were found in ACE2(-/-) mice in the first week of CS exposure. CS exposure increased pulmonary ACE and ACE2 activities in WT mice, and significantly increased ACE in ACE2(-/-) mice. Furthermore, the activity of pulmonary MMPs was decreased in CS-exposed WT mice, whereas this activity was increased in ACE2(-/-) mice. CS exposure increased the pulmonary p-p38, p-JNK and p-ERK1/2 level in all mice. In ACE2(-/-) mice, a significant increase p-STAT3 signaling was detected; however, no effect was observed on the p-STAT3 level in WT mice. Our results support the hypothesis that ACE2 deficiency influences MMPs activation and STAT3 phosphorylation signaling to promote more pulmonary inflammation in the development of lung injury.

  15. Studies on the formation and stability of a complex between Streptomyces proteinaceous metalloprotease inhibitor and thermolysin.

    Science.gov (United States)

    Kunugi, S; Yanagi, Y; Oda, K

    1999-02-01

    The effects of certain physicochemical parameters on the formation and stability of a complex between Streptomyces proteinaceous metalloprotease inhibitor (SMPI) and thermolysin were investigated. SMPI had its lowest Ki value at a pH of around 6.5 (similar to the pH dependence of the kcat/K(m) of thermolysin catalysis), reflecting the splitting mechanism of the SMPI inhibition of thermolysin. This Ki increased with an increase in pressure, and in (Ki-1) was almost linear with respect to pressure. The volume of the reaction (delta Vcomp), which is the volume change accompanying enzyme-inhibitor complex formation, was calculated as +8.1 +/- 0.3 mL.mol-1, which has a sign opposite to delta Vcomp for neutral peptide inhibitors and acyl-peptide substrates. The temperature dependence of Ki-1 gave the reaction enthalpy (delta Hcomp) and reaction entropy (delta Scomp) of the complex formation as 34.6 +/- 1.4 kJ.mol-1 and 298 +/- 5 J.mol-1.K-1, respectively. These positive reaction volumes and reaction entropies were related to the electrostatic interactions and ionic strength dependence of Ki which corresponded to the key ionic interaction during complex formation. Complex formation with SMPI stabilized thermolysin against pressure perturbation as observed by the changes in the Trp fluorescence of thermolysin with increasing pressure. Thermal stability, however, was affected very little by complex formation with SMPI. Phosphoramidon, Cbz-Phe-Gly-NH2 and Cbz-Phe also positively affected the pressure-tolerance of thermolysin, in the following order: Cbz-Gly-Phe-NH2 thermolysin was localized to the reactive site.

  16. Substrate recognition by the zinc metalloprotease effector NleC from enteropathogenic Escherichia coli.

    Science.gov (United States)

    Giogha, Cristina; Lung, Tania Wong Fok; Mühlen, Sabrina; Pearson, Jaclyn S; Hartland, Elizabeth L

    2015-12-01

    Upon infection of epithelial cells, enteropathogenic Escherichia coli suppresses host cell inflammatory signalling in a type III secretion system (T3SS) dependent manner. Two key T3SS effector proteins involved in this response are NleE and NleC. NleC is a zinc metalloprotease effector that degrades the p65 subunit of NF-κB. Although the site of p65 cleavage by NleC is now well described, other areas of interaction have not been precisely defined. Here we constructed overlapping truncations of p65 to identify regions required for NleC cleavage. We determined that NleC cleaved both p65 and p50 within the Rel homology domain (RHD) and that two motifs, E22IIE25 and P177VLS180 , within the RHD of p65 were important for recognition and binding by NleC. Alanine substitution of one or both of these motifs protected p65 from binding and degradation by NleC. The E22IIE25 and P177VLS180 motifs were located within the structurally distinct N-terminal subdomain of the RHD involved in DNA binding by p65 on adjacent, parallel strands. Although these motifs have not been recognized previously, both were needed for the correct localization and function of p65. In summary, this work has identified two regions of p65 within the RHD needed for binding and cleavage by NleC and provides further insight into the molecular basis of substrate recognition by a T3SS effector. © 2015 John Wiley & Sons Ltd.

  17. Plasmatic ADAMTS-13 metalloprotease and von Willebrand factor in children with cyanotic congenital heart disease

    Directory of Open Access Journals (Sweden)

    R.P.S. Soares

    2013-05-01

    Full Text Available Changes in plasma von Willebrand factor concentration (VWF:Ag and ADAMTS-13 activity (the metalloprotease that cleaves VWF physiologically have been reported in several cardiovascular disorders with prognostic implications. We therefore determined the level of these proteins in the plasma of children with cyanotic congenital heart disease (CCHD undergoing surgical treatment. Forty-eight children were enrolled (age 0.83 to 7.58 years. Measurements were performed at baseline and 48 h after surgery. ELISA, collagen-binding assays and Western blotting were used to estimate antigenic and biological activities, and proteolysis of VWF multimers. Preoperatively, VWF:Ag and ADAMTS-13 activity were decreased (65 and 71% of normal levels considered as 113 (105-129 U/dL and 91 ± 24% respectively, P < 0.003 and correlated (r = 0.39, P = 0.0064. High molecular weight VWF multimers were not related, suggesting an interaction of VWF with cell membranes, followed by proteolytic cleavage. A low preoperative ADAMTS-13 activity, a longer activated partial thromboplastin time and the need for cardiopulmonary bypass correlated with postoperative bleeding (P < 0.05. Postoperatively, ADAMTS-13 activity increased but less extensively than VWF:Ag (respectively, 2.23 and 2.83 times baseline, P < 0.0001, resulting in an increased VWF:Ag/ADAMTS-13 activity ratio (1.20 to 1.54, respectively, pre- and postoperative median values, P = 0.0029. ADAMTS-13 consumption was further confirmed by decreased ADAMTS-13 antigenic concentration (0.91 ± 0.30 to 0.70 ± 0.25 µg/mL, P < 0.0001 and persistent proteolysis of VWF multimers. We conclude that, in pediatric CCHD, changes in circulating ADAMTS-13 suggest enzyme consumption, associated with abnormal structure and function of VWF.

  18. Plasmatic ADAMTS-13 metalloprotease and von Willebrand factor in children with cyanotic congenital heart disease

    Energy Technology Data Exchange (ETDEWEB)

    Soares, R.P.S. [Fundação Pró-Sangue Hemocentro de São Paulo, São Paulo, SP (Brazil); Bydlowski, S.P.; Nascimento, N.M. [Laboratório de Investigação Médica-31, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP (Brazil); Thomaz, A.M.; Bastos, E.N.M.; Lopes, A.A. [Faculdade de Medicina, Instituto do Coração, Universidade de São Paulo, São Paulo, SP (Brazil)

    2013-04-05

    Changes in plasma von Willebrand factor concentration (VWF:Ag) and ADAMTS-13 activity (the metalloprotease that cleaves VWF physiologically) have been reported in several cardiovascular disorders with prognostic implications. We therefore determined the level of these proteins in the plasma of children with cyanotic congenital heart disease (CCHD) undergoing surgical treatment. Forty-eight children were enrolled (age 0.83 to 7.58 years). Measurements were performed at baseline and 48 h after surgery. ELISA, collagen-binding assays and Western blotting were used to estimate antigenic and biological activities, and proteolysis of VWF multimers. Preoperatively, VWF:Ag and ADAMTS-13 activity were decreased (65 and 71% of normal levels considered as 113 (105-129) U/dL and 91 ± 24% respectively, P < 0.003) and correlated (r = 0.39, P = 0.0064). High molecular weight VWF multimers were not related, suggesting an interaction of VWF with cell membranes, followed by proteolytic cleavage. A low preoperative ADAMTS-13 activity, a longer activated partial thromboplastin time and the need for cardiopulmonary bypass correlated with postoperative bleeding (P < 0.05). Postoperatively, ADAMTS-13 activity increased but less extensively than VWF:Ag (respectively, 2.23 and 2.83 times baseline, P < 0.0001), resulting in an increased VWF:Ag/ADAMTS-13 activity ratio (1.20 to 1.54, respectively, pre- and postoperative median values, P = 0.0029). ADAMTS-13 consumption was further confirmed by decreased ADAMTS-13 antigenic concentration (0.91 ± 0.30 to 0.70 ± 0.25 µg/mL, P < 0.0001) and persistent proteolysis of VWF multimers. We conclude that, in pediatric CCHD, changes in circulating ADAMTS-13 suggest enzyme consumption, associated with abnormal structure and function of VWF.

  19. TGF-β1 modulates the homeostasis between MMPs and MMP inhibitors through p38 MAPK and ERK1/2 in highly invasive breast cancer cells

    International Nuclear Information System (INIS)

    Gomes, Luciana R; Terra, Letícia F; Wailemann, Rosângela AM; Labriola, Leticia; Sogayar, Mari C

    2012-01-01

    Metastasis is the main factor responsible for death in breast cancer patients. Matrix metalloproteinases (MMPs) and their inhibitors, known as tissue inhibitors of MMPs (TIMPs), and the membrane-associated MMP inhibitor (RECK), are essential for the metastatic process. We have previously shown a positive correlation between MMPs and their inhibitors expression during breast cancer progression; however, the molecular mechanisms underlying this coordinate regulation remain unknown. In this report, we investigated whether TGF-β1 could be a common regulator for MMPs, TIMPs and RECK in human breast cancer cell models. The mRNA expression levels of TGF-β isoforms and their receptors were analyzed by qRT-PCR in a panel of five human breast cancer cell lines displaying different degrees of invasiveness and metastatic potential. The highly invasive MDA-MB-231 cell line was treated with different concentrations of recombinant TGF-β1 and also with pharmacological inhibitors of p38 MAPK and ERK1/2. The migratory and invasive potential of these treated cells were examined in vitro by transwell assays. In general, TGF-β2, TβRI and TβRII are over-expressed in more aggressive cells, except for TβRI, which was also highly expressed in ZR-75-1 cells. In addition, TGF-β1-treated MDA-MB-231 cells presented significantly increased mRNA expression of MMP-2, MMP-9, MMP-14, TIMP-2 and RECK. TGF-β1 also increased TIMP-2, MMP-2 and MMP-9 protein levels but downregulated RECK expression. Furthermore, we analyzed the involvement of p38 MAPK and ERK1/2, representing two well established Smad-independent pathways, in the proposed mechanism. Inhibition of p38MAPK blocked TGF-β1-increased mRNA expression of all MMPs and MMP inhibitors analyzed, and prevented TGF-β1 upregulation of TIMP-2 and MMP-2 proteins. Moreover, ERK1/2 inhibition increased RECK and prevented the TGF-β1 induction of pro-MMP-9 and TIMP-2 proteins. TGF-β1-enhanced migration and invasion capacities were blocked by p

  20. Active site specificity profiling datasets of matrix metalloproteinases (MMPs 1, 2, 3, 7, 8, 9, 12, 13 and 14

    Directory of Open Access Journals (Sweden)

    Ulrich Eckhard

    2016-06-01

    Full Text Available The data described provide a comprehensive resource for the family-wide active site specificity portrayal of the human matrix metalloproteinase family. We used the high-throughput proteomic technique PICS (Proteomic Identification of protease Cleavage Sites to comprehensively assay 9 different MMPs. We identified more than 4300 peptide cleavage sites, spanning both the prime and non-prime sides of the scissile peptide bond allowing detailed subsite cooperativity analysis. The proteomic cleavage data were expanded by kinetic analysis using a set of 6 quenched-fluorescent peptide substrates designed using these results. These datasets represent one of the largest specificity profiling efforts with subsequent structural follow up for any protease family and put the spotlight on the specificity similarities and differences of the MMP family. A detailed analysis of this data may be found in Eckhard et al. (2015 [1]. The raw mass spectrometry data and the corresponding metadata have been deposited in PRIDE/ProteomeXchange with the accession number http://www.ebi.ac.uk/pride/archive/projects/PXD002265.

  1. Hinokitiol Exerts Anticancer Activity through Downregulation of MMPs 9/2 and Enhancement of Catalase and SOD Enzymes: In Vivo Augmentation of Lung Histoarchitecture.

    Science.gov (United States)

    Huang, Chien-Hsun; Jayakumar, Thanasekaran; Chang, Chao-Chien; Fong, Tsorng-Harn; Lu, Shing-Hwa; Thomas, Philip Aloysius; Choy, Cheuk-Sing; Sheu, Joen-Rong

    2015-09-25

    Melanoma is extremely resistant to chemotherapy and the death rate is increasing hastily worldwide. Extracellular matrix promotes the migration and invasion of tumor cells through the production of matrix metalloproteinase (MMP)-2 and -9. Evidence has shown that natural dietary antioxidants are capable of inhibiting cancer cell growth. Our recent studies showed that hinokitiol, a natural bioactive compound, inhibited vascular smooth muscle cell proliferation and platelets aggregation. The present study is to investigate the anticancer efficacy of hinokitiol against B16-F10 melanoma cells via modulating tumor invasion factors MMPs, antioxidant enzymes in vitro. An in vivo mice model of histological investigation was performed to study the patterns of elastic and collagen fibers. Hinokitiol inhibited the expression and activity of MMPs-2 and -9 in B16-F10 melanoma cells, as measured by western blotting and gelatin zymography, respectively. An observed increase in protein expression of MMPs 2/9 in melanoma cells was significantly inhibited by hinokitiol. Notably, hinokitiol (1-5 μM) increased the activities of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) from the reduction in melanoma cells. Also, hinokitiol (2-10 µM) concentration dependently reduced in vitro Fenton reaction induced hydroxyl radical (OH·) formation. An in vivo study showed that hinokitiol treatment increased elastic fibers (EF), collagens dispersion, and improved alveolar alterations in the lungs of B16/F10 injected mice. Overall, our findings propose that hinokitiol may be a potent anticancer candidate through down regulation of MMPs 9/2, reduction of OH· production and enhancement of antioxidant enzymes SOD and CAT.

  2. Solution structure and dynamics of C-terminal regulatory domain of Vibrio vulnificus extracellular metalloprotease

    Energy Technology Data Exchange (ETDEWEB)

    Yun, Ji-Hye; Kim, Heeyoun [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Park, Jung Eun [Department of Biotechnology, College of Natural Sciences, Chosun University, Gwangju 501-759 (Korea, Republic of); Lee, Jung Sup, E-mail: jsplee@mail.chosun.ac.kr [Department of Biotechnology, College of Natural Sciences, Chosun University, Gwangju 501-759 (Korea, Republic of); Lee, Weontae, E-mail: wlee@spin.yonsei.ac.kr [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of)

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer We have determined solution structures of vEP C-terminal regulatory domain. Black-Right-Pointing-Pointer vEP C-ter100 has a compact {beta}-barrel structure with eight anti-parallel {beta}-strands. Black-Right-Pointing-Pointer Solution structure of vEP C-ter100 shares its molecular topology with that of the collagen-binding domain of collagenase. Black-Right-Pointing-Pointer Residues in the {beta}3 region of vEP C-ter100 might be important in putative ligand/receptor binding. Black-Right-Pointing-Pointer vEP C-ter100 interacts strongly with iron ion. -- Abstract: An extracellular metalloprotease (vEP) secreted by Vibrio vulnificus ATCC29307 is a 45-kDa proteolytic enzyme that has prothrombin activation and fibrinolytic activities during bacterial infection. The action of vEP could result in clotting that could serve to protect the bacteria from the host defense machinery. Very recently, we showed that the C-terminal propeptide (C-ter100), which is unique to vEP, is involved in regulation of vEP activity. To understand the structural basis of this function of vEP C-ter100, we have determined the solution structure and backbone dynamics using multidimensional nuclear magnetic resonance spectroscopy. The solution structure shows that vEP C-ter100 is composed of eight anti-parallel {beta}-strands with a unique fold that has a compact {beta}-barrel formation which stabilized by hydrophobic and hydrogen bonding networks. Protein dynamics shows that the overall structure, including loops, is very rigid and stabilized. By structural database analysis, we found that vEP C-ter100 shares its topology with that of the collagen-binding domain of collagenase, despite low sequence homology between the two domains. Fluorescence assay reveals that vEP C-ter100 interacts strongly with iron (Fe{sup 3+}). These findings suggest that vEP protease might recruit substrate molecules, such as collagen, by binding at C-ter100 and that vEP participates

  3. Sulforaphane inhibits IL-1β-induced proliferation of rheumatoid arthritis synovial fibroblasts and the production of MMPs, COX-2, and PGE2.

    Science.gov (United States)

    Choi, Yun Jung; Lee, Won-Seok; Lee, Eun-Gyeong; Sung, Myung-Soon; Yoo, Wan-Hee

    2014-10-01

    This study was performed to define the effects of sulforaphane on interleukin-1β (IL-1β)-induced proliferation of rheumatoid arthritis synovial fibroblasts (RASFs), the expression of matrix metalloproteinases (MMPs) and cyclooxygenase (COX), and the production of prostaglandin E2 (PGE2) by RASFs. The proliferation of RASFs was evaluated with CCK-8 reagent in the presence of IL-1β with/without sulforaphane. The expression of MMPs, tissue inhibitor of metalloproteinase-1, COXs, intracellular mitogen-activated protein kinase signalings, including p-ERK, p-p38, p-JNK, and nuclear factor-kappaB (NF-kB), and the production of PGE2 were examined by Western blotting or semi-quantitative RT-PCR and ELISA. Sulforaphane inhibits unstimulated and IL-1β-induced proliferation of RASFs; the expression of MMP-1, MMP-3, and COX-2 mRNA and protein; and the PGE2 production induced by IL-1β. Sulforaphane also inhibits the phosphorylation of ERK-1/2, p-38, and JNK and activation of NF-kB by IL-1β. These results indicate that sulforaphane inhibits the proliferation of synovial fibroblasts, the expression of MMPs and COX-2, and the production of PGE2, which are involved in synovitis and destruction of RA, and suggest that sulforaphane might be a new therapeutic agent for RA.

  4. α-Solanine Inhibits Invasion of Human Prostate Cancer Cell by Suppressing Epithelial-Mesenchymal Transition and MMPs Expression

    Directory of Open Access Journals (Sweden)

    Kun-Hung Shen

    2014-08-01

    Full Text Available α-Solanine, a naturally occurring steroidal glycoalkaloid found in nightshade (Solanum nigrum Linn., was found to inhibit proliferation and induce apoptosis of tumor cells. However, the mechanism involved in suppression of cancer cell metastasis by α-solanine remains unclear. This study investigates the suppression mechanism of α-solanine on motility of the human prostate cancer cell PC-3. Results show that α-solanine reduces the viability of PC-3 cells. When treated with non-toxic doses of α-solanine, cell invasion is markedly suppressed by α-solanine. α-Solanine also significantly elevates epithelial marker E-cadherin expression, while it concomitantly decreases mesenchymal marker vimentin expression, suggesting it suppresses epithelial-mesenchymal transition (EMT. α-Solanine reduces the mRNA level of matrix metalloproteinase-2 (MMP-2, MMP-9 and extracellular inducer of matrix metalloproteinase (EMMPRIN, but increases the expression of reversion-inducing cysteine-rich protein with kazal motifs (RECK, and tissue inhibitor of metalloproteinase-1 (TIMP-1 and TIMP-2. Immunoblotting assays indicate α-solanine is effective in suppressing the phosphorylation of phosphatidylinositide-3 kinase (PI3K, Akt and ERK. Moreover, α-solanine downregulates oncogenic microRNA-21 (miR-21 and upregulates tumor suppressor miR-138 expression. Taken together, the results suggest that inhibition of PC-3 cell invasion by α-solanine may be, at least in part, through blocking EMT and MMPs expression. α-Solanine also reduces ERK and PI3K/Akt signaling pathways and regulates expression of miR-21 and miR-138. These findings suggest an attractive therapeutic potential of α-solanine for suppressing invasion of prostate cancer cell.

  5. Modulation of u-PA, MMPs and their inhibitors by a novel nutrient mixture in human lung cancer and mesothelioma cell lines.

    Science.gov (United States)

    Roomi, M Waheed; Kalinovsky, Tatiana; Niedzwiecki, Aleksandra; Rath, Matthias

    2013-06-01

    Lung cancer, the most prevalent cancer worldwide and malignant mesothelioma are highly aggressive tumors that are characterized by high levels of matrix metalloproteinase (MMP)-2 and -9 secretion. Proteases play a key role in tumor cell invasion and metastasis by digesting the basement membrane and ECM components. Strong clinical and experimental evidence demonstrates association of elevated levels of u-PA and MMPs with cancer progression, metastasis and shortened patient survival. MMP activities are regulated by specific tissue inhibitors of metalloproteinases (TIMPS). Our main objective was to study the effect of a nutrient mixture (NM) on the activity of u-PA, MMPs and TIMPs on human lung and malignant mesothelioma (MM) cell lines. Human lung cancer (A-549 and Calu-3) and malignant mesothelioma (MSTO-211H) cell lines were cultured in their respective media and treated at confluence with NM at 0, 50, 100, 250, 500 and 1000 µg/ml. Analysis of u-PA activity was carried out by fibrin zymography, MMPs by gelatinase zymography and TIMPs by reverse zymography. Both lung cancer cell lines expressed u-PA, which was inhibited by NM in a dose-dependent manner. However, no bands corresponding to u-PA were detected for the MSTO-211H MM cell line. On gelatinase zymography, A-549 cells showed one band corresponding to MMP-2 and induction of MMP-9 with PMA (100 ng/ml) treatment. MSTO-211H showed two bands, an intense band corresponding to MMP-2 and a faint band corresponding to MMP-9; MMP-9 was enhanced significantly with PMA treatment. NM inhibited their expression in both cell lines in a dose-dependent manner. Calu-3 showed no MMP-2 or MMP-9 expression. Activity of TIMPs was upregulated by NM in all cancer cell lines in a dose-dependent manner. Analysis revealed a positive correlation between u-PA and MMPs and a negative correlation between u-PA/MMPs and TIMPs. These findings suggest the therapeutic potential of NM in the treatment of lung and mesothelioma cancers.

  6. A phage display delected 7-mer peptide inhibitor of the tannerella forsythia metalloprotease-like enzyme karilysin can be truncated to ser-trp-phe-pro

    DEFF Research Database (Denmark)

    Skottrup, Peter Durand; Sørensen, Grete; Ksiazek, Miroslaw

    2012-01-01

    Tannerella forsythia is a gram-negative bacteria, which is strongly associated with the development of periodontal disease. Karilysin is a newly identified metalloprotease-like enzyme, that is secreted from T. forsythia. Karilysin modulates the host immune response and is therefore considered a l...

  7. The urokinase plasminogen activator receptor-associated protein/endo180 is coexpressed with its interaction partners urokinase plasminogen activator receptor and matrix metalloprotease-13 during osteogenesis

    DEFF Research Database (Denmark)

    Engelholm, L H; Nielsen, B S; Netzel-Arnett, Sarah

    2001-01-01

    The urokinase plasminogen activator receptor-associated protein/Endo180 (uPARAP/Endo180) is a newly discovered member of the macrophage mannose receptor family that was reported to interact with ligand-bound urokinase plasminogen activator receptor (uPAR), matrix metalloprotease-13 (MMP-13), and ...

  8. Expression of matrix metalloprotease-2, -7 and -9 on human colon, liver and bile duct cell lines by enteric and gastric Helicobacter species.

    Science.gov (United States)

    Yanagisawa, Naoko; Geironson, Linda; Al-Soud, Waleed Abu; Ljungh, Sa

    2005-05-01

    Gastric and enteric Helicobacter species have been associated with malignant and inflammatory diseases of the stomach, liver, gall bladder and intestine. Matrix metalloproteinases (MMPs) participate in degradation of extracellular matrix, which allows bacteria to come in contact with and interact with the cells. Enhanced level of MMPs facilitates metastasis and cell invasion of tumor cells by removal of physical barriers, as well as modulation of biologic activities of the proteins residing in the extracellular matrix. The aim of this study was to evaluate the effect of gastric and enteric Helicobacter on induction of MMPs in hepatocytes and epithelial cells of gall bladder and colon. Human hepatocytes HepG2, gall bladder epithelial cells TFK-1, and colon epithelial cells HT29 were infected with strains of H. pylori cagA+, cagE+, H. pylori cagA-, cagE-, H. pullorum, H. cholecystus, H. bilis and H. hepaticus. Protein levels of MMPs were analyzed by enzyme-linked immunosorbent assay and immunohistochemistry. Reverse transcription-quantitative polymerase chain reaction was used to study mRNA levels. Increased expression of MMP-2 and MMP-9 was observed on HepG2, TFK-1 and HT29 infected with H. pylori cagA+, cagE+ and H. cholecystus strains. H. pylori cagA+, cagE+, H. cholecystus, H. pullorum, H. bilis and H. hepaticus strains increased expression of MMP-7 on HT29, compared to uninfected control cells. The effect of MMP upregulation on HepG2, TFK-1 and HT29 was bacterial dose dependent. H. pylori cagA-, cagE- strain did not increase expression of MMPs. Inducible MMPs on colon and bile duct epithelial cells as well as hepatocytes may play an important role in facilitating invasion and progression of cancer by Helicobacter species colonizing the hepatobiliary and gastrointestinal tract.

  9. GRP78 protects a disintegrin and metalloprotease 17 against protein-disulfide isomerase A6 catalyzed inactivation.

    Science.gov (United States)

    Schäfer, Miriam; Granato, Daniela C; Krossa, Sebastian; Bartels, Anne-Kathrin; Yokoo, Sami; Düsterhöft, Stefan; Koudelka, Tomas; Scheidig, Axel J; Tholey, Andreas; Paes Leme, Adriana F; Grötzinger, Joachim; Lorenzen, Inken

    2017-11-01

    The shedding of ectodomains is a crucial mechanism in many physiological and pathological events. A disintegrin and metalloprotease-17 (ADAM17) is a key sheddase involved in essential processes, such as development, regeneration, and immune defense. ADAM17 exists in two conformations which differ in their disulfide connection in the membrane-proximal domain (MPD). Protein-disulfide isomerases (PDIs) on the cell surface convert the open MPD into a rigid closed form, which corresponds to inactive ADAM17. ADAM17 is expressed in its open activatable form in the endoplasmic reticulum (ER) and consequently must be protected against ER-resident PDI activity. Here, we show that the chaperone 78-kDa glucose-regulated protein (GRP78) protects the MPD against PDI-dependent disulfide-bond isomerization by binding to this domain and, thereby, preventing ADAM17 inhibition. © 2017 Federation of European Biochemical Societies.

  10. Abnormalities in soluble CD147 / MMPs / TIMPs axis in Ankylosing Spondylitis patients with and without a history of Acute Anterior Uveitis / Anomalii ale axei CD147 solubil / MMPs / TIMPs la pacienții cu spondilită anchilozantă cu sau fără uveită acută anterioară

    Directory of Open Access Journals (Sweden)

    Mitulescu Traian Costin

    2014-12-01

    Full Text Available Spondilita Anchilozantă (SA este prototipul formei axiale a spondiloartritelor. În pofida studiilor extinse, sunt încă incomplet înțelese mecansimele complexe legate de procesele celulare și moleculare anormale din SA. Printre mediatorii inflamației, cum ar fi citokinele proinflamatoare, NOS-2, chemokinele, care conduc la inflamație, metaloproteinazele de matrice (MMPs joacă un rol important în procesele inflamatoare care caracterizează SA. De aceea, ne-am propus să evaluăm dacă perturbări ale homeostaziei inductorului extracelular de MMPs (EMMPRIN/CD147, MMPs și inhibitorilor tisulari ai MMPs (TIMPs joacă un rol în evoluția SA în special la pacienții care au în istoricul lor Uveită Acută Anterioară (UAA. În acest scop seruri de la pacienți cu SA și de la donatori sănătoși (DS au fost analizate pentru nivelurile de CD147 solubil (sCD147, MMP-3 și TIMP-1 prin tehnica imunoenzimatica ELISA și pentru activitatea gelatinazelor MMP-2 si MMP-9 folosind gelatin zimografia. Rezultatele experimentale au arătat că nivelurile de sCD147, MMP-3 si TIMP-1 sunt semnificativ crescute la pacienții cu SA comparativ cu DS. sCD147 ca și raportul MMP-2/sCD147 a diferențiat pacienții cu UAA de cei fără UAA în istoricul lor. La pacienții cu SA rapoartele MMP-2/sCD147, MMP-3/sCD147 și MMP-3/TIMP-1 au sugerat dezechilibrul dintre MMPs și reglatorii lor. Aceste rezultate sugerează că rapoartele MMPs/sCD147 pot deveni biomarkeri potențiali pentru întărirea caracterizării pacienților cu SA și pentru a prognoza evoluția bolii. Corelațiile pozitive și negative dintre anumite caracteristici experimentale și/sau clinice ale pacienților cu SA și terapie subliniază de asemenea utilitatea evaluării acestor biomarkeri pentru a identifica o terapie individualizată și eficientă.

  11. Differential metalloprotease content and activity of three Loxosceles spider venoms revealed using two-dimensional electrophoresis approaches.

    Science.gov (United States)

    Trevisan-Silva, Dilza; Bednaski, Aline Viana; Gremski, Luiza Helena; Chaim, Olga Meiri; Veiga, Silvio Sanches; Senff-Ribeiro, Andrea

    2013-12-15

    Loxosceles bites have been associated with characteristic dermonecrotic lesions with gravitational spreading and systemic manifestations. Venom primarily comprises peptides and protein molecules (5-40 kDa) with multiple biological activities. Although poorly studied, metalloproteases have been identified in venoms of several Loxosceles species, presenting proteolytic effects on extracellular matrix components. The characterization of an Astacin-like protease (LALP) in Loxosceles intermedia venom was the first report of an Astacin family member as a component of animal venom. Recently, these proteases were described as a gene family in L. intermedia, Loxosceles laeta and Loxosceles gaucho. Herein, the whole venom complexity of these three Loxosceles species was analyzed using two-dimensional electrophoresis (2DE). Subproteomes of LALPs were explored through 2DE immunostaining using anti-LALP1 antibodies and 2DE gelatin zymogram. Proteins presented molecular masses ranging from 24 to 29 kDa and the majority of these molecules had basic or neutral isoelectric points (6.89-9.93). Likewise, the measurement of gelatinolytic effects of Loxosceles venom using fluorescein-gelatin showed that the three venoms have distinct proteolytic activities. The metalloprotease fibrinogenolytic activities were also evaluated. All venoms showed fibrinogenolytic activity with different proteolytic effects on Aα and Bβ chains of fibrinogen. The results reported herein suggest that the LALP family is larger than indicated in previously published data and that the complex profile of the gelatinolytic activity reflects their relevance in loxoscelism. Furthermore, our investigation implicates the brown spider venom as a source of Astacin-like proteases for use in loxoscelism studies, cell biology research and biotechnological applications. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Liver Fibrosis in HCV Monoinfected and HIV/HCV Coinfected Patients: Dysregulation of Matrix Metalloproteinases (MMPs and Their Tissue Inhibitors TIMPs and Effect of HCV Protease Inhibitors

    Directory of Open Access Journals (Sweden)

    Tiziana Latronico

    2016-03-01

    Full Text Available An imbalance between matrix metalloproteinases (MMPs and tissue inhibitors of metalloproteinases (TIMPs may contribute to liver fibrosis in patients with hepatitis C (HCV infection. We measured the circulating levels of different MMPs and TIMPs in HCV monoinfected and HIV/HCV coinfected patients and evaluated the potential for anti-HCV therapy to modulate MMP and TIMP levels in HCV subjects. We analyzed 83 plasma samples from 16 HCV monoinfected patients undergoing dual or triple anti-HCV therapy, 15 HIV/HCV coinfected patients with undetectable HIV load, and 10 healthy donors (HD. Levels of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-10, TIMP-1, and TIMP-2 were measured by a SearchLight Multiplex Immunoassay Kit. MMP-2 and MMP-9 were the highest expressed MMPs among all the analyzed samples and their levels significantly increased in HCV monoinfected and HIV/HCV coinfected subjects compared to HD. TIMP-1 levels were significantly higher in HCV and HIV/HCV subjects compared to HD and were correlated with liver stiffness. These findings raise the possibility of using circulating TIMP-1 as a non-invasive marker of liver fibrosis in HCV infection. A longitudinal study demonstrated that MMP-9 levels significantly decreased (40% reduction from baseline in patients receiving dual as well as triple direct-acting antivirals (DAA anti-HCV therapy, which had no effect on MMP-2, TIMP-1, and TIMP-2. As the dysregulation of MMP-2 and MMP-9 may reflect inflammatory processes in the liver, the decrease of MMP-9 following HCV protease inhibitor treatment suggests a positive effect on the reduction of liver inflammation.

  13. Pinus densiflora extract protects human skin fibroblasts against UVB-induced photoaging by inhibiting the expression of MMPs and increasing type I procollagen expression

    Directory of Open Access Journals (Sweden)

    Hoe-Yune Jung

    2014-01-01

    Full Text Available Exposure to ultraviolet (UV light can cause skin photoaging, which is associated with upregulation of matrix metalloproteinases (MMPs and downregulation of collagen synthesis. It has been reported that MMPs, especially MMP-1, MMP-3 and MMP-9, decrease the elasticity of the dermis by degrading collagen. In this study, we assessed the effects of Pinus densiflora extract (PDE on photoaging and investigated its mechanism of action in human skin fibroblast (Hs68 cells after UVB exposure using real-time polymerase chain reaction, Western blot analysis, and enzymatic activity assays. PDE exhibited an antioxidant activity and inhibited elastase activities in vitro. We also found that PDE inhibited UVB-induced cytotoxicity, MMP-1 production and expression of MMP-1, -3 and -9 mRNA in Hs68 cells. In addition, PDE decreased UVB-induced MMP-2 activity and MMP-2 mRNA expression. Moreover, PDE prevented the decrease of type I procollagen mediated by exposure to UVB irradiation, an effect that is linked to the upregulation and downregulation of Smad3 and Smad7, respectively. Another effect of UV irradiation is to stimulate activator protein 1 (AP-1 activity via overexpression of c-Jun/c-Fos, which, in turn, upregulates MMP-1, -3, and -9. In this study, we found that PDE suppressed UV-induced c-Jun and c-Fos mRNA expression. Taken together, these results demonstrate that PDE regulates UVB-induced expression of MMPs and type I procollagen synthesis by inhibiting AP-1 activity and restoring impaired Smad signaling, suggesting that PDE may be useful as an effective anti-photoaging agent.

  14. Progressive Increase of Matrix Metalloprotease-9 and Interleukin-8 Serum Levels during Carcinogenic Process in Human Colorectal Tract

    Science.gov (United States)

    Biasi, Fiorella; Guina, Tina; Maina, Marco; Nano, Mario; Falcone, Alessandro; Aroasio, Emiliano; Saracco, Giorgio Maria; Papotti, Mauro; Leonarduzzi, Gabriella; Poli, Giuseppe

    2012-01-01

    Background Inflammatory reactions, known to promote tumor growth and invasion, have been found associated with colorectal carcinoma (CRC). Macrophages are the chief component of the inflammatory infiltration that occurs early in the progression from non-invasive to malignant tumor, with a switch from the pro-inflammatory phenotype to the tumor-promoting phenotype. Tumor and stroma are additional sources of inflammation-related molecules. The study aimed to evaluate, during colorectal carcinogenesis from benign to malignant phases: i) the trend of serum levels of IL-8, IL-6, TGFβ1, VEGF and MMPs; ii) the parallel trend of CRP serum levels; iii) derangement of the principal TGFβ1 receptors (TGFβ1RI/RII) in tumor tissues. Methodology/Principal findings 96 patients with colon adenomas or CRC at different stages of progression, and 17 controls, were recruited. Serum IL-8, IL-6, TGFβ1, VEGF, MMPs and CRP levels were analyzed before endoscopy or surgery. TGFβ1 receptors were evaluated in adenoma biopsies and surgically-removed colorectal adenocarcinomas. Serum levels of IL-8 in adenocarcinoma patients were increased from stage II, when also the enzymatic activity of MMP-9 increased. Of note, the increasing trend of the two serum markers was found significantly correlated. Trend of serum CRP was also very similar to that of IL-8 and MMP-9, but just below statistical significance. TGFβ1 levels were lower at stage III CRC, while IL-6 and VEGF levels had no significant variations. In tissue specimens, TGFβ1 receptors were already absent in about 50% of adenomas, and this percentage of missing receptors markedly increased in CRC stages III and IV. Conclusions Combined quantification of serum IL-8, MMP-9 and CRP, appears a reliable and advanced index of inflammation-related processes during malignant phase of colorectal carcinogenesis, since these molecules remain within normal range in colorectal adenoma bearing patients, while consistently increase in the blood of CRC

  15. Progressive increase of matrix metalloprotease-9 and interleukin-8 serum levels during carcinogenic process in human colorectal tract.

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    Fiorella Biasi

    Full Text Available BACKGROUND: Inflammatory reactions, known to promote tumor growth and invasion, have been found associated with colorectal carcinoma (CRC. Macrophages are the chief component of the inflammatory infiltration that occurs early in the progression from non-invasive to malignant tumor, with a switch from the pro-inflammatory phenotype to the tumor-promoting phenotype. Tumor and stroma are additional sources of inflammation-related molecules. The study aimed to evaluate, during colorectal carcinogenesis from benign to malignant phases: i the trend of serum levels of IL-8, IL-6, TGFβ1, VEGF and MMPs; ii the parallel trend of CRP serum levels; iii derangement of the principal TGFβ1 receptors (TGFβ1RI/RII in tumor tissues. METHODOLOGY/PRINCIPAL FINDINGS: 96 patients with colon adenomas or CRC at different stages of progression, and 17 controls, were recruited. Serum IL-8, IL-6, TGFβ1, VEGF, MMPs and CRP levels were analyzed before endoscopy or surgery. TGFβ1 receptors were evaluated in adenoma biopsies and surgically-removed colorectal adenocarcinomas. Serum levels of IL-8 in adenocarcinoma patients were increased from stage II, when also the enzymatic activity of MMP-9 increased. Of note, the increasing trend of the two serum markers was found significantly correlated. Trend of serum CRP was also very similar to that of IL-8 and MMP-9, but just below statistical significance. TGFβ1 levels were lower at stage III CRC, while IL-6 and VEGF levels had no significant variations. In tissue specimens, TGFβ1 receptors were already absent in about 50% of adenomas, and this percentage of missing receptors markedly increased in CRC stages III and IV. CONCLUSIONS: Combined quantification of serum IL-8, MMP-9 and CRP, appears a reliable and advanced index of inflammation-related processes during malignant phase of colorectal carcinogenesis, since these molecules remain within normal range in colorectal adenoma bearing patients, while consistently increase in

  16. Piper retrofractum Vahl. Extract, as a PPARδ and AMPK Activator, Suppresses UVB-Induced Photoaging through Mitochondrial Biogenesis and MMPs Inhibition in Human Dermal Fibroblasts and Hairless Mice

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    Jungon Yun

    2018-01-01

    Full Text Available Photoaging occurs by UVB-irradiation and involves production of reactive oxygen species (ROS and overexpression of matrix metalloproteinases (MMPs, leading to extracellular matrix damage. Piper retrofractum Vahl. is used as a traditional medicine for antiflatulence, expectorant, sedative, and anti-irritant; however, its antiphotoaging effect has not yet been studied. The current study investigated the antiphotoaging effect of standardized Piper retrofractum extract (PRE on UVB-damaged human dermal fibroblasts and hairless mouse skin. PRE treatment activated the peroxisome proliferator-activated receptor delta (PPARδ and the adenosine monophosphate-activated protein kinase (AMPK, consequently upregulating mitochondrial synthesis and reducing ROS production. Additionally, PRE inhibited MMPs expression via suppressing mitogen-activated protein kinase (MAPK and activator protein-1 (AP-1. PRE downregulated UVB-induced inflammatory reactions by inhibiting the nuclear factor-kappa B (NF-κB activity. PRE also enhanced transforming growth factor-beta (TGF-β and the Smad signaling pathway, thereby promoting procollagen gene transcription. Furthermore, oral administration of PRE (300 mg/kg/day similarly regulated the signaling pathways and increased antioxidant enzyme expression, thus attenuating physiological deformations, such as wrinkle formation and erythema response. Collectively, these results suggest that PRE acts as a potent antiphotoaging agent via PPARδ and AMPK activation.

  17. Ubiquitin-Like Protein SAMP1 and JAMM/MPN+ Metalloprotease HvJAMM1 Constitute a System for Reversible Regulation of Metabolic Enzyme Activity in Archaea.

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    Shiyun Cao

    Full Text Available Ubiquitin/ubiquitin-like (Ub/Ubl proteins are involved in diverse cellular processes by their covalent linkage to protein substrates. Here, we provide evidence for a post-translational modification system that regulates enzyme activity which is composed of an archaeal Ubl protein (SAMP1 and a JAMM/MPN+ metalloprotease (HvJAMM1. Molybdopterin (MPT synthase activity was found to be inhibited by covalent linkage of SAMP1 to the large subunit (MoaE of MPT synthase. HvJAMM1 was shown to cleave the covalently linked inactive form of SAMP1-MoaE to the free functional individual SAMP1 and MoaE subunits of MPT synthase, suggesting reactivation of MPT synthase by this metalloprotease. Overall, this study provides new insight into the broad idea that Ub/Ubl modification is a post-translational process that can directly and reversibly regulate the activity of metabolic enzymes. In particular, we show that Ub/Ubl linkages on the active site residues of an enzyme (MPT synthase can inhibit its catalytic activity and that the enzyme can be reactivated through cleavage by a JAMM/MPN+ metalloprotease.

  18. A disintegrin and metalloprotease (ADAM)10 is highly expressed in hepatocellular carcinoma and is associated with tumour progression.

    Science.gov (United States)

    Zhang, Wei; Liu, Songyang; Liu, Kuai; Wang, Yingchao; Ji, Bai; Zhang, Xuechun; Liu, Yahui

    2014-06-01

    A disintegrin and metalloprotease (ADAM)10 has been implicated in the progression of various solid tumours. Little is known, however, about its role in hepatocellular carcinoma (HCC). The aim of the present study was to evaluate the protein and transcript level expression of ADAM10 in HCC patients. Samples of HCC and adjacent noncancerous liver tissue were taken during liver resection surgery. Immunostaining was used to measure ADAM10 protein expression levels and quantitative reverse- transcription polymerase chain reaction was used to measure ADAM10 mRNA expression levels. Levels of ADAM10 were compared, and a survival analysis undertaken. In total, 98 HCC patient samples were studied. There were significant associations between protein levels of ADAM10 and tumour grade, amount of tumour differentiation, tumour size and the presence of metastasis. Furthermore, ADAM10 protein expression was significantly associated with shortened patient survival. ADAM10 is strongly expressed in a large proportion of HCC cases, which is in agreement with findings in other tumour entities. Expression of ADAM10 may serve as a useful molecular marker for HCC. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

  19. Association of MMP - 12 polymorphisms with severe and very severe COPD: A case control study of MMPs - 1, 9 and 12 in a European population.

    LENUS (Irish Health Repository)

    Haq, Imran

    2010-01-15

    Abstract Background Genetic factors play a role in chronic obstructive pulmonary disease (COPD) but are poorly understood. A number of candidate genes have been proposed on the basis of the pathogenesis of COPD. These include the matrix metalloproteinase (MMP) genes which play a role in tissue remodelling and fit in with the protease - antiprotease imbalance theory for the cause of COPD. Previous genetic studies of MMPs in COPD have had inadequate coverage of the genes, and have reported conflicting associations of both single nucleotide polymorphisms (SNPs) and SNP haplotypes, plausibly due to under-powered studies. Methods To address these issues we genotyped 26 SNPs, providing comprehensive coverage of reported SNP variation, in MMPs- 1, 9 and 12 from 977 COPD patients and 876 non-diseased smokers of European descent and evaluated their association with disease singly and in haplotype combinations. We used logistic regression to adjust for age, gender, centre and smoking history. Results Haplotypes of two SNPs in MMP-12 (rs652438 and rs2276109), showed an association with severe\\/very severe disease, corresponding to GOLD Stages III and IV. Conclusions Those with the common A-A haplotype for these two SNPs were at greater risk of developing severe\\/very severe disease (p = 0.0039) while possession of the minor G variants at either SNP locus had a protective effect (adjusted odds ratio of 0.76; 95% CI 0.61 - 0.94). The A-A haplotype was also associated with significantly lower predicted FEV1 (42.62% versus 44.79%; p = 0.0129). This implicates haplotypes of MMP-12 as modifiers of disease severity.

  20. IL-17A promotes the migration and invasiveness of cervical cancer cells by coordinately activating MMPs expression via the p38/NF-κB signal pathway.

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    Minjuan Feng

    Full Text Available IL-17A plays an important role in many inflammatory diseases and cancers. We aimed to examine the effect of IL-17A on the invasion of cervical cancer cells and study its related mechanisms.Wound healing and matrigel transwell assays were used to examine the effect of IL-17A on cervical cancer cell migration and invasion by a panel of cervical cancer cell lines. The levels of matrix metalloproteinases (MMPs and tissue inhibitor of metalloproteinases (TIMPs were investigated using western blotting. The activity of p38 and nuclear factor-kappa B (NF-κB signal pathway was detected too.Here, we showed that IL-17A could promote the migration and invasion of cervical cancer cells. Further molecular analysis showed that IL-17A could up-regulate the expressions and activities of MMP2 and MMP9, and down-regulate the expressions of TIMP-1 and TIMP-2. Furthermore, IL-17A also activates p38 signal pathway and increased p50 and p65 nuclear expression. In addition, treatment of cervical cancer cells with the pharmacological p38/NF-κB signal pathway inhibitors, SB203580 and PDTC, potently restored the roles of invasion and upregulation of MMPs induced by IL-17A.IL-17A could promote the migration and invasion of cervical cancer cell via up-regulating MMP2 and MMP9 expression, and down-regulating TIMP-1 and TIMP-2 expression via p38/NF-κB signal pathway. IL-17A may be a potential target to improve the prognosis for patients with cervical cancer.

  1. CpxR-Dependent Thermoregulation of Serratia marcescens PrtA Metalloprotease Expression and Its Contribution to Bacterial Biofilm Formation.

    Science.gov (United States)

    Bruna, Roberto E; Molino, María Victoria; Lazzaro, Martina; Mariscotti, Javier F; García Véscovi, Eleonora

    2018-04-15

    PrtA is the major secreted metalloprotease of Serratia marcescens Previous reports implicate PrtA in the pathogenic capacity of this bacterium. PrtA is also clinically used as a potent analgesic and anti-inflammatory drug, and its catalytic properties attract industrial interest. Comparatively, there is scarce knowledge about the mechanisms that physiologically govern PrtA expression in Serratia In this work, we demonstrate that PrtA production is derepressed when the bacterial growth temperature decreases from 37°C to 30°C. We show that this thermoregulation occurs at the transcriptional level. We determined that upstream of prtA , there is a conserved motif that is directly recognized by the CpxR transcriptional regulator. This feature is found along Serratia strains irrespective of their isolation source, suggesting an evolutionary conservation of CpxR-dependent regulation of PrtA expression. We found that in S. marcescen s, the CpxAR system is more active at 37°C than at 30°C. In good agreement with these results, in a cpxR mutant background, prtA is derepressed at 37°C, while overexpression of the NlpE lipoprotein, a well-known CpxAR-inducing condition, inhibits PrtA expression, suggesting that the levels of the activated form of CpxR are increased at 37°C over those at 30°C. In addition, we establish that PrtA is involved in the ability of S. marcescens to develop biofilm. In accordance, CpxR influences the biofilm phenotype only when bacteria are grown at 37°C. In sum, our findings shed light on regulatory mechanisms that fine-tune PrtA expression and reveal a novel role for PrtA in the lifestyle of S. marcescens IMPORTANCE We demonstrate that S. marcescens metalloprotease PrtA expression is transcriptionally thermoregulated. While strongly activated below 30°C, its expression is downregulated at 37°C. We found that in S. marcescens , the CpxAR signal transduction system, which responds to envelope stress and bacterial surface adhesion, is

  2. The Disintegrin and Metalloprotease ADAM12 Is Associated with TGF-β-Induced Epithelial to Mesenchymal Transition

    Science.gov (United States)

    Ruff, Michaël; Leyme, Anthony; Le Cann, Fabienne; Bonnier, Dominique; Le Seyec, Jacques; Chesnel, Franck; Fattet, Laurent; Rimokh, Ruth; Baffet, Georges; Théret, Nathalie

    2015-01-01

    The increased expression of the Disintegrin and Metalloprotease ADAM12 has been associated with human cancers, however its role remain unclear. We have previously reported that ADAM12 expression is induced by the transforming growth factor, TGF-β and promotes TGF-β-dependent signaling through interaction with the type II receptor of TGF-β. Here we explore the implication of ADAM12 in TGF-β-mediated epithelial to mesenchymal transition (EMT), a key process in cancer progression. We show that ADAM12 expression is correlated with EMT markers in human breast cancer cell lines and biopsies. Using a non-malignant breast epithelial cell line (MCF10A), we demonstrate that TGF-β-induced EMT increases expression of the membrane-anchored ADAM12L long form. Importantly, ADAM12L overexpression in MCF10A is sufficient to induce loss of cell-cell contact, reorganization of actin cytoskeleton, up-regulation of EMT markers and chemoresistance. These effects are independent of the proteolytic activity but require the cytoplasmic tail and are specific of ADAM12L since overexpression of ADAM12S failed to induce similar changes. We further demonstrate that ADAM12L-dependent EMT is associated with increased phosphorylation of Smad3, Akt and ERK proteins. Conversely, inhibition of TGF-β receptors or ERK activities reverses ADAM12L-induced mesenchymal phenotype. Together our data demonstrate that ADAM12L is associated with EMT and contributes to TGF-β-dependent EMT by favoring both Smad-dependent and Smad-independent pathways. PMID:26407179

  3. YghJ, the secreted metalloprotease of pathogenic E. coli induces hemorrhagic fluid accumulation in mouse ileal loop.

    Science.gov (United States)

    Tapader, Rima; Bose, Dipro; Pal, Amit

    2017-04-01

    YghJ, also known as SslE (Secreted and surface associated lipoprotein) is a cell surface associated and secreted lipoprotein harbouring M60 metalloprotease domain. Though the gene is known to be conserved among both pathogenic and commensal Escherichia coli isolates, the expression and secretion of YghJ was found to be higher among diverse E. coli pathotypes. YghJ, secreted from intestinal pathogens such as enterotoxigenic E. coli (ETEC) and enteropathogenic E. coli (EPEC) has been demonstrated to possess mucinase activity and hence facilitates colonization of these enteric pathogens to intestinal epithelial cells. Importantly, YghJ is also reported to be secreted from extraintestinal pathogenic E. coli isolates. In our previous study we have shown that YghJ, purified from a neonatal septicemic E. coli isolate could trigger induction of various proinflammatory cytokines in vitro. This led us to investigate the role of YghJ in causing in vivo tissue hemorrhage. In the present study, we validate the earlier in vitro finding and have showed that YghJ can cause extensive tissue damage in mouse ileum and is also able to induce significant fluid accumulation in a dose dependent manner in a mouse ileal loop (MIL) assay. Hence, our present study not only confirms the pathogenic potential of YghJ in sepsis pathophysiology but also indicates the enterotoxic ability of YghJ which makes it an important virulence determinant of intestinal pathogenic E. coli. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. The Disintegrin and Metalloprotease ADAM12 Is Associated with TGF-β-Induced Epithelial to Mesenchymal Transition.

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    Michaël Ruff

    Full Text Available The increased expression of the Disintegrin and Metalloprotease ADAM12 has been associated with human cancers, however its role remain unclear. We have previously reported that ADAM12 expression is induced by the transforming growth factor, TGF-β and promotes TGF-β-dependent signaling through interaction with the type II receptor of TGF-β. Here we explore the implication of ADAM12 in TGF-β-mediated epithelial to mesenchymal transition (EMT, a key process in cancer progression. We show that ADAM12 expression is correlated with EMT markers in human breast cancer cell lines and biopsies. Using a non-malignant breast epithelial cell line (MCF10A, we demonstrate that TGF-β-induced EMT increases expression of the membrane-anchored ADAM12L long form. Importantly, ADAM12L overexpression in MCF10A is sufficient to induce loss of cell-cell contact, reorganization of actin cytoskeleton, up-regulation of EMT markers and chemoresistance. These effects are independent of the proteolytic activity but require the cytoplasmic tail and are specific of ADAM12L since overexpression of ADAM12S failed to induce similar changes. We further demonstrate that ADAM12L-dependent EMT is associated with increased phosphorylation of Smad3, Akt and ERK proteins. Conversely, inhibition of TGF-β receptors or ERK activities reverses ADAM12L-induced mesenchymal phenotype. Together our data demonstrate that ADAM12L is associated with EMT and contributes to TGF-β-dependent EMT by favoring both Smad-dependent and Smad-independent pathways.

  5. Insecticidal activity of the metalloprotease AprA occurs through suppression of host cellular and humoral immunity.

    Science.gov (United States)

    Lee, Seung Ah; Jang, Seong Han; Kim, Byung Hyun; Shibata, Toshio; Yoo, Jinwook; Jung, Yunjin; Kawabata, Shun-Ichiro; Lee, Bok Luel

    2018-04-01

    The biochemical characterization of virulence factors from entomopathogenic bacteria is important to understand entomopathogen-insect molecular interactions. Pseudomonas entomophila is a typical entomopathogenic bacterium that harbors virulence factors against several insects. However, the molecular actions of these factors against host innate immune responses are not clearly elucidated. In this study, we observed that bean bugs (Riptortus pedestris) that were injected with P. entomophila were highly susceptible to this bacterium. To determine how P. entomophila counteracts the host innate immunity to survive within the insect, we purified a highly enriched protein with potential host insect-killing activity from the culture supernatant of P. entomophila. Then, a 45-kDa protein was purified to homogeneity and identified as AprA which is an alkaline zinc metalloprotease of the genus Pseudomonas by liquid chromatography mass spectrometry (LC-MS). Purified AprA showed a pronounced killing effect against host insects and suppressed both host cellular and humoral innate immunity. Furthermore, to show that AprA is an important insecticidal protein of P. entomophila, we used an aprA-deficient P. entomophila mutant strain (ΔaprA). When ΔaprA mutant cells were injected to host insects, this mutant exhibited extremely attenuated virulence. In addition, the cytotoxicity against host hemocytes and the antimicrobial peptide-degrading ability of the ΔaprA mutant were greatly decreased. These findings suggest that AprA functions as an important insecticidal protein of P. entomophila via suppression of host cellular and humoral innate immune responses. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Ex Vivo and In Vivo Imaging and Biodistribution of Aptamers Targeting the Human Matrix MetalloProtease-9 in Melanomas.

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    David Kryza

    Full Text Available The human Matrix MetalloProtease-9 (hMMP-9 is overexpressed in tumors where it promotes the release of cancer cells thus contributing to tumor metastasis. We raised aptamers against hMMP-9, which constitutes a validated marker of malignant tumors, in order to design probes for imaging tumors in human beings. A chemically modified RNA aptamer (F3B, fully resistant to nucleases was previously described. This compound was subsequently used for the preparation of F3B-Cy5, F3B-S-acetylmercaptoacetyltriglycine (MAG and F3B-DOTA. The binding properties of these derivatives were determined by surface plasmon resonance and electrophoretic mobility shift assay. Optical fluorescence imaging confirmed the binding to hMMP-9 in A375 melanoma bearing mice. Quantitative biodistribution studies were performed at 30 min, 1h and 2 h post injection of 99mTc-MAG-aptamer and 111In-DOTA-F3B. 99mTc radiolabeled aptamer specifically detected hMMP-9 in A375 melanoma tumors but accumulation in digestive tract was very high. Following i.v. injection of 111In-DOTA-F3B, high level of radioactivity was observed in kidneys and bladder but digestive tract uptake was very limited. Tumor uptake was significantly (student t test, p<0.05 higher for 111In-DOTA-F3B with 2.0%ID/g than for the 111In-DOTA-control oligonucleotide (0.7%ID/g with tumor to muscle ratio of 4.0. Such difference in tumor accumulation has been confirmed by ex vivo scintigraphic images performed at 1h post injection and by autoradiography, which revealed the overexpression of hMMP-9 in sections of human melanomas. These results demonstrate that F3B aptamer is of interest for detecting hMMP-9 in melanoma tumor.

  7. Characterization of an extensin-modifying metalloprotease: N-terminal processing and substrate cleavage pattern of Pectobacterium carotovorum Prt1.

    Science.gov (United States)

    Feng, Tao; Nyffenegger, Christian; Højrup, Peter; Vidal-Melgosa, Silvia; Yan, Kok-Phen; Fangel, Jonatan Ulrik; Meyer, Anne S; Kirpekar, Finn; Willats, William G; Mikkelsen, Jørn D

    2014-12-01

    Compared to other plant cell wall-degrading enzymes, proteases are less well understood. In this study, the extracellular metalloprotease Prt1 from Pectobacterium carotovorum (formerly Erwinia carotovora) was expressed in Escherichia coli and characterized with respect to N-terminal processing, thermal stability, substrate targets, and cleavage patterns. Prt1 is an autoprocessing protease with an N-terminal signal pre-peptide and a pro-peptide which has to be removed in order to activate the protease. The sequential cleavage of the N-terminus was confirmed by mass spectrometry (MS) fingerprinting and N-terminus analysis. The optimal reaction conditions for the activity of Prt1 on azocasein were at pH 6.0, 50 °C. At these reaction conditions, K M was 1.81 mg/mL and k cat was 1.82 × 10(7) U M(-1). The enzyme was relatively stable at 50 °C with a half-life of 20 min. Ethylenediaminetetraacetic acid (EDTA) treatment abolished activity; Zn(2+) addition caused regain of the activity, but Zn(2+)addition decreased the thermal stability of the Prt1 enzyme presumably as a result of increased proteolytic autolysis. In addition to casein, the enzyme catalyzed degradation of collagen, potato lectin, and plant extensin. Analysis of the cleavage pattern of different substrates after treatment with Prt1 indicated that the protease had a substrate cleavage preference for proline in substrate residue position P1 followed by a hydrophobic residue in residue position P1' at the cleavage point. The activity of Prt1 against plant cell wall structural proteins suggests that this enzyme might become an important new addition to the toolbox of cell-wall-degrading enzymes for biomass processing.

  8. Matrix metalloproteases as maestros for the dual role of LPS- and IL-10-stimulated macrophages in cancer cell behaviour

    International Nuclear Information System (INIS)

    Cardoso, Ana P.; Pinto, Marta L.; Pinto, Ana T.; Pinto, Marta T.; Monteiro, Cátia; Oliveira, Marta I.; Santos, Susana G.; Relvas, João B.; Seruca, Raquel; Mantovani, Alberto; Mareel, Marc; Barbosa, Mário A.; Oliveira, Maria J.

    2015-01-01

    The interactions established between macrophages and cancer cells are largely dependent on instructions from the tumour microenvironment. Macrophages may differentiate into populations with distinct inflammatory profiles, but knowledge on their role on cancer cell activities is still very scarce. In this work, we investigated the influence of pro-inflammatory (LPS-stimulated) and anti-inflammatory (IL-10-stimulated) macrophages on gastric and colorectal cancer cell invasion, motility/migration, angiogenesis and proteolysis, and the associated molecular mechanisms. Following exposure of gastric and colon cancer cell lines to LPS- and IL-10-stimulated human macrophages, either by indirect contact or conditioned media, we analyzed the effect of the different macrophage populations on cancer cell invasion, migration, motility and phosphorylation status of EGFR and several interacting partners. Cancer-cell induced angiogenesis upon the influence of conditioned media from both macrophage populations was assessed using the chick embryo chorioallantoic membrane assay. MMP activities were evaluated by gelatin zymograhy. Our results show that IL-10-stimulated macrophages are more efficient in promoting in vitro cancer cell invasion and migration. In addition, soluble factors produced by these macrophages enhanced in vivo cancer cell-induced angiogenesis, as opposed to their LPS-stimulated counterparts. We further demonstrate that differences in the ability of these macrophage populations to stimulate invasion or angiogenesis cannot be explained by the EGFR-mediated signalling, since both LPS- and IL-10-stimulated macrophages similarly induce the phosphorylation of cancer cell EGFR, c-Src, Akt, ERK1/2, and p38. Interestingly, both populations exert distinct proteolytic activities, being the IL-10-stimulated macrophages the most efficient in inducing matrix metalloprotease (MMP)-2 and MMP-9 activities. Using a broad-spectrum MMP inhibitor, we demonstrated that proteolysis was

  9. Astacin-like metalloproteases are a gene family of toxins present in the venom of different species of the brown spider (genus Loxosceles).

    Science.gov (United States)

    Trevisan-Silva, Dilza; Gremski, Luiza H; Chaim, Olga M; da Silveira, Rafael B; Meissner, Gabriel O; Mangili, Oldemir C; Barbaro, Katia C; Gremski, Waldemiro; Veiga, Silvio S; Senff-Ribeiro, Andrea

    2010-01-01

    Brown spiders have a worldwide distribution, and their venom has a complex composition containing many different molecules. Herein, we report the existence of a family of astacin-like metalloprotease toxins in Loxosceles intermedia venom, as well as in the venom of different species of Loxosceles. Using a cDNA library from the L. intermedia venom gland, we cloned two novel cDNAs encoding astacin-like metalloprotease toxins, LALP2 and LALP3. Using an anti-serum against the previously described astacin-like toxin in L. intermedia venom (LALP1), we detected the presence of immunologically-related toxins in the venoms of L. intermedia, Loxosceles laeta, and Loxosceles gaucho. Zymographic experiments showed gelatinolytic activity of crude venoms of L. intermedia, L. laeta, and L. gaucho (which could be inhibited by the divalent metal chelator 1,10-phenanthroline) at electrophoretic mobilities identical to those reported for immunological cross-reactivity. Moreover, mRNAs extracted from L. laeta and L. gaucho venom glands were screened for astacin-like metalloproteases, and cDNAs obtained using LALP1-specific primers were sequenced, and their deduced amino acid sequences confirmed they were members of the astacin family with the family signatures (HEXXHXXGXXHE and MXY), LALP4 and LALP5, respectively. Sequence comparison of deduced amino acid sequences revealed that LALP2, LALP3, LALP4, and LALP5 are related to the astacin family. This study identified the existence of gene family of astacin-like toxins in the venoms of brown spiders and raises the possibility that these molecules are involved in the deleterious effects triggered by the venom. 2009 Elsevier Masson SAS. All rights reserved.

  10. Norcantharidin inhibits tumor growth and vasculogenic mimicry of human gallbladder carcinomas by suppression of the PI3-K/MMPs/Ln-5γ2 signaling pathway

    International Nuclear Information System (INIS)

    Zhang, Jing-Tao; Sun, Wei; Zhang, Wen-Zhong; Ge, Chun-Yan; Liu, Zhong-Yan; Zhao, Ze-Ming; Lu, Xing-Sui; Fan, Yue-Zu

    2014-01-01

    Vasculogenic mimicry (VM) is a novel tumor blood supply in some highly aggressive malignant tumors. Recently, we reported VM existed in gallbladder carcinomas (GBCs) and the formation of the special passage through the activation of the PI3K/MMPs/Ln-5γ2 signaling pathway. GBC is a highly aggressive malignant tumor with disappointing treatments and a poor prognosis. Norcantharidin (NCTD) has shown to have multiple antitumor activities against GBCs, etc; however the exact mechanism is not thoroughly elucidated. In this study, we firstly investigated the anti-VM activity of NCTD as a VM inhibitor for GBCs and its underlying mechanisms. In vitro and in vivo experiments to determine the effects of NCTD on proliferation, invasion, migration, VM formation, hemodynamic and tumor growth of GBC-SD cells and xenografts were respectively done by proliferation, invasion, migration assays, H&E staining and CD31-PAS double stainings, optic/electron microscopy, tumor assay, and dynamic micro-MRA. Further, immunohistochemistry, immunofluorescence, Western blotting and RT-PCR were respectively used to examine expression of VM signaling-related markers PI3-K, MMP-2, MT1-MMP and Ln-5γ2 in GBC-SD cells and xenografts in vitro and in vivo. After treatment with NCTD, proliferation, invasion, migration of GBC-SD cells were inhibited; GBC-SD cells and xenografts were unable to form VM-like structures; tumor center-VM region of the xenografts exhibited a decreased signal in intensity; then cell or xenograft growth was inhibited. Whereas all of untreated GBC-SD cells and xenografts formed VM-like structures with the same conditions; the xenograft center-VM region exhibited a gradually increased signal; and facilitated cell or xenograft growth. Furthermore, expression of MMP-2 and MT1-MMP products from sections/supernates of 3-D matrices and the xenografts, and expression of PI3-K, MMP-2, MM1-MMP and Ln-5γ2 proteins/mRNAs of the xenografts were all decreased in NCTD or TIMP-2 group; (all P

  11. PARP-1 depletion in combination with carbon ion exposure significantly reduces MMPs activity and overall increases TIMPs expression in cultured HeLa cells

    International Nuclear Information System (INIS)

    Ghorai, Atanu; Sarma, Asitikantha; Chowdhury, Priyanka; Ghosh, Utpal

    2016-01-01

    Hadron therapy is an innovative technique where cancer cells are precisely killed leaving surrounding healthy cells least affected by high linear energy transfer (LET) radiation like carbon ion beam. Anti-metastatic effect of carbon ion exposure attracts investigators into the field of hadron biology, although details remain poor. Poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors are well-known radiosensitizer and several PARP-1 inhibitors are in clinical trial. Our previous studies showed that PARP-1 depletion makes the cells more radiosensitive towards carbon ion than gamma. The purpose of the present study was to investigate combining effects of PARP-1 inhibition with carbon ion exposure to control metastatic properties in HeLa cells. Activities of matrix metalloproteinases-2, 9 (MMP-2, MMP-9) were measured using the gelatin zymography after 85 MeV carbon ion exposure or gamma irradiation (0- 4 Gy) to compare metastatic potential between PARP-1 knock down (HsiI) and control cells (H-vector - HeLa transfected with vector without shRNA construct). Expression of MMP-2, MMP-9, tissue inhibitor of MMPs such as TIMP-1, TIMP-2 and TIMP-3 were checked by immunofluorescence and western blot. Cell death by trypan blue, apoptosis and autophagy induction were studied after carbon ion exposure in each cell-type. The data was analyzed using one way ANOVA and 2-tailed paired-samples T-test. PARP-1 silencing significantly reduced MMP-2 and MMP-9 activities and carbon ion exposure further diminished their activities to less than 3 % of control H-vector. On the contrary, gamma radiation enhanced both MMP-2 and MMP-9 activities in H-vector but not in HsiI cells. The expression of MMP-2 and MMP-9 in H-vector and HsiI showed different pattern after carbon ion exposure. All three TIMPs were increased in HsiI, whereas only TIMP-1 was up-regulated in H-vector after irradiation. Notably, the expressions of all TIMPs were significantly higher in HsiI than H-vector at 4 Gy. Apoptosis was

  12. Aminopeptidase N1 (EtAPN1), an M1 metalloprotease of the apicomplexan parasite Eimeria tenella, participates in parasite development.

    Science.gov (United States)

    Gras, Simon; Byzia, Anna; Gilbert, Florence B; McGowan, Sheena; Drag, Marcin; Silvestre, Anne; Niepceron, Alisson; Lecaille, Fabien; Lalmanach, Gilles; Brossier, Fabien

    2014-07-01

    Aminopeptidases N are metalloproteases of the M1 family that have been reported in numerous apicomplexan parasites, including Plasmodium, Toxoplasma, Cryptosporidium, and Eimeria. While investigating the potency of aminopeptidases as therapeutic targets against coccidiosis, one of the most important avian diseases caused by the genus Eimeria, we identified and characterized Eimeria tenella aminopeptidase N1 (EtAPN1). Its inhibition by bestatin and amastatin, as well as its reactivation by divalent ions, is typical of zinc-dependent metalloproteases. EtAPN1 shared a similar sequence, three-dimensional structure, and substrate specificity and similar kinetic parameters with A-M1 from Plasmodium falciparum (PfA-M1), a validated target in the treatment of malaria. EtAPN1 is synthesized as a 120-kDa precursor and cleaved into 96-, 68-, and 38-kDa forms during sporulation. Further, immunolocalization assays revealed that, similar to PfA-M1, EtAPN1 is present during the intracellular life cycle stages in both the parasite cytoplasm and the parasite nucleus. The present results support the hypothesis of a conserved role between the two aminopeptidases, and we suggest that EtAPN1 might be a valuable target for anticoccidiosis drugs. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  13. Oral administration of Aloe vera gel powder prevents UVB-induced decrease in skin elasticity via suppression of overexpression of MMPs in hairless mice.

    Science.gov (United States)

    Saito, Marie; Tanaka, Miyuki; Misawa, Eriko; Yao, Ruiquing; Nabeshima, Kazumi; Yamauchi, Kouji; Abe, Fumiaki; Yamamoto, Yuki; Furukawa, Fukumi

    2016-07-01

    This study reports the effects of oral Aloe vera gel powder (AVGP) containing Aloe sterols on skin elasticity and the extracellular matrix in ultraviolet B (UVB)-irradiated hairless mice. Ten-week-old hairless mice were fed diets containing 0.3% AVGP for 8 weeks and irradiated UVB for 6 weeks. Mice treated with AVGP showed significant prevention of the UVB-induced decrease in skin elasticity. To investigate the mechanism underlying this suppression of skin elasticity loss, we measured the expression of matrix metalloproteinase (MMP)-2, -9, and -13. AVGP prevented both the UVB-induced increases in MMPs expressions. Moreover, we investigated hyaluronic acid (HA) content of mice dorsal skin and gene expression of HA synthase-2 (Has2). In the results, AVGP oral administration prevented UVB-induced decreasing in skin HA content and Has2 expression and attenuates the UVB-induced decrease in serum adiponectin, which promotes Has2 expression. These results suggested that AVGP has the ability to prevent the skin photoaging.

  14. CS5931, a Novel Polypeptide in Ciona savignyi, Represses Angiogenesis via Inhibiting Vascular Endothelial Growth Factor (VEGF and Matrix Metalloproteinases (MMPs

    Directory of Open Access Journals (Sweden)

    Ge Liu

    2014-03-01

    Full Text Available CS5931 is a novel polypeptide from Ciona savignyi with anticancer activities. Previous study in our laboratory has shown that CS5931 can induce cell death via mitochondrial apoptotic pathway. In the present study, we found that the polypeptide could inhibit angiogenesis both in vitro and in vivo. CS5931 inhibited the proliferation, migration and formation of capillary-like structures of HUVECs (Human Umbilical Vein Endothelial Cell in a dose-dependent manner. Additionally, CS5931 repressed spontaneous angiogenesis of the zebrafish vessels. Further studies showed that CS5931 also blocked vascular endothelial growth factor (VEGF production but without any effect on its mRNA expression. Moreover, CS5931 reduced the expression of matrix metalloproteinases (MMP-2 and MMP-9 both on protein and mRNA levels in HUVEC cells. We demonstrated that CS5931 possessed strong anti-angiogenic activity both in vitro and in vivo, possible via VEGF and MMPs. This study indicates that CS5931 has the potential to be developed as a novel therapeutic agent as an inhibitor of angiogenesis for the treatment of cancer.

  15. MicroRNA-21 promotes bone mesenchymal stem cells migration in vitro by activating PI3K/Akt/MMPs pathway.

    Science.gov (United States)

    Lv, Chen; Yang, Shengwu; Chen, Xin; Zhu, Xiongbai; Lin, Wenjun; Wang, Lu; Huang, Zhengxiang; Wang, Mingyue; Tu, Guanjun

    2017-12-01

    MicroRNA-21 (miR-21) contributes to anti-apoptosis in bone marrow mesenchymal stem cells (BMSC), but its role in the migration of BMSCs remains vague. The aim of this study was to determine the possible effect of miR-21 on regulating BMSCs directional migration and the expression of MMP-2/MMP-9 in BMSCs in vitro. BMSCs were successfully infected with miR-21-up lentivirus. Cell migration using Transwell assay indicated that upregulated expression of miR-21 could significantly promote BMSCs migration. Western blot analysis indicated that miR-21 significantly upregulated the expression of MMP-2 and MMP-9, which were related to metastasis-associated genes. GM6001, the specific MMPs inhibitor, abrogated the upregulated expression of MMP-2/MMP-9 and abolished the positive effect of miR-21 on promoting BMSCs migration. Meanwhile, miR-21 significantly enhanced Akt phosphorylation, as measured by Western blot analysis. LY294002, an inhibitor of Akt activation, abrogated the phosphorylation of Akt and abolished the positive effect of miR-21 on promoting BMSCs migration and upregulating MMP-2/MMP-9 expression. These results suggest that miR-21 contributes to BMSCs migration by upregulating MMP-2/MMP-9, potentially via the PI3K/Akt pathway. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Dynamic expression profiles of MMPs/TIMPs and collagen deposition in mechanically unloaded rat heart: implications for left ventricular assist device support-induced cardiac alterations.

    Science.gov (United States)

    Wang, Lu; Xu, Yu-Xian; Du, Xiao-Jie; Sun, Quan-Ge; Tian, Ying-Jun

    2013-09-01

    Left ventricular assist devices (LVADs) ameliorate heart failure by reducing preload and afterload. However, extracellular matrix (ECM) deposition after application of LVADs is not clearly defined. The purpose of the present study was to investigate ECM remodeling after mechanical unloading in a rat heart transplant model. Sixty male Lewis rats were subjected to abdominal heterotopic heart transplantation, and the transplanted hearts were pressure- and volume-unloaded. The age- and weight- matched male Lewis rats who had undergone open thoracic surgeries were used as the control. Left ventricle ECM accumulation and the expression/activity of matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases (TIMPs) were measured on the third, seventh, and fourteenth days after transplantation/sham surgery. Compared with the control group, myocardial ECM deposition significantly increased on the seventh and fourteenth days after heart transplantation (P < 0.05) and peaked on the 14th day. The gelatinase activity as well as mRNA expression of MMP-2 and MMP-9 significantly increased after transplantation (P < 0.05). Both mRNA and protein levels of TIMP-1 and TIMP-2 significantly increased compared with those of the control group. Mechanical unloading may lead to adverse remodeling of the ECM of the left ventricle. The underlying mechanism may due to the imbalance of the MMP/TIMP system, especially the remarkable upregulation of TIMPs in the pressure and volume unloaded heart.

  17. A smallest 6 kda metalloprotease, mini-matrilysin, in living world: a revolutionary conserved zinc-dependent proteolytic domain- helix-loop-helix catalytic zinc binding domain (ZBD

    Directory of Open Access Journals (Sweden)

    Yu Wei-Hsuan

    2012-05-01

    Full Text Available Abstract Background The Aim of this study is to study the minimum zinc dependent metalloprotease catalytic folding motif, helix B Met loop-helix C, with proteolytic catalytic activities in metzincin super family. The metzincin super family share a catalytic domain consisting of a twisted five-stranded β sheet and three long α helices (A, B and C. The catalytic zinc is at the bottom of the cleft and is ligated by three His residues in the consensus sequence motif, HEXXHXXGXXH, which is located in helix B and part of the adjacent Met turn region. An interesting question is - what is the minimum portion of the enzyme that still possesses catalytic and inhibitor recognition?” Methods We have expressed a 60-residue truncated form of matrilysin which retains only the helix B-Met turn-helix C region and deletes helix A and the five-stranded β sheet which form the upper portion of the active cleft. This is only 1/4 of the full catalytic domain. The E. coli derived 6 kDa MMP-7 ZBD fragments were purified and refolded. The proteolytic activities were analyzed by Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay and CM-transferrin zymography analysis. SC44463, BB94 and Phosphoramidon were computationally docked into the 3day structure of the human MMP7 ZBD and TAD and thermolysin using the docking program GOLD. Results This minimal 6 kDa matrilysin has been refolded and shown to have proteolytic activity in the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay. Triton X-100 and heparin are important factors in the refolding environment for this mini-enzyme matrilysin. This minienzyme has the proteolytic activity towards peptide substrate, but the hexamer and octamer of the mini MMP-7 complex demonstrates the CM-transferrin proteolytic activities in zymographic analysis. Peptide digestion is inhibited by SC44463, specific MMP7 inhibitors, but not phosphorimadon. Interestingly, the mini MMP-7 can be processed by autolysis and producing ~ 6

  18. Abnormalities in the zinc-metalloprotease-BDNF axis may contribute to megalencephaly and cortical hyperconnectivity in young autism spectrum disorder patients.

    Science.gov (United States)

    Koh, Jae-Young; Lim, Joon Seo; Byun, Hyae-Ran; Yoo, Min-Heui

    2014-09-03

    Whereas aberrant brain connectivity is likely the core pathology of autism-spectrum disorder (ASD), studies do not agree as to whether hypo- or hyper-connectivity is the main underlying problem. Recent functional imaging studies have shown that, in most young ASD patients, cerebral cortical regions appear hyperconnected, and cortical thickness/brain size is increased. Collectively, these findings indicate that developing ASD brains may exist in an altered neurotrophic milieu. Consistently, some ASD patients, as well as some animal models of ASD, show increased levels of brain-derived neurotrophic factor (BDNF). However, how BDNF is upregulated in ASD is unknown. To address this question, we propose the novel hypothesis that a putative zinc-metalloprotease-BDNF (ZMB) axis in the forebrain plays a pivotal role in the development of hyperconnectivity and megalencephaly in ASD. We have previously demonstrated that extracellular zinc at micromolar concentrations can rapidly increase BDNF levels and phosphorylate the receptor tyrosine kinase TrkB via the activation of metalloproteases. The role of metalloproteases in ASD is still uncertain, but in fragile X syndrome, a monogenic disease with an autistic phenotype, the levels of MMP are increased. Early exposure to lipopolysaccharides (LPS) and other MMP activators such as organic mercurials also have been implicated in ASD pathogenesis. The resultant increases in BDNF levels at synapses, especially those involved in the zinc-containing, associative glutamatergic system may produce abnormal brain circuit development. Various genetic mutations that lead to ASD are also known to affect BDNF signaling: some down-regulate, and others up-regulate it. We hypothesize that, although both up- and down-regulation of BDNF may induce autism symptoms, only BDNF up-regulation is associated with the hyperconnectivity and large brain size observed in most young idiopathic ASD patients. To test this hypothesis, we propose to examine the

  19. A smallest 6 kda metalloprotease, mini-matrilysin, in living world: a revolutionary conserved zinc-dependent proteolytic domain- helix-loop-helix catalytic zinc binding domain (ZBD).

    Science.gov (United States)

    Yu, Wei-Hsuan; Huang, Po-Tsang; Lou, Kuo-Long; Yu, Shuan-Su C; Lin, Chen

    2012-05-29

    The Aim of this study is to study the minimum zinc dependent metalloprotease catalytic folding motif, helix B Met loop-helix C, with proteolytic catalytic activities in metzincin super family. The metzincin super family share a catalytic domain consisting of a twisted five-stranded β sheet and three long α helices (A, B and C). The catalytic zinc is at the bottom of the cleft and is ligated by three His residues in the consensus sequence motif, HEXXHXXGXXH, which is located in helix B and part of the adjacent Met turn region. An interesting question is - what is the minimum portion of the enzyme that still possesses catalytic and inhibitor recognition?" We have expressed a 60-residue truncated form of matrilysin which retains only the helix B-Met turn-helix C region and deletes helix A and the five-stranded β sheet which form the upper portion of the active cleft. This is only 1/4 of the full catalytic domain. The E. coli derived 6 kDa MMP-7 ZBD fragments were purified and refolded. The proteolytic activities were analyzed by Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay and CM-transferrin zymography analysis. SC44463, BB94 and Phosphoramidon were computationally docked into the 3 day structure of the human MMP7 ZBD and TAD and thermolysin using the docking program GOLD. This minimal 6 kDa matrilysin has been refolded and shown to have proteolytic activity in the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay. Triton X-100 and heparin are important factors in the refolding environment for this mini-enzyme matrilysin. This minienzyme has the proteolytic activity towards peptide substrate, but the hexamer and octamer of the mini MMP-7 complex demonstrates the CM-transferrin proteolytic activities in zymographic analysis. Peptide digestion is inhibited by SC44463, specific MMP7 inhibitors, but not phosphorimadon. Interestingly, the mini MMP-7 can be processed by autolysis and producing ~ 6 ~ 7 kDa fragments. Thus, many of the functions of the enzyme are

  20. Moojenactivase, a novel pro-coagulant PIIId metalloprotease isolated from Bothrops moojeni snake venom, activates coagulation factors II and X and induces tissue factor up-regulation in leukocytes.

    Science.gov (United States)

    Sartim, Marco A; Costa, Tassia R; Laure, Helen J; Espíndola, Milena S; Frantz, Fabiani G; Sorgi, Carlos A; Cintra, Adélia C O; Arantes, Eliane C; Faccioli, Lucia H; Rosa, José C; Sampaio, Suely V

    2016-05-01

    Coagulopathies following snakebite are triggered by pro-coagulant venom toxins, in which metalloproteases play a major role in envenomation-induced coagulation disorders by acting on coagulation cascade, platelet function and fibrinolysis. Considering this relevance, here we describe the isolation and biochemical characterization of moojenactivase (MooA), a metalloprotease from Bothrops moojeni snake venom, and investigate its involvement in hemostasis in vitro. MooA is a glycoprotein of 85,746.22 Da, member of the PIIId group of snake venom metalloproteases, composed of three linked disulfide-bonded chains: an N-glycosylated heavy chain, and two light chains. The venom protease induced human plasma clotting in vitro by activating on both blood coagulation factors II (prothrombin) and X, which in turn generated α-thrombin and factor Xa, respectively. Additionally, MooA induced expression of tissue factor (TF) on the membrane surface of peripheral blood mononuclear cells (PBMC), which led these cells to adopt pro-coagulant characteristics. MooA was also shown to be involved with production of the inflammatory mediators TNF-α, IL-8 and MCP-1, suggesting an association between MooA pro-inflammatory stimulation of PBMC and TF up-regulation. We also observed aggregation of washed platelets when in presence of MooA; however, the protease had no effect on fibrinolysis. Our findings show that MooA is a novel hemostatically active metalloprotease, which may lead to the development of coagulopathies during B. moojeni envenomation. Moreover, the metalloprotease may contribute to the development of new diagnostic tools and pharmacological approaches applied to hemostatic disorders.

  1. Inhibitory properties of the anti-bothropic complex from Didelphis albiventris serum on toxic and pharmacological actions of metalloproteases and myotoxins from Bothrops asper venom.

    Science.gov (United States)

    Trento, E P; Garcia, O S; Rucavado, A; França, S C; Batalini, C; Arantes, E C; Giglio, J R; Soares, A M

    2001-12-01

    Anti-bothropic complex (ABC) was isolated from the serum of the South American opossum (Didelphis albiventris) by single-step affinity chromatography using a Sepharose-immobilized metalloprotease (BaP1) from Bothrops asper as the binding protein. Biochemical characterization of ABC showed the presence of two glycosylated subunits of 43 and 45 kDa, respectively, with an isoelectric point asper venom. In addition to the anti-hemorrhagic and anti-proteolytic activities, ABC also showed anti-myotoxic, anti-lethal, and anti-edematogenic effects against myotoxic phospholipases A(2) isolated from the same venom. Moreover, it had inhibitory effects on the phospholipase A(2) activity of the crude venom as well as the isolated venom phospholipases A(2).

  2. Amyloid-beta (25-35) peptide induces the release of pro-matrix metalloprotease 9 (pro-MMP-9) from human neutrophils.

    Science.gov (United States)

    Achilli, Cesare; Ciana, Annarita; Minetti, Giampaolo

    2014-12-01

    Alzheimer's disease (AD) is a degenerative process of the brain, leading to increasing impairment of cognitive functions, and is associated with accumulation in the brain of several amyloid-beta (Aβ) peptides (as amyloid plaques), including Aβ25-35. Neutrophils, the most abundant immune cell type infiltrated in the brain of AD patients, accumulate behind amyloid plaques. Aβ peptides can trigger activation of chemotaxis and oxidative burst in neutrophils, suggesting a role in modulating the neuroinflammation process. We have shown that Aβ25-35 can induce the release from human neutrophils of pro-MMP-9, a metalloprotease involved in the onset of inflammation, corroborating the hypothesis of the involvement of infiltrated neutrophils in the inflammatory processes, which occur in the AD brain.

  3. Haemorrhagic snake venom metalloproteases and human ADAMs cleave LRP5/6, which disrupts cell-cell adhesions in vitro and induces haemorrhage in vivo.

    Science.gov (United States)

    Seo, Tadahiko; Sakon, Taketo; Nakazawa, Shiori; Nishioka, Asuka; Watanabe, Kohei; Matsumoto, Kaori; Akasaka, Mari; Shioi, Narumi; Sawada, Hitoshi; Araki, Satohiko

    2017-06-01

    Snake venom metalloproteases (SVMPs) are members of the a disintegrin and metalloprotease (ADAM) family of proteins, as they possess similar domains. SVMPs are known to elicit snake venom-induced haemorrhage; however, the target proteins and cleavage sites are not known. In this work, we identified a target protein of vascular apoptosis-inducing protein 1 (VAP1), an SVMP, relevant to its ability to induce haemorrhage. VAP1 disrupted cell-cell adhesions by relocating VE-cadherin and γ-catenin from the cell-cell junction to the cytosol, without inducing proteolysis of VE-cadherin. The Wnt receptors low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) are known to promote catenin relocation, and are rendered constitutively active in Wnt signalling by truncation. Thus, we examined whether VAP1 cleaves LRP5/6 to induce catenin relocation. Indeed, we found that VAP1 cleaved the extracellular region of LRP6 and LRP5. This cleavage removes four inhibitory β-propeller structures, resulting in activation of LRP5/6. Recombinant human ADAM8 and ADAM12 also cleaved LRP6 at the same site. An antibody against a peptide including the LRP6-cleavage site inhibited VAP1-induced VE-cadherin relocation and disruption of cell-cell adhesions in cultured cells, and blocked haemorrhage in mice in vivo. Intriguingly, animals resistant to the effects of haemorrhagic snake venom express variants of LRP5/6 that lack the VAP1-cleavage site, or low-density lipoprotein receptor domain class A domains involved in formation of the constitutively active form. The results validate LRP5/6 as physiological targets of ADAMs. Furthermore, they indicate that SVMP-induced cleavage of LRP5/6 causes disruption of cell-cell adhesion and haemorrhage, potentially opening new avenues for the treatment of snake bites. © 2017 Federation of European Biochemical Societies.

  4. VanT, a homologue of Vibrio harveyi LuxR, regulates serine, metalloprotease, pigment, and biofilm production in Vibrio anguillarum.

    Science.gov (United States)

    Croxatto, Antony; Chalker, Victoria J; Lauritz, Johan; Jass, Jana; Hardman, Andrea; Williams, Paul; Cámara, Miguel; Milton, Debra L

    2002-03-01

    Vibrio anguillarum possesses at least two N-acylhomoserine lactone (AHL) quorum-sensing circuits, one of which is related to the luxMN system of Vibrio harveyi. In this study, we have cloned an additional gene of this circuit, vanT, encoding a V. harveyi LuxR-like transcriptional regulator. A V. anguillarum Delta vanT null mutation resulted in a significant decrease in total protease activity due to loss of expression of the metalloprotease EmpA, but no changes in either AHL production or virulence. Additional genes positively regulated by VanT were identified from a plasmid-based gene library fused to a promoterless lacZ. Three lacZ fusions (serA::lacZ, hpdA-hgdA::lacZ, and sat-vps73::lacZ) were identified which exhibited decreased expression in the Delta vanT strain. SerA is similar to 3-phosphoglycerate dehydrogenases and catalyzes the first step in the serine-glycine biosynthesis pathway. HgdA has identity with homogentisate dioxygenases, and HpdA is homologous to 4-hydroxyphenylpyruvate dioxygenases (HPPDs) involved in pigment production. V. anguillarum strains require an active VanT to produce high levels of an L-tyrosine-induced brown color via HPPD, suggesting that VanT controls pigment production. Vps73 and Sat are related to Vibrio cholerae proteins encoded within a DNA locus required for biofilm formation. A V. anguillarum Delta vanT mutant and a mutant carrying a polar mutation in the sat-vps73 DNA locus were shown to produce defective biofilms. Hence, a new member of the V. harveyi LuxR transcriptional activator family has been characterized in V. anguillarum that positively regulates serine, metalloprotease, pigment, and biofilm production.

  5. Análise in vitro da expressão de proteínas da matriz extracelular (MEC) e de metaloproteinases da matriz (MMPs) em células-tronco adultas de polpa dentária humana

    OpenAIRE

    Sueli Patricia Harumi Miyagi

    2008-01-01

    Células-tronco adultas podem ser isoladas de vários tecidos, dentre eles a polpa dentária humana, tecido originado na papila dentária do dente em desenvolvimento. Estas linhagens multipotentes podem ser estudadas sob vários aspectos, como na elucidação da histogênese de tumores. O objetivo deste estudo foi inferir a histogênese do mixoma odontogênico, neoplasia odontogênica benigna, analisando a expressão de proteínas da matriz extracelular (MEC) e de metaloproteinases da matriz (MMPs) em cél...

  6. Mucus Detachment by Host Metalloprotease Meprin β Requires Shedding of Its Inactive Pro-form, which Is Abrogated by the Pathogenic Protease RgpB

    Directory of Open Access Journals (Sweden)

    Rielana Wichert

    2017-11-01

    Full Text Available The host metalloprotease meprin β is required for mucin 2 (MUC2 cleavage, which drives intestinal mucus detachment and prevents bacterial overgrowth. To gain access to the cleavage site in MUC2, meprin β must be proteolytically shed from epithelial cells. Hence, regulation of meprin β shedding and activation is important for physiological and pathophysiological conditions. Here, we demonstrate that meprin β activation and shedding are mutually exclusive events. Employing ex vivo small intestinal organoid and cell culture experiments, we found that ADAM-mediated shedding is restricted to the inactive pro-form of meprin β and is completely inhibited upon its conversion to the active form at the cell surface. This strict regulation of meprin β activity can be overridden by pathogens, as demonstrated for the bacterial protease Arg-gingipain (RgpB. This secreted cysteine protease potently converts membrane-bound meprin β into its active form, impairing meprin β shedding and its function as a mucus-detaching protease.

  7. Enhancement of 9α-Hydroxy-4-androstene-3,17-dione Production from Soybean Phytosterols by Deficiency of a Regulated Intramembrane Proteolysis Metalloprotease in Mycobacterium neoaurum.

    Science.gov (United States)

    Xiong, Liang-Bin; Sun, Wan-Ju; Liu, Yong-Jun; Wang, Feng-Qing; Wei, Dong-Zhi

    2017-12-06

    Modification of the sterol catabolism pathway in mycobacteria may result in the accumulation of some valuable steroid pharmaceutical intermediates, such as 9α-hydroxy-4-androstene-3,17-dione (9-OHAD). In previous work, sigma factor D (SigD) was identified as a negative factor of the 9-OHAD production in Mycobacterium neoaurum. Here, the deficiency of rip1 putatively coding for a regulated intramembrane proteolysis metalloprotease (Rip1), which could cleave the negative regulator of SigD (anti-SigD), enhanced the transcription of some key genes (choM1, kshA, and hsd4A) in the sterol catabolic pathway. Furthermore, the deletion of rip1 increased the consumption of phytosterols by 37.8% after 96 h of growth in M. neoaurum. The production of 9-OHAD in the engineered M. neoaurumΔkstD1ΔkstD2ΔkstD3Δrip1 (MnΔk123Δrip1) strain was ultimately increased by 27.3% compared to that in its parental strain M. neoaurumΔkstD1ΔkstD2ΔkstD3 (MnΔk123). This study further confirms the important role of SigD-related factors in the catabolism of sterols.

  8. A disintegrin and metalloprotease-17 and galectin-9 are important regulators of local 4-1BB activity and disease outcome in rheumatoid arthritis

    DEFF Research Database (Denmark)

    Nielsen, Morten Aagaard; Andersen, Thomas; Etzerodt, Anders

    2016-01-01

    OBJECTIVE: Co-stimulatory T cell cytokines are important in the progression of RA. This study investigates the interplay between 4-1BB, a disintegrin and metalloprotease-17 (ADAM17) and galectin-9 (Gal-9) in RA. METHODS: Stimulated mononuclear cells from patients with chronic RA (n = 12) were co......-incubated with tissue inhibitor of metalloproteinase, 4-1BB ligand and Gal-9. Plasma samples were examined for soluble 4-1BB (s4-1BB) in newly diagnosed, treatment-naïve patients with RA (n = 97). The 28-joint DAS with CRP (28DAS-CRP), total Sharp score, erosion score and joint space narrowing were used to evaluate...... treatment outcome serially over a 2-year period. RESULTS: RA CD4(+) and CD8(+) synovial T cells express high levels of 4-1BB. The addition of TNF-α to cultured synovial mononuclear cells increased shedding of 4-1BB. 4-1BB ligand only increased TNF-α shedding in combination with Gal-9. RNA interference...

  9. A phage display selected 7-mer peptide inhibitor of the Tannerella forsythia metalloprotease-like enzyme Karilysin can be truncated to Ser-Trp-Phe-Pro.

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    Peter Durand Skottrup

    Full Text Available Tannerella forsythia is a gram-negative bacteria, which is strongly associated with the development of periodontal disease. Karilysin is a newly identified metalloprotease-like enzyme, that is secreted from T. forsythia. Karilysin modulates the host immune response and is therefore considered a likely drug target. In this study peptides were selected towards the catalytic domain from Karilysin (Kly18 by phage display. The peptides were linear with low micromolar binding affinities. The two best binders (peptide14 and peptide15, shared the consensus sequence XWFPXXXGGG. A peptide15 fusion with Maltose Binding protein (MBP was produced with peptide15 fused to the N-terminus of MBP. The peptide15-MBP was expressed in E. coli and the purified fusion-protein was used to verify Kly18 specific binding. Chemically synthesised peptide15 (SWFPLRSGGG could inhibit the enzymatic activity of both Kly18 and intact Karilysin (Kly48. Furthermore, peptide15 could slow down the autoprocessing of intact Kly48 to Kly18. The WFP motif was important for inhibition and a truncation study further demonstrated that the N-terminal serine was also essential for Kly18 inhibition. The SWFP peptide had a Ki value in the low micromolar range, which was similar to the intact peptide15. In conclusion SWFP is the first reported inhibitor of Karilysin and can be used as a valuable tool in structure-function studies of Karilysin.

  10. The hookworm tissue inhibitor of metalloproteases (Ac-TMP-1 modifies dendritic cell function and induces generation of CD4 and CD8 suppressor T cells.

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    Carmen Cuéllar

    Full Text Available Hookworm infection is a major cause of disease burden for humans. Recent studies have described hookworm-related immunosuppression in endemic populations and animal models. A Tissue Inhibitor of Metalloproteases (Ac-TMP-1 has been identified as one of the most abundant proteins released by the adult parasite. We investigated the effect of recombinant Ac-TMP-1 on dendritic cell (DC and T cell function. Splenic T cells from C57BL/6 mice injected with Ac-TMP-1 showed reduced proliferation to restimulation with anti CD3 or bystander antigens such as OVA. Incubation of bone marrow-derived DCs with Ac-TMP-1 decreased MHC Class I and, especially, Class II expression but increased CD86 and IL-10 expression. Co-incubation of splenic T cells with DCs pulsed with Ac-TMP-1 induced their differentiation into CD4+ and, particularly, CD8+ CD25+Foxp3+ T cells that expressed IL-10. These cells were able to suppress proliferation of naïve and activated CD4+ T cells by TGF-Beta-dependent (CD4+ suppressors or independent (CD8+ suppressors mechanisms. Priming of DCs with non-hookworm antigens, such as OVA, did not result in the generation of suppressor T cells. These data indicate that Ac-TMP-1 initiates the development of a regulatory response through modifications in DC function and generation of suppressor T cells. This is the first report to propose a role of suppressor CD8+ T cells in gastrointestinal helminthic infections.

  11. Specific fluorogenic substrates for neprilysin (neutral endopeptidase, EC 3.4.24.11 which are highly resistant to serine- and metalloproteases

    Directory of Open Access Journals (Sweden)

    M.A.S. Medeiros

    1997-10-01

    Full Text Available Two intramolecularly quenched fluorogenic peptides containing o-aminobenzoyl (Abz and ethylenediamine 2,4-dinitrophenyl (EDDnp groups at amino- and carboxyl-terminal amino acid residues, Abz-DArg-Arg-Leu-EDDnp (Abz-DRRL-EDDnp and Abz-DArg-Arg-Phe-EDDnp (Abz-DRRF-EDDnp, were selectively hydrolyzed by neutral endopeptidase (NEP, enkephalinase, neprilysin, EC 3.4.24.11 at the Arg-Leu and Arg-Phe bonds, respectively. The kinetic parameters for the NEP-catalyzed hydrolysis of Abz-DRRL-EDDnp and Abz-DRRF-EDDnp were Km = 2.8 µM, kcat = 5.3 min-1, kcat/Km = 2 min-1 µM-1 and Km = 5.0 µM, kcat = 7.0 min-1, kcat/Km = 1.4 min-1 µM-1, respectively. The high specificity of these substrates was demonstrated by their resistance to hydrolysis by metalloproteases [thermolysin (EC 3.4.24.2, angiotensin-converting enzyme (ACE; EC 3.4.24.15], serineproteases [trypsin (EC 3.4.21.4, a-chymotrypsin (EC 3.4.21.1] and proteases present in tissue homogenates from kidney, lung, brain and testis. The blocked amino- and carboxyl-terminal amino acids protected these substrates against the action of aminopeptidases, carboxypeptidases and ACE. Furthermore, DR amino acids ensured total protection of Abz-DRRL-EDDnp and Abz-DRRF-EDDnp against the action of thermolysin and trypsin. Leu-EDDnp and Phe-EDDnp were resistant to hydrolysis by a-chymotrypsin. The high specifity of these substrates suggests their use for specific NEP assays in crude enzyme preparations

  12. Processing of the major autolysin of E. faecalis, AtlA, by the zinc-metalloprotease, GelE, impacts AtlA septal localization and cell separation.

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    Emily K Stinemetz

    Full Text Available AtlA is the major peptidoglycan hydrolase of Enterococcus faecalis involved in cell division and cellular autolysis. The secreted zinc metalloprotease, gelatinase (GelE, has been identified as an important regulator of cellular function through post-translational modification of protein substrates. AtlA is a known target of GelE, and their interplay has been proposed to regulate AtlA function. To study the protease-mediated post-translational modification of AtlA, monoclonal antibodies were developed as research tools. Flow cytometry and Western blot analysis suggests that in the presence of GelE, surface-bound AtlA exists primarily as a N-terminally truncated form whereas in the absence of GelE, the N-terminal domain of AtlA is retained. We identified the primary GelE cleavage site occurring near the transition between the T/E rich Domain I and catalytic region, Domain II via N-terminal sequencing. Truncation of AtlA had no effect on the peptidoglycan hydrolysis activity of AtlA. However, we observed that N-terminal cleavage was required for efficient AtlA-mediated cell division while unprocessed AtlA was unable to resolve dividing cells into individual units. Furthermore, we observed that the processed AtlA has the propensity to localize to the cell septum on wild-type cells whereas unprocessed AtlA in the ΔgelE strain were dispersed over the cell surface. Combined, these results suggest that AtlA septum localization and subsequent cell separation can be modulated by a single GelE-mediated N-terminal cleavage event, providing new insights into the post-translation modification of AtlA and the mechanisms governing chaining and cell separation.

  13. Isoform-specific interactions between meprin metalloproteases and the catalytic subunit of protein kinase A: significance in acute and chronic kidney injury

    Science.gov (United States)

    Niyitegeka, Jean-Marie V.; Bastidas, Adam C.; Newman, Robert H.; Taylor, Susan S.

    2014-01-01

    Meprin metalloproteases are abundantly expressed in the brush-border membranes of kidney proximal tubules. Meprins are implicated in ischemia-reperfusion (IR)-induced renal injury and diabetic nephropathy. The protein kinase A (PKA) signaling pathway modulates extracellular matrix metabolism in diabetic kidneys. The present study evaluated isoform-specific interactions between the catalytic subunit of PKA (PKA C) and meprins. To this end, cytosolic-enriched kidney proteins from meprin αβ double knockout mice, and purified forms of recombinant mouse PKA Cα, Cβ1, and Cβ2, were incubated with activated forms of either homomeric meprin A or meprin B. The cleaved protein products were subjected to SDS-PAGE and analyzed by Coomassie staining and Western blot analysis. While meprin A only cleaved PKA Cβ1, meprin B cleaved all three PKA C isoforms. Analysis of the proteolytic fragments by mass spectrometry revealed that meprin A and B cleave the PKA C isoforms at defined sites, resulting in unique cleavage products. Michaelis-Menten enzyme kinetics demonstrated that meprin B-mediated cleavage of PKA Cα occurs at a rate consistent with that of other physiologically relevant meprin substrates. Meprin cleavage decreased the kinase activity of PKA Cα, Cβ1, and Cβ2. PKA C levels were higher in diabetic kidneys, with evidence of in vivo fragmentation in wild-type diabetic kidneys. Confocal microscopy showed localization of meprin A in the glomeruli of diabetic kidneys. At 3 h post-IR, PKA C levels in proximal tubules decreased compared with distal tubules, which lack meprins. These data suggest that meprins may impact kidney injury, in part, via modulation of PKA signaling pathways. PMID:25354939

  14. Mycobacterium tuberculosis Zinc Metalloprotease-1 Elicits Tuberculosis-specific Humoral Immune Response Independent of Mycobacterial Load in Pulmonary and Extra-Pulmonary Tuberculosis Patients

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    Mani Harika eVemula

    2016-03-01

    Full Text Available Conventionally, facultative intracellular pathogen, Mycobacterium tuberculosis (M.tb, the tuberculosis (TB causing bacilli in human is cleared by cell-mediated immunity (CMI with CD4+ T cells playing instrumental role in protective immunity, while antibody-mediated immunity (AMI is considered non-protective. This longstanding convention has been challenged with recent evidences of increased susceptibility of hosts with compromised AMI and monoclonal antibodies conferring passive protection against TB and other intracellular pathogens. Therefore, novel approaches towards vaccine development include strategies aiming at induction of humoral response along with CMI. This necessitates the identification of mycobacterial proteins with properties of immunomodulation and strong immunogenicity. In this study, we determined the immunogenic potential of M.tb Zinc metalloprotease-1 (Zmp1, a secretory protein essential for intracellular survival and pathogenesis of M.tb. We observed that Zmp1 was secreted by in vitro grown M.tb under granuloma-like stress conditions (acidic, oxidative, iron deficiency and nutrient deprivation and generated Th2 cytokine microenvironment upon exogenous treatment of Peripheral Blood Mononulear Cells (PBMCs with recombinant Zmp1 (rZmp1. This was supported by recording specific and robust humoral response in TB patients in a cohort of 295. The anti-Zmp1 titers were significantly higher in TB patients (n=121 as against healthy control (n=62, household contacts (n=89 and non-specific infection controls (n=23. A significant observation of the study is the presence of equally high titers of anti-Zmp1 antibodies in a range of patients with high bacilli load (sputum bacilli load of 300+ per mL to paucibacillary smear-negative pulmonary tuberculosis (PTB cases. This clearly indicated the potential of Zmp1 to evoke an effective humoral response independent of mycobacterial load. Such mycobacterial proteins can be explored as antigen

  15. ICAM-3, radiation resistance gene, activates PI3K/Akt-CREB-MMPs pathway and promotes migration/invasion of the human non-small cell lung cancer cell NCI-H1299

    International Nuclear Information System (INIS)

    Park, Jong Kuk; Park, Seon Ho; Hong, Sung Hee; Um, Hong Duck; Yoo, Young Do

    2008-01-01

    Cancer cell is characterized by various distinctive functions difference from normal cell. The one of specific properties of cancer is invasion and metastasis. Invasion and metastasis is a multi-step process involving over-expression of proteolytic enzymes such as matrix metalloproteinases (MMPs) and critically dependent on the ability of cells to move away from the primary tumor to gain access to the vascular or lymphatic systems which disperses cells to distant sites, where they can grow in a permissive microenvironment at a secondary location. All of these processes are critically dependent upon the ability of cancer cells to breach the basement membrane and to migrate through neighboring tissues. Cancer cell invasion is an important, tightly regulated process that is related with development, immune response and wound healing. This invasive response is dependent on activation of signaling pathways that result in both short-term and long-term cellular responses. The gene expressions of the cancer cell invasion related-proteolytic enzymes are regulated at the transcriptional level (through AP-1 and NF-kB via mitogen activated protein kinases (MAPKs) and PI3K-Akt pathways) and post-transcriptional levels, and the protein level via their activators or inhibitors, and their cell surface localization. Therefore, the related proteins such as MMPs, MAPK, PI3K, Akt and their regulatory pathway have been considered as promising targets for anti-cancer drugs. In previous reports, Intercellular adherin molecule-3 (ICAM-3) showed increase of radio-resistance and proliferation. We have made ICAM-3 overexpressed cancer cells which shows elevated level of invasion compared with normal cancer cells and its invasion capacity was down regulated with treatment of specific inhibitor for PI3K. These results suggest that ICAM-3 related invasion is associated with PI3K signaling pathway

  16. A novel assay for extracellular matrix remodeling associated with liver fibrosis

    DEFF Research Database (Denmark)

    Barascuk, N; Veidal, S S; Larsen, L

    2010-01-01

    Accumulation of extracellular matrix (ECM) components and increased matrix-metalloprotease (MMPs) activity are hallmarks of fibrosis. We developed an ELISA for quantification of MMP-9 derived collagen type III (CO3) degradation....

  17. IL-1β-induced matrix metalloproteinase-13 is activated by a disintegrin and metalloprotease-28-regulated proliferation of human osteoblast-like cells

    Energy Technology Data Exchange (ETDEWEB)

    Ozeki, Nobuaki; Kawai, Rie; Yamaguchi, Hideyuki; Hiyama, Taiki; Kinoshita, Katsue; Hase, Naoko; Nakata, Kazuhiko [Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya, Aichi 464-8651 (Japan); Kondo, Ayami [Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto, Chikusa-ku, Nagoya, Aichi 464-8650 (Japan); Mogi, Makio, E-mail: makio@dpc.agu.ac.jp [Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto, Chikusa-ku, Nagoya, Aichi 464-8650 (Japan); Nakamura, Hiroshi [Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya, Aichi 464-8651 (Japan)

    2014-04-15

    We reported previously that matrix metalloproteinase (MMP)-13 accelerates bone remodeling in oral periradicular lesions, and indicated a potentially unique role for MMP-13 in wound healing and regeneration of alveolar bone. The ADAM (a disintegrin and metalloprotease) family is a set of multifunctional cell surface and secreted glycoproteins, of which ADAM-28 has been localized in bone and bone-like tissues. In this study, we show that interleukin (IL)-1β induces the expression of MMP-13 and ADAM-28 in homogeneous α7 integrin-positive human skeletal muscle stem cell (α7{sup +}hSMSC)-derived osteoblast-like (α7{sup +}hSMSC-OB) cells, and promotes proliferation while inhibiting apoptosis in these cells. At higher concentrations, however, IL-1β failed to induce the expression of these genes and caused an increase in apoptosis. We further employed ADAM-28 small interfering RNA (siRNA) to investigate whether IL-1β-induced MMP-13 expression is linked to this IL-1β-mediated changes in cell proliferation and apoptosis. Silencing ADAM-28 expression potently suppressed IL-1β-induced MMP-13 expression and activity, decreased cell proliferation and increased apoptosis in α7{sup +}hSMSC-OB cells. In contrast, MMP-13 siRNA had no effect on ADAM-28 expression, suggesting ADAM-28 regulates MMP-13. Exogenous MMP-13 induced α7{sup +}hSMSC-OB cell proliferation and could rescue ADAM-28 siRNA-induced apoptosis, and we found that proMMP-13 is partially cleaved into its active form by ADAM-28 in vitro. Overall, our results suggest that IL-1β-induced MMP-13 expression and changes in cell proliferation and apoptosis in α7{sup +}hSMSC-OB cells are regulated by ADAM-28. - Highlights: • IL-1β induces the MMP-13 and ADAM-28 expression in human osteoblast-like cells. • IL-1β-induced MMP-13 expression increases proliferation and decreased apoptosis. • MMP-13 expression induced by IL-1β is regulated by ADAM-28. • proMMP-13 appears to be cleaved into its active form via

  18. IL-1β-induced matrix metalloproteinase-13 is activated by a disintegrin and metalloprotease-28-regulated proliferation of human osteoblast-like cells

    International Nuclear Information System (INIS)

    Ozeki, Nobuaki; Kawai, Rie; Yamaguchi, Hideyuki; Hiyama, Taiki; Kinoshita, Katsue; Hase, Naoko; Nakata, Kazuhiko; Kondo, Ayami; Mogi, Makio; Nakamura, Hiroshi

    2014-01-01

    We reported previously that matrix metalloproteinase (MMP)-13 accelerates bone remodeling in oral periradicular lesions, and indicated a potentially unique role for MMP-13 in wound healing and regeneration of alveolar bone. The ADAM (a disintegrin and metalloprotease) family is a set of multifunctional cell surface and secreted glycoproteins, of which ADAM-28 has been localized in bone and bone-like tissues. In this study, we show that interleukin (IL)-1β induces the expression of MMP-13 and ADAM-28 in homogeneous α7 integrin-positive human skeletal muscle stem cell (α7 + hSMSC)-derived osteoblast-like (α7 + hSMSC-OB) cells, and promotes proliferation while inhibiting apoptosis in these cells. At higher concentrations, however, IL-1β failed to induce the expression of these genes and caused an increase in apoptosis. We further employed ADAM-28 small interfering RNA (siRNA) to investigate whether IL-1β-induced MMP-13 expression is linked to this IL-1β-mediated changes in cell proliferation and apoptosis. Silencing ADAM-28 expression potently suppressed IL-1β-induced MMP-13 expression and activity, decreased cell proliferation and increased apoptosis in α7 + hSMSC-OB cells. In contrast, MMP-13 siRNA had no effect on ADAM-28 expression, suggesting ADAM-28 regulates MMP-13. Exogenous MMP-13 induced α7 + hSMSC-OB cell proliferation and could rescue ADAM-28 siRNA-induced apoptosis, and we found that proMMP-13 is partially cleaved into its active form by ADAM-28 in vitro. Overall, our results suggest that IL-1β-induced MMP-13 expression and changes in cell proliferation and apoptosis in α7 + hSMSC-OB cells are regulated by ADAM-28. - Highlights: • IL-1β induces the MMP-13 and ADAM-28 expression in human osteoblast-like cells. • IL-1β-induced MMP-13 expression increases proliferation and decreased apoptosis. • MMP-13 expression induced by IL-1β is regulated by ADAM-28. • proMMP-13 appears to be cleaved into its active form via ADAM-28

  19. Metalloprotease NleC suppresses host NF-κB/inflammatory responses by cleaving p65 and interfering with the p65/RPS3 interaction.

    Directory of Open Access Journals (Sweden)

    Andrea Hodgson

    2015-03-01

    Full Text Available Attaching/Effacing (A/E pathogens including enteropathogenic Escherichia coli (EPEC, enterohemorrhagic E. coli (EHEC and the rodent equivalent Citrobacter rodentium are important causative agents of foodborne diseases. Upon infection, a myriad of virulence proteins (effectors encoded by A/E pathogens are injected through their conserved type III secretion systems (T3SS into host cells where they interfere with cell signaling cascades, in particular the nuclear factor kappaB (NF-κB signaling pathway that orchestrates both innate and adaptive immune responses for host defense. Among the T3SS-secreted non-LEE-encoded (Nle effectors, NleC, a metalloprotease, has been recently elucidated to modulate host NF-κB signaling by cleaving NF-κB Rel subunits. However, it remains elusive how NleC recognizes NF-κB Rel subunits and how the NleC-mediated cleavage impacts on host immune responses in infected cells and animals. In this study, we show that NleC specifically targets p65/RelA through an interaction with a unique N-terminal sequence in p65. NleC cleaves p65 in intestinal epithelial cells, albeit a small percentage of the molecule, to generate the p65¹⁻³⁸ fragment during C. rodentium infection in cultured cells. Moreover, the NleC-mediated p65 cleavage substantially affects the expression of a subset of NF-κB target genes encoding proinflammatory cytokines/chemokines, immune cell infiltration in the colon, and tissue injury in C. rodentium-infected mice. Mechanistically, the NleC cleavage-generated p65¹⁻³⁸ fragment interferes with the interaction between p65 and ribosomal protein S3 (RPS3, a 'specifier' subunit of NF-κB that confers a subset of proinflammatory gene transcription, which amplifies the effect of cleaving only a small percentage of p65 to modulate NF-κB-mediated gene expression. Thus, our results reveal a novel mechanism for A/E pathogens to specifically block NF-κB signaling and inflammatory responses by cleaving a

  20. Occurrence and evolution of the paralogous zinc metalloproteases IgA1 protease, ZmpB, ZmpC, and ZmpD in Streptococcus pneumoniae and related commensal species.

    Science.gov (United States)

    Bek-Thomsen, Malene; Poulsen, Knud; Kilian, Mogens

    2012-01-01

    The distribution, genome location, and evolution of the four paralogous zinc metalloproteases, IgA1 protease, ZmpB, ZmpC, and ZmpD, in Streptococcus pneumoniae and related commensal species were studied by in silico analysis of whole genomes and by activity screening of 154 representatives of 20 species. ZmpB was ubiquitous in the Mitis and Salivarius groups of the genus Streptococcus and in the genera Gemella and Granulicatella, with the exception of a fragmented gene in Streptococcus thermophilus, the only species with a nonhuman habitat. IgA1 protease activity was observed in all members of S. pneumoniae, S. pseudopneumoniae, S. oralis, S. sanguinis, and Gemella haemolysans, was variably present in S. mitis and S. infantis, and absent in S. gordonii, S. parasanguinis, S. cristatus, S. oligofermentans, S. australis, S. peroris, and S. suis. Phylogenetic analysis of 297 zmp sequences and representative housekeeping genes provided evidence for an unprecedented selection for genetic diversification of the iga, zmpB, and zmpD genes in S. pneumoniae and evidence of very frequent intraspecies transfer of entire genes and combination of genes. Presumably due to their adaptation to a commensal lifestyle, largely unaffected by adaptive mucosal immune factors, the corresponding genes in commensal streptococci have remained conserved. The widespread distribution and significant sequence diversity indicate an ancient origin of the zinc metalloproteases predating the emergence of the humanoid species. zmpB, which appears to be the ancestral gene, subsequently duplicated and successfully diversified into distinct functions, is likely to serve an important but yet unknown housekeeping function associated with the human host. The paralogous zinc metalloproteases IgA1 protease, ZmpB, ZmpC, and ZmpD have been identified as crucial for virulence of the human pathogen Streptococcus pneumoniae. This study maps the presence of the corresponding genes and enzyme activities in S

  1. The proteasomal Rpn11 metalloprotease suppresses tombusvirus RNA recombination and promotes viral replication via facilitating assembly of the viral replicase complex.

    Science.gov (United States)

    Prasanth, K Reddisiva; Barajas, Daniel; Nagy, Peter D

    2015-03-01

    RNA viruses co-opt a large number of cellular proteins that affect virus replication and, in some cases, viral genetic recombination. RNA recombination helps viruses in an evolutionary arms race with the host's antiviral responses and adaptation of viruses to new hosts. Tombusviruses and a yeast model host are used to identify cellular factors affecting RNA virus replication and RNA recombination. In this study, we have examined the role of the conserved Rpn11p metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates, in tombusvirus replication and recombination in Saccharomyces cerevisiae and plants. Depletion or mutations of Rpn11p lead to the rapid formation of viral RNA recombinants in combination with reduced levels of viral RNA replication in yeast or in vitro based on cell extracts. Rpn11p interacts with the viral replication proteins and is recruited to the viral replicase complex (VRC). Analysis of the multifunctional Rpn11p has revealed that the primary role of Rpn11p is to act as a "matchmaker" that brings the viral p92(pol) replication protein and the DDX3-like Ded1p/RH20 DEAD box helicases into VRCs. Overexpression of Ded1p can complement the defect observed in rpn11 mutant yeast by reducing TBSV recombination. This suggests that Rpn11p can suppress tombusvirus recombination via facilitating the recruitment of the cellular Ded1p helicase, which is a strong suppressor of viral recombination, into VRCs. Overall, this work demonstrates that the co-opted Rpn11p, which is involved in the assembly of the functional proteasome, also functions in the proper assembly of the tombusvirus VRCs. RNA viruses evolve rapidly due to genetic changes based on mutations and RNA recombination. Viral genetic recombination helps viruses in an evolutionary arms race with the host's antiviral responses and facilitates adaptation of viruses to new hosts. Cellular factors affect viral RNA recombination, although the role

  2. Occurrence and Evolution of the Paralogous Zinc Metalloproteases IgA1 Protease, ZmpB, ZmpC, and ZmpD in Streptococcus pneumoniae and Related Commensal Species

    Science.gov (United States)

    Bek-Thomsen, Malene; Poulsen, Knud; Kilian, Mogens

    2012-01-01

    ABSTRACT The distribution, genome location, and evolution of the four paralogous zinc metalloproteases, IgA1 protease, ZmpB, ZmpC, and ZmpD, in Streptococcus pneumoniae and related commensal species were studied by in silico analysis of whole genomes and by activity screening of 154 representatives of 20 species. ZmpB was ubiquitous in the Mitis and Salivarius groups of the genus Streptococcus and in the genera Gemella and Granulicatella, with the exception of a fragmented gene in Streptococcus thermophilus, the only species with a nonhuman habitat. IgA1 protease activity was observed in all members of S. pneumoniae, S. pseudopneumoniae, S. oralis, S. sanguinis, and Gemella haemolysans, was variably present in S. mitis and S. infantis, and absent in S. gordonii, S. parasanguinis, S. cristatus, S. oligofermentans, S. australis, S. peroris, and S. suis. Phylogenetic analysis of 297 zmp sequences and representative housekeeping genes provided evidence for an unprecedented selection for genetic diversification of the iga, zmpB, and zmpD genes in S. pneumoniae and evidence of very frequent intraspecies transfer of entire genes and combination of genes. Presumably due to their adaptation to a commensal lifestyle, largely unaffected by adaptive mucosal immune factors, the corresponding genes in commensal streptococci have remained conserved. The widespread distribution and significant sequence diversity indicate an ancient origin of the zinc metalloproteases predating the emergence of the humanoid species. zmpB, which appears to be the ancestral gene, subsequently duplicated and successfully diversified into distinct functions, is likely to serve an important but yet unknown housekeeping function associated with the human host. PMID:23033471

  3. Análisis de MMPs en fluidos orales en el diagnóstico complementario de las enfermedades periodontales Oral-fluid MMP analysis in the complementary diagnosis of periodontal diseases

    Directory of Open Access Journals (Sweden)

    P Hernández

    2012-12-01

    Full Text Available La periodontitis constituye la infección bacteriana más prevalente a nivel mundial y representa un factor de riesgo para diversas patologías sistémicas. El estado de inflamación y destrucción periodontal se manifiestan a través de la presencia de biomarcadores en el suero y fluidos orales, tales como el fluido gingival crevicular (FGC, saliva y enjuague oral. Enzimas como las metaloproteinasas de matriz (MMP y mieloperoxidasa, constituyen biomarcadores potenciales para ensayos moleculares complementarios a la clínica de uso en el sillón dental. A continuación se presenta una revisión de la literatura respecto de la aplicación potencial del análisis de metaloproteinasas de matriz extracelular (MMPs en el diagnóstico complementario de las enfermedades periodontales. Se ha demostrado que los niveles de MMP-9, -13 y particularmente de MMP-8, se asocian con el grado de inflamación periodontal, y pueden diferenciar entre sujetos sanos, con gingivitis, periodontitis y peri-implantitis, mientras que la mejoría de los parámetros clínicos en respuesta al tratamiento periodontal se asocia con la reducción de la activación y niveles de estas enzimas en FGC, como así también en el suero. Se concluye que la determinación, particularmente de MMP-8 en fluidos orales presenta un elevado potencial como complemento de los métodos clínicos tradicionales para identificar a los pacientes con periodontitis o en riesgo de desarrollar la enfermedad, monitorear fases del tratamiento y mejoría de signos periodontales e incluso evaluar el estado de inflamación sistémica.Periodontal disease is the most common bacterial infection worldwide and it can contribute to enhance the risk for the development of several systemic diseases. The status of periodontal inflammation and destruction can be reflected in biomarker measurement in serum and oral fluids, like gingival crevicular fluid (GCF, saliva and mouth-rinse. Some enzymes, such as matrix

  4. Complex regulation of AprA metalloprotease in Pseudomonas fluorescens M114: evidence for the involvement of iron, the ECF sigma factor, PbrA and pseudobactin M114 siderophore.

    Science.gov (United States)

    Maunsell, Bláithín; Adams, Claire; O'Gara, Fergal

    2006-01-01

    In the soil bacterium Pseudomonas fluorescens M114, extracellular proteolytic activity and fluorescent siderophore (pseudobactin M114) production were previously shown to be co-ordinately negatively regulated in response to environmental iron levels. An iron-starvation extracytoplasmic function sigma factor, PbrA, required for the transcription of siderophore biosynthetic genes, was also implicated in M114 protease regulation. The current study centred on the characterization and genetic regulation of the gene(s) responsible for protease production in M114. A serralysin-type metalloprotease gene, aprA, was identified and found to encode the major, if not only, extracellular protease produced by this strain. The expression of aprA and its protein product were found to be subject to complex regulation. Transcription analysis confirmed that PbrA was required for full aprA transcription under low iron conditions, while the ferric uptake regulator, Fur, was implicated in aprA repression under high iron conditions. Interestingly, the iron regulation of AprA was dependent on culture conditions, with PbrA-independent AprA-mediated proteolytic activity observed on skim milk agar supplemented with yeast extract, when supplied with iron or purified pseudobactin M114. These effects were not observed on skim milk agar without yeast extract. PbrA-independent aprA expression was also observed from a truncated transcriptional fusion when grown in sucrose asparagine tryptone broth supplied with iron or purified pseudobactin M114. Thus, experimental evidence suggested that iron mediated its effects via transcriptional activation by PbrA under low iron conditions, while an as-yet-unidentified sigma factor(s) may be required for the PbrA-independent aprA expression and AprA proteolytic activity induced by siderophore and iron.

  5. Expression of matrix metalloproteases-2 and -9 in horse hoof laminae after intestinal obstruction, with or without Hydrocortisone treatment Expressão de metaloproteinases 2 e 9 no tecido laminar do casco de equinos após obstrução intestinal e tratamento com hidrocortisona

    Directory of Open Access Journals (Sweden)

    Luciane Maria Laskoski

    2013-01-01

    Full Text Available Twenty horses were used in the experiment, for composed control group, (Cg instrumented group, (Ig;without intestinal obstruction, treated group (Tg;submitted to intestinal obstruction and hydrocortisone treatment and non-treated group (Ntg;submitted to intestinal obstruction without treatment. Immunohistochemistry and zymography techniques were used for researches on MMPs 2 and 9 in horse hoof laminae. There was an increase in the expression of MMP-2 in animals of Tg and Ntg. MMP-9 increased on animals from groups Ntg and Ig, however there was no rise of this MMP on the Tg when compared to the other groups in the immunohistochemistry analysis. Based on the results, it was observed that the intestinal injury caused by enterotomy and intestinal obstruction raise the quantities of MMPs in the hoof laminae.Vinte cavalos foram usados no experimento: para compor o grupo controle (Cg, grupo instrumentado, Ig (sem obstrução intestinal, grupo tratado, Tg (submetidos à obstrução intestinal e tratamento com hidrocortisona e grupo não tratado, Ntg (submetidos à obstrução intestinal, sem tratamento. Técnicas de zimografia e imunoistoquímica foram utilizadas para pesquisa de MMP-2 e MMP-9 no tecido laminar do casco dos equinos. Houve um aumento na expressão de MMP-2 nos animais dos grupos Tg e Ntg. A MMP-9 aumentou nos animais dos grupos Ig e Ntg. Houve aumento desta MMP no Tg quando comparado aos demais grupos na análise por zimografia. Observou-se que a injúria intestinal, causada pela enterotomia e obstrução intestinal, eleva a quantidade de MMPs no tecido laminar do casco.

  6. Partial purification and characterization of metalloprotease of ...

    African Journals Online (AJOL)

    USER

    2013-07-31

    Jul 31, 2013 ... The supplementation of partially purified enzyme preparation in detergents such as Rin and Wheel significantly improved their cleansing efficiency as blood and fish curry stains on the cloth disappeared within 15 min (Figure 6). Our finding go hand in hand with earlier findings on Bacillus licheniformis ...

  7. Therapeutic applications of collagenase (metalloproteases: A review

    Directory of Open Access Journals (Sweden)

    Hamzeh Alipour

    2016-11-01

    Full Text Available Non-invasive therapeutic methods have recently been used in medical sciences. Enzymes have shown high activity at very low concentrations in laboratories and pharmaceutical, enabling them to play crucial roles in different biological phenomena related to living organism, especially human medicine. Recently, using the therapeutic methods based on non-invasive approaches has been emphasized in medical society. Researchers have focused on producing medicines and tools reducing invasive procedures in medical. Collagenases are proteins which catalyze chemical processes and break the peptide bonds in collagen. Collagen may be generated more than the required amount or produced in unsuitable sites or may not degrade after a certain time. In such cases, using an injectable collagenase or its ointment can be helpful in collagen degradation. In both in vitro and in vivo tests, it has been revealed that collagenases have several therapeutic properties in wound healing, burns, nipple pain and some diseases including intervertebral disc herniation, keloid, cellulite, lipoma among others. This review describes the therapeutic application of collagenase in medical sciences and the process for its production using novel methods, paving the way for more effective and safe applications of collagenases.

  8. How MMPs Impact Bone Responses to Metastatic Prostate Cancer

    Science.gov (United States)

    2011-04-30

    Matrisian LM. Matrix metalloproteinase-7-dependent release of tumor necrosis factor-alpha in a model of herniated disc resorption. J Clin Investig 2000;105...or disease. For example, in nonpathologic conditions such as herniated disc resorption, macrophage-derived MMP-7 is critical for the resorption of the... herniated disc and in mammary and prostate involution; MMP-7 processing of FasL is important for initiating apoptosis (22, 23, 35). More often

  9. Zymography and reverse zymography for detecting MMPs and TIMPs.

    Science.gov (United States)

    Hawkes, Susan P; Li, Hongxia; Taniguchi, Gary T

    2010-01-01

    Zymography is the electrophoretic separation of proteins through a polyacrylamide gel containing a proteolytic substrate. After denaturing (but nonreducing) electrophoresis, proteins are renatured and incubated in an appropriate buffer for proteolytic activity. Clear zones of lysis in the stained gel indicated active proteinases. Reverse zymography is a similar technique to detect proteinase inhibitors. After renaturing of proteins, the gel is incubated with metalloproteinases which digest the substrate incorporated into the gel. Inhibitors are shown as dark zones of inhibition against a clear background upon staining.

  10. Understanding the Mechanisms Through Which Matrix Metalloproteinases (MMPs) Contribute to Breast Cancer-Associated Osteolytic Lesions

    Science.gov (United States)

    2009-03-01

    not shown). Similar levels of osteoprotegerin (OPG), a soluble decoy receptor of RANKL, were found in the wild-type and MMP-7 null animals and were...Tan, H.  L., Kelley, M.  J., Dunstan, C. R., Burgess, T., Elliott, R., Colombero, A.,  Elliott,  G.,  Scully,  S.,  et  al.  (1998).  Osteoprotegerin

  11. Fluorogenic MMP activity assay for plasma including MMPs complexed to α2-macroglobulin

    NARCIS (Netherlands)

    Beekman, B.; Drijfhout, J.W.; Ronday, H.K.; TeKoppele, J.M.

    1999-01-01

    Elevated MMP activities are implicated in tissue degradation in, e.g., arthritis and cancer. The present study was designed to measure MMP enzyme activity in plasma. Free active MMP is unlikely to be present in plasma: upon entering the circulation, active MMP is expected to be captured by the

  12. Active MMPs captured by alpha2Macroglobulin as a marker of disease activity in rheumatoid arthritis

    NARCIS (Netherlands)

    Tchetverikov, I.; Verzijl, N.; Huizinga, T.W.J.; TeKoppele, J.M.; Hanemaaijer, R.; Groot, J. de

    2003-01-01

    Objective. The aim of the present study was to analyze α2Macroglobulin/MMP (α2M/MMP) complex formation and to investigate whether MMP activity in α2M/MMP complexes in serum can be used as a disease marker in rheumatoid arthritis (RA). Methods. High and low molecular weight (H/LMW) substrates and

  13. Infrared optical imaging of matrix metalloproteinases (MMPs up regulation following ischemia reperfusion is ameliorated by hypothermia

    Directory of Open Access Journals (Sweden)

    Barber Philip A

    2012-06-01

    Full Text Available Abstract Background We investigated the use of a new MMP activatable probe MMPSense™ 750 FAST (MMPSense750 for in-vivo visualization of early MMP activity in ischemic stroke. Following middle cerebral artery occlusion (MCAO optical imaging was performed. Near-infrared (NIR fluorescent images of MMPSense activation were acquired using an Olympus fluorescent microscope, 1.25x objective, a CCD camera and an appropriate filter cube for detecting the activated probe with peak excitation and emission at 749 and 775 nm, respectively. Images were acquired starting at 2 or 24 hours after reperfusion over the ipsilateral and contralateral cortex before and for 3 hours after, MMPSense750 was injected. Results Increased intensities ipsilaterally were observed following MMPSense750 injection with ischemic injury but not in sham animals. There were significant ipsilateral and contralateral differences at 15 minutes (P Conclusions Matrix-metalloproteinase upregulation in ischemia reperfusion can be imaged acutely in-vivo with NIRF using MMPSense750. Hypothermia attenuated both the optical increase in intensity after MMPSense750 and the increase in MMP-9 protein expression supporting the proof of concept that NIRF imaging using MMPSense can be used to assess potential therapeutic strategies for stroke treatment.

  14. In vivo imaging of matrix metalloprotease 12 and matrix metalloprotease 13 activities in the mouse model of collagen-induced arthritis

    DEFF Research Database (Denmark)

    Lim, Ngee Han; Meinjohanns, Ernst; Bou-Gharios, George

    2014-01-01

    inhibitor GM6001 and specific synthetic inhibitors of MMP-12 and MMP-13. The probes were used to follow these enzyme activities in the collagen-induced arthritis (CIA) model in vivo. Results. The MMP-12- and MMP-13-activity probes (MMP12ap and MMP13ap, respectively) discriminated between the two enzymatic...... activities. The in vivo activation of these probes was inhibited by GM6001 and by their respective specific inhibitors. In the CIA model, MMP12ap activation peaked 5 days after disease onset and showed strong correlation with disease severity during this time (r = 0.85; p...

  15. Reappraisal of quantitative gel zymography for matrix metalloproteinases.

    Science.gov (United States)

    Prescimone, Tommaso; Tognotti, Danika; Caselli, Chiara; Cabiati, Manuela; D'Amico, Andrea; Del Ry, Silvia; Giannessi, Daniela

    2014-09-01

    The determination of matrix metalloproteases (MMPs) is relevant in many pathophysiological conditions, especially if associated with extracellular matrix remodeling; however, the results obtained are closely linked to the method used and are not directly comparable. The aim of this study was to perform a reappraisal of quantitative gel zymography technique for MMPs in human plasma, to use for comparison with commercially available ELISA and in those experimental conditions where the MMP active form needs to be revealed. The critical methodological parameters of zymography were checked and a comparison with a routinely used ELISA was performed. Sensitivity and reproducibility levels of zymography are suitable for detection of MMP-9 in human plasma, providing results closely related to those obtained by ELISA. Analytical parameters of zymography were suitable for detection of MMPs in human plasma. Quantitative zymography for MMPs is an alternative method for comparing the results of ELISA widely employed for MMP determination, thus reducing the discrepancies between laboratories regarding gelatinase assay. © 2014 Wiley Periodicals, Inc.

  16. Prediction of metalloproteinase family based on the concept of Chou's pseudo amino acid composition using a machine learning approach.

    Science.gov (United States)

    Mohammad Beigi, Majid; Behjati, Mohaddeseh; Mohabatkar, Hassan

    2011-12-01

    Matrix metalloproteinase (MMPs) and disintegrin and metalloprotease (ADAMs) belong to the zinc-dependent metalloproteinase family of proteins. These proteins participate in various physiological and pathological states. Thus, prediction of these proteins using amino acid sequence would be helpful. We have developed a method to predict these proteins based on the features derived from Chou's pseudo amino acid composition (PseAAC) server and support vector machine (SVM) as a powerful machine learning approach. With this method, for ADAMs and MMPs families, an overall accuracy and Matthew's correlation coefficient (MCC) of 95.89 and 0.90% were achieved respectively. Furthermore, the method is able to predict two major subclasses of MMP family; Furin-activated secreted MMPs and Type II trans-membrane; with MCC of 0.89 and 0.91%, respectively. The overall accuracy for Furin-activated secreted MMPs and Type II trans-membrane was 98.18 and 99.07, respectively. Our data demonstrates an effective classification of Metalloproteinase family based on the concept of PseAAC and SVM.

  17. The xenoestrogens biphenol-A and nonylphenol differentially regulate metalloprotease-mediated shedding of EGFR ligands.

    Science.gov (United States)

    Urriola-Muñoz, Paulina; Li, Xue; Maretzky, Thorsten; McIlwain, David R; Mak, Tak W; Reyes, Juan G; Blobel, Carl P; Moreno, Ricardo D

    2018-03-01

    The xenoestrogens bisphenol-A (BPA) and nonylphenol (NP) are endocrine disruptors used in the plastic polymer industry to manufacture different products for human use. Previous studies have suggested a role of these compounds in the shedding of signaling molecules, such as tumor necrosis factor α (TNF-α). The aim of this work was to evaluate the effect of BPA and NP on the sheddase ADAM17 and its newly discovered regulators iRhom1 and iRhom2 in the release of EGFR-ligands. We report that BPA and NP can stimulate the release of the ADAM17-substrates HB-EGF and TGF-α. In cells lacking ADAM17 (Adam17 -/- mEFs) BPA-stimulated release of HB-EGF, but not TGF-α, was strongly reduced, whereas NP-stimulated shedding of HB-EGF and TGF-α was completely abolished. Inactivation of both ADAM17 and the related ADAM10 (Adam10/17 -/- mEFs) completely prevented the release of these substrates. In the absence of iRhom1, BPA- or NP-stimulated release of HB-EGF or TGF-α was comparable to wild-type control mEFs, conversely the BPA-induced release of HB-EGF was abolished in iRhom2 -/- mEFs. The defect in shedding of HB-EGF in iRhom2 -/- mEF cells could be rescued by overexpressing iRhom2. Interestingly, the NP-stimulated release of HB-EGF was not affected by the absence of iRhom2, suggesting that NP could potentially activate both ADAM10 and ADAM17. We tested this hypothesis using betacellulin (BTC), an EGFR-ligand that is a substrate for ADAM10. We found that NP, but not BPA stimulated the release of BTC in Adam17 -/- , iRhom2 -/- , or iRhom1/2 -/- , but not in Adam10/17 -/- cells. Taken together, our results suggest that BPA and NP stimulate the release of EGFR-ligands by differentially activating ADAM17 or ADAM10. The identification of specific effects of these endocrine disruptors on ADAM10 and ADAM17 will help to provide a better understanding of their roles in cell signaling and proinflammatory processes, and provide new potential targets for treatment of reproductive or inflammatory diseases such as asthma or breast cancer that are promoted by xenoestrogens. © 2017 Wiley Periodicals, Inc.

  18. Statistical Optimization of Media Components for Production of Fibrinolytic Alkaline Metalloproteases from Xenorhabdus indica KB-3

    Directory of Open Access Journals (Sweden)

    Kumar Pranaw

    2014-01-01

    Full Text Available Xenorhabdus indica KB-3, a well-known protease producer, was isolated from its entomopathogenic nematode symbiont Steinernema thermophilum. Since medium constituents are critical to the protease production, the chemical components of the selected medium (soya casein digest broth were optimized by rotatable central composite design (RCCD using response surface methodology (RSM. The effects of all five chemical components (considered as independent variables, namely tryptone, soya peptone, dextrose, NaCl, and dipotassium phosphate, on protease production (dependent variable were studied, and it was found that tryptone and dextrose had maximum influence on protease production. The protease production was increased significantly by 66.31% under optimal medium conditions (tryptone—5.71, soya peptone—4.9, dextrose—1.45, NaCl—6.08, and dipotassium phosphate—0.47 in g/L. To best of knowledge, there are no reports on optimization of medium component for protease production by X. indica KB-3 using RSM and their application in fibrinolysis. This study will be useful for industrial processes for production of protease enzyme from X. indica KB-3 for its application in the field of agriculture and medicine.

  19. Spastic paraplegia and OXPHOS impairment caused by mutations in paraplegin, a nuclear-encoded mitochondrial metalloprotease.

    Science.gov (United States)

    Casari, G; De Fusco, M; Ciarmatori, S; Zeviani, M; Mora, M; Fernandez, P; De Michele, G; Filla, A; Cocozza, S; Marconi, R; Dürr, A; Fontaine, B; Ballabio, A

    1998-06-12

    Hereditary spastic paraplegia (HSP) is characterized by progressive weakness and spasticity of the lower limbs due to degeneration of corticospinal axons. We found that patients from a chromosome 16q24.3-linked HSP family are homozygous for a 9.5 kb deletion involving a gene encoding a novel protein, named Paraplegin. Two additional Paraplegin mutations, both resulting in a frameshift, were found in a complicated and in a pure form of HSP. Paraplegin is highly homologous to the yeast mitochondrial ATPases, AFG3, RCA1, and YME1, which have both proteolytic and chaperon-like activities at the inner mitochondrial membrane. Immunofluorescence analysis and import experiments showed that Paraplegin localizes to mitochondria. Analysis of muscle biopsies from two patients carrying Paraplegin mutations showed typical signs of mitochondrial OXPHOS defects, thus suggesting a mechanism for neurodegeneration in HSP-type disorders.

  20. Structural analysis of silanediols as transition-state-analogue inhibitors of the benchmark metalloprotease thermolysin.

    Science.gov (United States)

    Juers, Douglas H; Kim, Jaeseung; Matthews, Brian W; Sieburth, Scott McN

    2005-12-20

    Dialkylsilanediols have been found to be an effective functional group for the design of active-site-directed protease inhibitors, including aspartic (HIV protease) and metallo (ACE and thermolysin) proteases. The use of silanediols is predicated on its resemblance to the hydrated carbonyl transition-state structure of amide hydrolysis. This concept has been tested by replacing the presumed tetrahedral carbon of a thermolysin substrate with a silanediol group, resulting in an inhibitor with an inhibition constant K(i) = 40 nM. The structure of the silanediol bound to the active site of thermolysin was found to have a conformation very similar to that of a corresponding phosphonamidate inhibitor (K(i) = 10 nM). In both cases, a single oxygen is within bonding distance to the active-site zinc ion, mimicking the presumed tetrahedral transition state. There are binding differences that appear to be related to the presence or absence of protons on the oxygens attached to the silicon or phosphorus. This is the first crystal structure of an organosilane bound to the active site of a protease.

  1. A disintegrin and metalloprotease 10 (ADAM10) is a central regulator of murine liver tissue homeostasis

    Czech Academy of Sciences Publication Activity Database

    Muller, M.; Wetzel, S.; Koehn-Gaone, J.; Chalupský, Karel; Luellmann-Rauch, R.; Barikbin, R.; Bergmann, J.; Woehner, B.; Žbodáková, Olga; Leuschner, I.; Gregor, Martin; Tiegs, G.; Rose-John, S.; Sedláček, Radislav; Tirnitz-Parker, J.E.E.; Saftig, P.; Schmidt-Arras, D.

    2016-01-01

    Roč. 7, č. 14 (2016), s. 17431-17441 ISSN 1949-2553 R&D Projects: GA ČR GAP302/11/2048; GA ČR GAP303/10/2044; GA ČR GA13-01710S; GA ČR GA15-23858S; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:68378050 Keywords : ADAM10 * liver progenitor cell * c-Met * Notch * hepatocyte differentiation * Pathology Section Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.168, year: 2016

  2. SPINK9 Stimulates Metalloprotease/EGFR-Dependent Keratinocyte Migration via Purinergic Receptor Activation

    Czech Academy of Sciences Publication Activity Database

    Sperrhacke, M.; Fischer, J.; Wu, Z.H.; Klunder, S.; Sedláček, Radislav; Schroeder, J.M.; Meyer-Hoffert, U.; Reiss, K.

    2014-01-01

    Roč. 134, č. 6 (2014), s. 1645-1654 ISSN 0022-202X R&D Projects: GA ČR GAP303/10/2044 Institutional support: RVO:68378050 Keywords : SPINK * ADAM * keratinocyte Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 7.216, year: 2014

  3. Effect of a disintegrin and metalloprotease 33 (ADAM33 gene polymorphisms and smoking in COPD

    Directory of Open Access Journals (Sweden)

    Ayman H. Abd El-Zaher

    2012-10-01

    Conclusion: In conclusion, we found that polymorphisms in the SNPs (Q1 and S1 of ADAM33 gene are associated with COPD in the general population. In addition, smoker patients with GG genotype in (S1 and Q1 ADAM33 will have more pronounced decline in the pulmonary function test (FEV1.

  4. Identification and characterization of the Vibrio anguillarum prtV gene encoding a new metalloprotease

    Science.gov (United States)

    Mo, Zhaolan; Guo, Dongsheng; Mao, Yunxiang; Ye, Xuhong; Zou, Yuxia; Xiao, Peng; Hao, Bin

    2010-01-01

    We cloned and sequenced a prtV-like gene from Vibrio anguillarum M3 strain. This prtV gene encodes a putative protein of 918 amino acids, and is highly homologous to the V. cholerae prtV gene. We found that a prtV insertion mutant strain displayed lower gelatinase activity on gelatin agar, lower protease activity against azocasein, and lower activity for four glycosidases. This prtV mutant strain also had increased activity for two esterases in its extracellular products, as analyzed by the API ZYM system. In addition, the prtV mutant strain exhibited decreased growth in turbot intestinal mucus and reduced hemolytic activity on turbot erythrocytes. Infection experiments showed that the LD50 of the prtV mutant strain increased by at least 1 log compared to the wild-type in turbot fish. We propose that prtV plays an important role in the pathogenesis of V. anguillarum.

  5. Vibrio Zinc-Metalloprotease Causes Photoinactivation of Coral Endosymbionts and Coral Tissue Lesions

    NARCIS (Netherlands)

    Sussman, Meir; Mieog, Jos C.; Doyle, Jason; Victor, Steven; Willis, Bette L.; Bourne, David G.

    2009-01-01

    Background: Coral diseases are emerging as a serious threat to coral reefs worldwide. Of nine coral infectious diseases, whose pathogens have been characterized, six are caused by agents from the family Vibrionacae, raising questions as to their origin and role in coral disease aetiology.

  6. Osteoactivin regulates head and neck squamous cell carcinoma invasion by modulating matrix metalloproteases.

    Science.gov (United States)

    Arosarena, Oneida A; Barr, Eric W; Thorpe, Ryan; Yankey, Hilary; Tarr, Joseph T; Safadi, Fayez F

    2018-01-01

    Nearly 60% of patients with head and neck squamous cell carcinoma (HNSCC) die of metastases or locoregional recurrence. Metastasis is mediated by cancer cell migration and invasion, which are in part dependent on extracellular matrix degradation by matrix metalloproteinases. Osteoactivin (OA) overexpression plays a role in metastases in several malignancies, and has been shown to upregulate matrix metalloproteinase (MMP) expression and activity. To determine how OA modulates MMP expression and activity in HNSCC, and to investigate OA effects on cell invasion, we assessed effects of OA treatment on MMP mRNA and protein expression, as well as gelatinase and caseinolytic activity in HNSCC cell lines. We assessed the effects of OA gene silencing on MMP expression, gelatinase and caseinolytic activity, and cell invasion. OA treatment had differential effects on MMP mRNA expression. OA treatment upregulated MMP-10 expression in UMSCC14a (p = 0.0431) and SCC15 (p < 0.0001) cells, but decreased MMP-9 expression in UMSCC14a cells (p = 0.0002). OA gene silencing decreased MMP-10 expression in UMSCC12 cells (p = 0.0001), and MMP-3 (p = 0.0005) and -9 (p = 0.0036) expression in SCC25 cells. In SCC15 and SCC25 cells, OA treatment increased MMP-2 (p = 0.0408) and MMP-9 gelatinase activity (p < 0.0001), respectively. OA depletion decreased MMP-2 (p = 0.0023) and -9 (p < 0.0001) activity in SCC25 cells. OA treatment increased 70 kDa caseinolytic activity in UMSCC12 cells consistent with tissue type plasminogen activator (p = 0.0078). OA depletion decreased invasive capacity of UMSCC12 cells (p < 0.0001). OA's effects on MMP expression in HNSCC are variable, and may promote cancer cell invasion. © 2017 Wiley Periodicals, Inc.

  7. Relative contribution of matrix metalloprotease and cysteine protease activities to cytokine-stimulated articular cartilage degradation

    DEFF Research Database (Denmark)

    Sondergaard, B C; Henriksen, K; Wulf, H

    2006-01-01

    (GAG), hydroxyproline, and cross-linked C-telopeptide fragments of type II collagen (CTX-II), which were compared to immunohistochemical evaluations of proteoglycans and CTX-II. We assessed MMP expression by gelatine zymography and CK expression by immunohistochemistry. In vivo, CTX-II release...... was measured from CK-deficient mice. RESULTS: OSM and TNF-alpha combined induced significant (Pzymography, and CK expression...

  8. Characteristics of the biologically active 35-kDa metalloprotease virulence factor from Listeria monocytogenes

    NARCIS (Netherlands)

    Coffey, A; van den Burg, B; Veltman, R; Abee, T

    Listeria monocytogenes, a facultative intracellular pathogen, synthesizes an extracellular protease which is responsible for the maturation of phosphatidylcholine phospholipase C (lecithinase), a virulence factor involved in cell-to-cell spread. This work describes the environmental parameters

  9. Análisis de MMPs en fluidos orales en el diagnóstico complementario de las enfermedades periodontales

    OpenAIRE

    Hernández,P; Mäntylä,P; Tervahartiala,T; Sorsa,T; Hernández,M

    2012-01-01

    La periodontitis constituye la infección bacteriana más prevalente a nivel mundial y representa un factor de riesgo para diversas patologías sistémicas. El estado de inflamación y destrucción periodontal se manifiestan a través de la presencia de biomarcadores en el suero y fluidos orales, tales como el fluido gingival crevicular (FGC), saliva y enjuague oral. Enzimas como las metaloproteinasas de matriz (MMP) y mieloperoxidasa, constituyen biomarcadores potenciales para ensayos moleculares c...

  10. RKIP Inhibits Local Breast Cancer Invasion by Antagonizing the Transcriptional Activation of MMP13.

    Directory of Open Access Journals (Sweden)

    Ila Datar

    Full Text Available Raf Kinase Inhibitory Protein or RKIP was initially identified as a Raf-1 binding protein using the yeast 2-hybrid screen. RKIP inhibits the activation phosphorylation of MEK by Raf-1 by competitively inhibiting the binding of MEK to Raf-1 and thus exerting an inhibitory effect on the Raf-MEK-Erk pathway. RKIP has been identified as a metastasis suppressor gene. Expression of RKIP is low in cancer metastases. Although primary tumor growth remains unaffected, re- expression of RKIP inhibits cancer metastasis. Mechanistically, RKIP constrains metastasis by inhibiting angiogenesis, local invasion, intravasation, and colonization. The molecular mechanism of how RKIP inhibits these individual steps remains undefined. In our present study, using an unbiased PCR based screening and by analyzing DNA microarray expression datasets we observe that the expression of multiple metalloproteases (MMPs including MMP1, MMP3, MMP10 and MMP13 are negatively correlated with RKIP expression in breast cancer cell lines and clinical samples. Since expression of MMPs by cancer cells is important for cancer metastasis, we hypothesize that RKIP may mediate suppression of breast cancer metastasis by inhibiting multiple MMPs. We show that the expression signature of RKIP and MMPs is better at predicting high metastatic risk than the individual gene. Using a combination of loss- and gain-of-function approaches, we find that MMP13 is the cause of RKIP-mediated inhibition of local cancer invasion. Interestingly expression of MMP13 alone is not sufficient to reverse the inhibition of breast cancer cell metastasis to the lung due to the expression of RKIP. We find that RKIP negatively regulates MMP13 through the Erk2 signaling pathway and the repression of MMP13 by RKIP is transcription factor AP-1 independent. Together, our findings indicate that RKIP inhibits cancer cell invasion, in part, via MMP13 inhibition. These data also implicate RKIP in the regulation of MMP

  11. Increasing robustness and sensitivity of protein microarrays through microagitation and automation.

    Science.gov (United States)

    Hartmann, M; Toegl, A; Kirchner, R; Templin, M F; Joos, T O

    2006-03-30

    Assay systems that employ protein microarrays for the analysis of complex samples are powerful tools to generate a high amount of data from a limiting amount of sample. Due to miniaturization, these systems are susceptible to fluctuations during signal generation and the use of uniform conditions for sample incubation and during the assay procedure is required to get reproducible results. Diffusion limits may prevent constant conditions on all parts of the array and can lead to the decease of the sensitivity of the array. Therefore, we set-up an automated assay system integrating a novel microagitation device using surface acoustic wave (SAW) technology. Multiplexed assays for the detection of autoantibodies from human serum and sandwich immunoassay for the detection of matrix metalloproteases (MMPs) were used to evaluate the system. Diffusion-rate limited solid phase reactions were enhanced by microagitation using the SAW technology resulting in up to three-fold higher signals.

  12. Matrix metalloproteinases during and outside of migraine attacks without aura

    DEFF Research Database (Denmark)

    Ashina, M.; Tvedskov, J.F.; Thiesen, Kerstin Lipka

    2010-01-01

    Ashina M, Tvedskov JF, Lipka K, Bilello J, Penkowa M & Olesen J. Matrix metalloproteinases during and outside of migraine attacks without aura. Cephalalgia 2009. London. ISSN 0333-1024To test the hypothesis that permeability of the blood-brain barrier (BBB) is altered during migraine attack due...... to enhanced activation of matrix metalloproteinases (MMPs), we investigated MMP-3, MMP-9 and tissue inhibitor of metalloproteases (TIMP)-1 in the external jugular vein during and outside of migraine attacks in 21 patients with migraine without aura. In addition, we measured plasma levels of several other...... of MMP-3 in the external jugular (P = 0.002) and cubital (P = 0.008) vein during attacks compared with outside of attacks. We found no correlation of ictal or interictal MMP-3, MMP-9 and TIMP-1 to migraine duration and frequency analysed in 21 patients (P > 0.05). There was no difference between ictal...

  13. Analysis of the Specificity and Biochemical Characterization of Metalloproteases Isolated from Eupenicillium javanicum Using Fluorescence Resonance Energy Transfer Peptides

    Science.gov (United States)

    Hamin Neto, Youssef A. A.; de Oliveira, Lilian C. G.; de Oliveira, Juliana R.; Juliano, Maria A.; Juliano, Luiz; Arantes, Eliane C.; Cabral, Hamilton

    2017-01-01

    Enzymes have important features that may facilitate their application in industrial processes and have been used as alternatives to chemical catalysts. In particular, proteases can be isolated from microorganisms, which provide important sources of advantageous enzymes for industrial processes. For example, Eupenicillium javanicum is a filamentous fungus that has been shown to express industrially applicable enzymes and chemical components, such as antifungal compounds. The biotechnological potential of E. javanicum and proteases made us search a novel protease from this microorganism. The macromolecule was isolated, the main biochemical properties was evaluated, and the specificity of the protease subsites was determined. The protease was produced under solid-state bioprocess with wheat bran and isolated by two chromatography steps with yield of 27.5% and 12.4-fold purification. The molecular mass was estimated at 30 kDa. The N-terminal sequence of the first 20 amino acid residues was AVGAGYNASVALALEKALNN. The enzyme presented higher proteolytic activity at pH 6.0 and 60°C. The protease is stable at wide range of pH values and temperatures and in the presence of surfactants. The “primed” side of the catalytic site showed the highest catalytic efficiency of the enzyme isolated from E. javanicum. The S′1 subsite is responsible for catalyzing the protease reaction with substrates with tyrosine in P′1. These findings provide important insights into the biochemical characterization of a highly active protease from E. javanicum and may facilitate the development of industrial processes involving this protease. PMID:28119672

  14. Purification and Properties of an Insecticidal Metalloprotease Produced by Photorhabdus luminescens Strain 0805-P5G, the Entomopathogenic Nematode Symbiont

    Directory of Open Access Journals (Sweden)

    Feng-Chia Hsieh

    2012-12-01

    Full Text Available A total of 13 Photorhabdus luminescens strains were screened for proteolytic activity. The P. luminescens strain 0805-P5G had the highest activity on both skim milk and gelatin plates. The protease was purified to electrophoretical homogeneity by using a two-step column chromatographic procedure. It had a molecular weight of 51.8 kDa, as determined by MALDI-TOF mass spectrometry. The optimum pH, temperature, as well as pH and thermal stabilities were 8, 60 °C, 5–10, and 14–60 °C, respectively. It was completely inhibited by EDTA and 1,10-phenanthroline. Bioassay of the purified protease against Galleria mellonella by injection showed high insecticidal activity. The protease also showed high oral toxicity to the diamondback moth (Plutella xylostella of a Taiwan field-collected strain, but low toxicity to an American strain. To our knowledge, this is the first report to demonstrate that the purified protease of P. luminescens has direct toxicity to P. xylostella and biopesticide potentiality.

  15. Inhibition of neutrophil elastase and metalloprotease-9 of human adenocarcinoma gastric cells by chamomile (Matricaria recutita L.) infusion.

    Science.gov (United States)

    Bulgari, Michela; Sangiovanni, Enrico; Colombo, Elisa; Maschi, Omar; Caruso, Donatella; Bosisio, Enrica; Dell'Agli, Mario

    2012-12-01

    This study investigated whether the antiinflammatory effect of chamomile infusion at gastric level could be ascribed to the inhibition of metalloproteinase-9 and elastase. The infusions from capitula and sifted flowers (250-1500 µg/mL) and individual flavonoids (10 µM) were tested on phorbol 12-myristate 13-acetate-stimulated AGS cells and human neutrophil elastase. The results indicate that the antiinflammatory activity associated with chamomile infusions from both the capitula and sifted flowers is most likely due to the inhibition of neutrophil elastase and gastric metalloproteinase-9 activity and secretion; the inhibition occurring in a concentration dependent manner. The promoter activity was inhibited as well and the decrease of metalloproteinase-9 expression was found to be associated with the inhibition of NF-kB driven transcription. The results further indicate that the flavonoid-7-glycosides, major constituents of chamomile flowers, may be responsible for the antiinflammatory action of the chamomile infusion observed here. Copyright © 2012 John Wiley & Sons, Ltd.

  16. Bacillus licheniformis BlaR1 L3 Loop Is a Zinc Metalloprotease Activated by Self-Proteolysis

    Science.gov (United States)

    Sépulchre, Jérémy; Amoroso, Ana; Joris, Bernard

    2012-01-01

    In Bacillus licheniformis 749/I, BlaP β-lactamase is induced by the presence of a β-lactam antibiotic outside the cell. The first step in the induction mechanism is the detection of the antibiotic by the membrane-bound penicillin receptor BlaR1 that is composed of two functional domains: a carboxy-terminal domain exposed outside the cell, which acts as a penicillin sensor, and an amino-terminal domain anchored to the cytoplasmic membrane, which works as a transducer-transmitter. The acylation of BlaR1 sensor domain by the antibiotic generates an intramolecular signal that leads to the activation of the L3 cytoplasmic loop of the transmitter by a single-point cleavage. The exact mechanism of L3 activation and the nature of the secondary cytoplasmic signal launched by the activated transmitter remain unknown. However, these two events seem to be linked to the presence of a HEXXH zinc binding motif of neutral zinc metallopeptidases. By different experimental approaches, we demonstrated that the L3 loop binds zinc ion, belongs to Gluzincin metallopeptidase superfamily and is activated by self-proteolysis. PMID:22623956

  17. Characterization of an extensin-modifying metalloprotease: N-terminal processing and substrate cleavage pattern of Pectobacterium carotovorum Prt1

    DEFF Research Database (Denmark)

    Feng, Tao; Nyffenegger, Christian; Højrup, Peter

    2014-01-01

    (EDTA) treatment abolished activity; Zn2+ addition caused regain of the activity, but Zn2+addition decreased the thermal stability of the Prt1 enzyme presumably as a result of increased proteolytic autolysis. In addition to casein, the enzyme catalyzed degradation of collagen, potato lectin, and plant...

  18. Atherosclerotic arterial remodeling and the localization of macrophages and matrix metalloproteases 1, 2 and 9 in the human coronary artery

    NARCIS (Netherlands)

    Pasterkamp, G.; Schoneveld, A. H.; Hijnen, D. J.; de Kleijn, D. P.; Teepen, H.; van der Wal, A. C.; Borst, C.

    2000-01-01

    Atherosclerotic luminal narrowing is determined by plaque mass and the mode of geometrical remodeling. Recently, we reported that the type of atherosclerotic remodeling is associated with the presence of histological markers for plaque vulnerability. Inflammation and matrix degrading proteases

  19. Production of Vibrio vulnificus metalloprotease VvpE begins during the early growth phase: usefulness of gelatin-zymography.

    Science.gov (United States)

    Kim, Choon-Mee; Kang, Sang-Mee; Jeon, Ho-Jong; Shin, Sung-Heui

    2007-07-01

    Recent studies have demonstrated that expression of the vvpE gene begins during the early growth phase albeit at low levels. However, we found that the traditional protease assay method that is used to measure caseinolytic activity in culture supernatants is not suitable for the measurement of extracellular VvpE that is produced at low levels during the early growth phase. By using gelatin-zymography in place of the protease assay, we could specifically detect only VvpE of several proteases produced by Vibrio vulnificus. Moreover, we could sensitively measure VvpE produced at low levels during the early growth phase, which was consistent with transcription of the vvpE gene. The extracellular production of VvpE was reduced or delayed by mutation of the pilD gene which encodes for the type IV leader peptidase/N-methyltransferase associated with the type II general secretion system; the delayed production of VvpE was recovered by in trans complementation of the wild-type pilD gene. These results indicate that VvpE begins to be produced during the early growth phase via the PilD-mediated type II general secretion system, and that the use of gelatin-zymography is recommended as a simple method for the sensitive and specific detection of VvpE production.

  20. Retroviral delivery of TIMP-2 inhibits H-ras-induced migration and invasion in MCF10A human breast epithelial cells.

    Science.gov (United States)

    Ahn, Seong-Min; Jeong, Seo-Jin; Kim, Yeon-Soo; Sohn, Yeowon; Moon, Aree

    2004-04-15

    The matrix metalloproteases (MMPs) play important roles in invasion, metastasis and angiogenesis in various cell types. Tissue inhibitor of metalloprotease (TIMP)-2, an endogenous inhibitor of MMP-2, has been shown to inhibit invasion and metastasis. We have previously shown that MMP-2 is responsible for the H-ras-induced invasive and migrative phenotypes in MCF10A human breast epithelial cells. Here, we investigated the effect of TIMP-2 overexpression on migration and invasion in H-ras MCF10A cells. Human TIMP-2 gene was effectively introduced into H-ras MCF10A cells by retrovirus-mediated gene delivery. TIMP-2 overexpression mediated by retrovirus significantly inhibited migration as well as invasion of H-ras MCF10A cells in a dose-dependent manner. We also show the antiangiogenic effect of TIMP-2 gene delivery. Taken together, our study shows that retrovirus-mediated delivery of TIMP-2 efficiently inhibits metastatic progression of ras-transformed human breast epithelial cells, suggesting a potential use of the TIMP-2 gene therapy for the treatment of breast cancer.

  1. Inhibition of metastasis of B16F-10 melanoma cells in C57BL/6 mice by an extract of Calendula officinalis L flowers.

    Science.gov (United States)

    Preethi, Korangath C; Siveen, Kodappully S; Kuttan, Ramadasan; Kuttan, Girija

    2010-01-01

    To determine the effect of a Calendula officinalis flower extract on lung metastasis by B16F-10 melanoma cells in C57BL/6 mice. Male mice were injected with B16F-10 melanoma cells through the tail vein and simultaneously treated with C.officinalis flower extract. Parameters studied were lung tumor nodule count, life span of animals, gamma glutamyl transpeptidase activity, sialic acid, TNF-α, IL-1β, IL-6, IL-2, GM-CSF, VEGF and TIMP-1 levels in serum, and lung hydroxyproline, uronic acid and hexosamine levels, as well as histopathological features. Effects of C.officinalis on the expression of various genes involved in metastasis like matrix metalloproteases (MMPs), tissue inhibitor of metalloproteases (TIMPs), prolyl hydoxylase, lysyl oxidase, nm23, and proinflammatory cytokines were also investigated. Simultaneous administration of C. officinalis extract to tumor bearing C57BL/6 mice reduced the lung tumor nodules by 74% with 43.3% increase in life span. Elevated levels of hydroxyproline, uronic acid, hexosamine, serum sialic acid and γ-glutamyl transpeptidase in the metastatic controls were found to be significantly lowered in the C. officinalis treated animals. The extract also inhibited expression of MMP-2, MMP-9, prolyl hydroxylase and lysyl oxidase and activated TIMP-1 and TIMP-2 and downregulated proinflammatory cytokines. The present investigation indicated antimetastatic effects of Calendula officinalis flowers through the inhibition of key enzymes involved in processes of metastasis.

  2. The Multifunctional Role of the Pallilysin-Associated Treponema pallidum Protein, Tp0750, in Promoting Fibrinolysis and Extracellular Matrix Component Degradation

    Science.gov (United States)

    Houston, Simon; Russell, Shannon; Hof, Rebecca; Roberts, Alanna K.; Cullen, Paul; Irvine, Kyle; Smith, Derek S.; Borchers, Christoph H.; Tonkin, Michelle L.; Boulanger, Martin J.; Cameron, Caroline E.

    2014-01-01

    Summary The mechanisms that facilitate dissemination of the highly invasive spirochete, Treponema pallidum, are incompletely understood. Previous studies showed the treponemal metalloprotease pallilysin (Tp0751) possesses fibrin clot degradation capability, suggesting a role in treponemal dissemination. In the current study we report characterization of the functionally-linked protein Tp0750. Structural modelling predicts Tp0750 contains a von Willebrand factor type A (vWFA) domain, a protein-protein interaction domain commonly observed in extracellular matrix (ECM)-binding proteins. We report Tp0750 is a serine protease that degrades the major clot components fibrinogen and fibronectin. We also demonstrate Tp0750 cleaves a matrix metalloprotease (MMP) peptide substrate that is targeted by several MMPs, enzymes central to ECM remodelling. Through proteomic analyses we show Tp0750 binds the endothelial fibrinolytic receptor, annexin A2, in a specific and dose-dependent manner. These results suggest Tp0750 constitutes a multifunctional protein that is able to (1) degrade infection-limiting clots by both inhibiting clot formation through degradation of host coagulation cascade proteins and promoting clot dissolution by complexing with host proteins involved in the fibrinolytic cascade and (2) facilitate ECM degradation via MMP-like proteolysis of host components. We propose that through these activities Tp0750 functions in concert with pallilysin to enable T. pallidum dissemination. PMID:24303899

  3. [Pt(O,O'-acac)(γ-acac)(DMS)] alters SH-SY5Y cell migration and invasion by the inhibition of Na+/H+ exchanger isoform 1 occurring through a PKC-ε/ERK/mTOR Pathway.

    Science.gov (United States)

    Muscella, Antonella; Vetrugno, Carla; Calabriso, Nadia; Cossa, Luca Giulio; De Pascali, Sandra Angelica; Fanizzi, Francesco Paolo; Marsigliante, Santo

    2014-01-01

    We previously showed that [Pt(O,O'-acac)(γ-acac)(DMS)] ([Pt(acac)2(DMS)]) exerted substantial cytotoxic effects in SH-SY5Y neuroblastoma cells, and decreased metalloproteases (MMPs) production and cells migration in MCF-7 breast cancer cells. The ubiquitously distributed sodium-hydrogen antiporter 1 (NHE1) is involved in motility and invasion of many solid tumours. The present study focuses on the effects of [Pt(acac)2(DMS)] in SH-SY5Y cell migration and also on the possibility that NHE1 may be involved in such effect. After sublethal [Pt(acac)2(DMS)] treatment cell migration was examined by wounding assay and cell invasion by transwell assay. NHE1 activity was measured in BCECF-loaded SH-SY5Y as the rate of Na+-dependent intracellular pH recovery in response to an acute acid pulse. Gelatin zymography for MMP-2/9 activities, Western blottings of MMPs, MAPKs, mTOR, S6 and PKCs and small interfering RNAs to PKC-ε/-δ mRNA were performed. Sublethal concentrations of [Pt(acac)2(DMS)] decreases NHE1 activity, inhibits cell migration and invasion and decreases expression and activity of MMP-2 and -9. [Pt(acac)2(DMS)] administered to SH-SY5Y cells provokes the increment of ROS, generated by NADPH oxidase, responsible for the PKC-ε and PKC-δ activation. Whilst PKC-δ activates p38/MAPK, responsible for the inhibition of MMP-2 and -9 secretion, PKC-ε activates a pathway made of ERK1/2, mTOR and S6K responsible for the inhibition of NHE1 activity and cell migration. In conclusion, we have shown a drastic impairment in tumour cell metastatization in response to inhibition of NHE1 and MMPs activities by [Pt(acac)2(DMS)] occurring through a novel mechanism mediated by PKC-δ/-ε activation.

  4. Long Non-coding RNA LINC00339 Stimulates Glioma Vasculogenic Mimicry Formation by Regulating the miR-539-5p/TWIST1/MMPs Axis

    Directory of Open Access Journals (Sweden)

    Junqing Guo

    2018-03-01

    Full Text Available Glioma is recognized as a highly angiogenic malignant brain tumor. Vasculogenic mimicry (VM greatly restricts the therapeutic effect of anti-angiogenic tumor therapy for glioma patients. However, the molecular mechanisms of VM formation in glioma remain unclear. Here, we demonstrated that LINC00339 was upregulated in glioma tissue as well as in glioma cell lines. The expression of LINC00339 in glioma tissues was positively correlated with glioma VM formation. Knockdown of LINC00339 inhibited glioma cell proliferation, migration, invasion, and tube formation, meanwhile downregulating the expression of VM-related molecular MMP-2 and MMP-14. Furthermore, knockdown of LINC00339 significantly increased the expression of miR-539-5p. Both bioinformatics and luciferase reporter assay revealed that LINC00339 regulated the above effects via binding to miR-539-5p. Besides, overexpression of miR-539-5p resulted in decreased expression of TWIST1, a transcription factor known to play an oncogenic role in glioma and identified as a direct target of miR-539-5p. TWIST1 upregulated the promoter activities of MMP-2 and MMP-14. The in vivo study showed that nude mice carrying tumors with knockdown of LINC00339 and overexpression of miR-539-5p exhibited the smallest tumor volume through inhibiting VM formation. In conclusion, LINC00339 may be used as a novel therapeutic target for VM formation in glioma.

  5. [6]-Gingerol Prevents Disassembly of Cell Junctions and Activities of MMPs in Invasive Human Pancreas Cancer Cells through ERK/NF-κB/Snail Signal Transduction Pathway

    Directory of Open Access Journals (Sweden)

    Sung Ok Kim

    2013-01-01

    Full Text Available To study the effects of [6]-gingerol, a ginger phytochemical, on tight junction (TJ molecules, we investigated TJ tightening and signal transduction pathways in human pancreatic duct cell-derived cancer cell line PANC-1. The following methods were utilized: MTT assay to determine cytotoxicity; zymography to examine matrix metalloproteinase (MMP activities; transepithelial electrical resistance (TER and paracellular flux for TJ measurement; RT-PCR and immunoblotting for proteins related to TJ and invasion; and EMSA for NF-κB activity in PANC-1 cells. Results revealed that TER significantly increased and claudin 4 and MMP-9 decreased compared to those of the control. TJ protein levels, including zonula occludens (ZO- 1, occludin, and E-cadherin, increased in [6]-gingerol-treated cells, which correlated with a decrease in paracellular flux and MMP activity. Furthermore, NF-κB/Snail nuclear translocation was suppressed via downregulation of the extracellular signal-regulated kinase (ERK pathway in response to [6]-gingerol treatment. Moreover, treatment with U0126, an ERK inhibitor, completely blocked NF-κB activity. In conclusion, these findings demonstrate that [6]-gingerol regulates TJ-related proteins and suppresses invasion and metastasis through NF-κB/Snail inhibition via inhibition of the ERK pathway. Therefore, [6]-gingerol may suppress the invasive activity of PANC-1 cells.

  6. In vitro cadmium effects on ECM gene expression in human bronchial epithelial cells.

    Science.gov (United States)

    Baroni, Tiziano; Lilli, Cinzia; Bellucci, Catia; Luca, Giovanni; Mancuso, Francesca; Fallarino, Francesca; Falabella, Giulia; Arato, Iva; Calvitti, Mario; Marinucci, Lorella; Muzi, Giacomo; Dell'Omo, Marco; Gambelunghe, Angela; Bodo, Maria

    2015-03-01

    Occupational and environmental exposure to the heavy metal cadmium (Cd) and its inhalation from cigarette smoke are associated with emphysema. Many growth factors and extracellular matrix (ECM) cell signaling molecules are directly involved in the epithelial bronchial cell pathway. This study investigated the direct effects of Cd on the production of several ECM components in human bronchial epithelial cells (BEAS-2B) that were exposed in vitro for 48 h to sub-toxic and toxic concentrations of Cd. Gene expression of collagens, metalloproteases (MMPs), integrins, tenascin and vitronectin were quantified by RT-PCR. To study apoptosis cascade, annexin assay and cellular cytotoxicity by MTT assay were performed. We also investigated whether an imbalance in the TGFβ/TGFβ receptor (TGFβR) expression mediated Cd effects. The results showed the sub-toxic Cd dose significantly increased tenascin, vitronectin, β1 and β5 integrin gene expression. The toxic Cd dose decreased type IV and V collagen, α1, α2 and β3 integrins. Both Cd doses down-regulated type I collagen and up-regulated metalloproteases. Each Cd dose caused a different imbalance in the complex pattern of TGFβ and its receptors. No alteration in classic apoptotic marker protein expression was observed in presence of the sub-toxic dose of Cd, suggesting this metal alters ECM production without apoptotic activation. In conclusion, all these data show even sub-toxic Cd dose exposure alters the specific gene expression of several ECM components that are crucially implicated in the mechanical properties of lung parenchyma supporting the hypothesis that the mechanism underlying Cd-induced lung disease may involve downstream changes in TGFβ/TGFβR signaling. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Circulating levels of matrix proteases and their inhibitors in pregnant women with and without a history of recurrent pregnancy loss.

    Science.gov (United States)

    Anumba, Dilly O C; El Gelany, Saad; Elliott, Sarah L; Li, Tin C

    2010-06-16

    We have recently shown that serum relaxin-2 levels are attenuated in women with a history of recurrent pregnancy loss (RPL). We sought to determine whether a history of RPL is also associated with changes in serum matrix metalloproteases (MMPs) and tissue inhibitors of matrix metalloproteases (TIMP) -1 and -2. We obtained serum from 20 pregnant women with a history of RPL and 20 age-matched pregnant women with no history of RPL (NRPL) at 6-8, 10-12, 20, and 34 weeks gestation, and from cord blood. We quantified total serum concentrations of MMP-1, MMP-3, MMP-9 and TIMP-1 and TIMP-2 by ELISA. We determined whether these serum marker levels were associated with a history of RPL and delivery before 37 weeks gestation. There was no difference in the rates of miscarriage, preterm birth or prelabour rupture of fetal membranes between RPL and NRPL. However babies born to RPL were lighter than those born to NRPL. Serum MMP-1, 9, and TIMP-1 did not differ between RPL and NRPL but MMP-3 was higher in RPL vs. NRPL at 6-8 weeks (P history of RPL than either peptide separately - area under the ROC curves for RLX-2 0.79 (95% CI 0.57 to 0.92), TIMP-2 0.83 (95% CI 0.63 to 0.95), and for RLX-2:TIMP-2 ratio 0.92 (95% CI 0.74 to 0.99). Women with a history of RPL demonstrate increased serum TIMP-2 and reduced RLX-2 during a subsequent viable pregnancy. Determination of both markers in early pregnancy enhances the discrimination of women with a history of RPL. These observations suggest roles for these two peptides in early implantation and placental development. Whether these may prove to be reliable early predictive markers for subsequent pregnancy loss in the index pregnancy is unknown and will require further studies.

  8. The role of polymorphisms in ADAM33, a disintegrin and metalloprotease 33, in childhood asthma and lung function in two German populations

    Directory of Open Access Journals (Sweden)

    Klopp Norman

    2006-06-01

    Full Text Available Abstract Background ADAM33, the first asthma candidate gene identified by positional cloning, may be associated with childhood asthma, lung function decline and bronchial hyperresponsiveness. However, replication results have been inconclusive in smaller previous study populations probably due to inconsistencies in asthma phenotypes or yet unknown environmental influences. Thus, we tried to further elucidate the role of ADAM33 polymorphisms (SNPs in a genetic analysis of German case control and longitudinal populations. Methods Using MALDI-TOF, ten ADAM33 SNPs were genotyped in 1,872 children from the International Study of Asthma and Allergy in Childhood (ISAAC II in a case control setting and further 824 children from the longitudinal cohort Multicentre Study of Allergy (MAS. In both populations the effects of single SNPs and haplotypes were studied and a gene environment analysis with passive smoke exposure was performed using SAS/Genetics. Results No single SNP showed a significant association with doctor's diagnosis of asthma. A trend for somewhat more profound effects of ADAM33 SNPs was observed in individuals with asthma and BHR. Haplotype analyses suggested a minor effect of the ADAM33 haplotype H4 on asthma (p = 0.033 but not on BHR. Associations with non atopic asthma and baseline lung function were identified but no interaction with passive smoke exposure could be detected. Conclusion The originally reported association between ADAM33 polymorphisms and asthma and BHR could not be confirmed. However, our data may suggest a complex role of ADAM33 polymorphisms in asthma ethiology, especially in non atopic asthma.

  9. Inhibition of Metalloprotease Botulinum Serotype A from a Pseudo-Peptide Binding Mode to a Small Molecule that is Active in Primary Neurons

    National Research Council Canada - National Science Library

    Burnett, James C; Ruthel, Gordon; Stegmann, Christian M; Panchal, Rekha G; Nguyen, Tam L; Hermone, Ann R; Stafford, Robert G; Lane, Douglas J; Kenny, Tara A; McGarth, Connor F

    2007-01-01

    An efficient research strategy integrating empirically-guided, structure-based modeling and chemoinformatics was used to discover potent small molecule inhibitors of the botulinum neurotoxin serotype A light chain...

  10. Analysis of the Specificity and Biochemical Characterization of Metalloproteases Isolated fromEupenicillium javanicumUsing Fluorescence Resonance Energy Transfer Peptides.

    Science.gov (United States)

    Hamin Neto, Youssef A A; de Oliveira, Lilian C G; de Oliveira, Juliana R; Juliano, Maria A; Juliano, Luiz; Arantes, Eliane C; Cabral, Hamilton

    2016-01-01

    Enzymes have important features that may facilitate their application in industrial processes and have been used as alternatives to chemical catalysts. In particular, proteases can be isolated from microorganisms, which provide important sources of advantageous enzymes for industrial processes. For example, Eupenicillium javanicum is a filamentous fungus that has been shown to express industrially applicable enzymes and chemical components, such as antifungal compounds. The biotechnological potential of E. javanicum and proteases made us search a novel protease from this microorganism. The macromolecule was isolated, the main biochemical properties was evaluated, and the specificity of the protease subsites was determined. The protease was produced under solid-state bioprocess with wheat bran and isolated by two chromatography steps with yield of 27.5% and 12.4-fold purification. The molecular mass was estimated at 30 kDa. The N-terminal sequence of the first 20 amino acid residues was AVGAGYNASVALALEKALNN. The enzyme presented higher proteolytic activity at pH 6.0 and 60°C. The protease is stable at wide range of pH values and temperatures and in the presence of surfactants. The "primed" side of the catalytic site showed the highest catalytic efficiency of the enzyme isolated from E. javanicum . The S' 1 subsite is responsible for catalyzing the protease reaction with substrates with tyrosine in P' 1 . These findings provide important insights into the biochemical characterization of a highly active protease from E. javanicum and may facilitate the development of industrial processes involving this protease.

  11. Identification, recombinant production and partial biochemical characterization of an extracellular cold-active serine-metalloprotease from an Antarctic Pseudomonas isolate

    Directory of Open Access Journals (Sweden)

    Natalia Fullana

    2017-08-01

    Full Text Available Cold-adapted enzymes are generally derived from psychrophilic microorganisms and have features that make them very attractive for industrial and biotechnological purposes. In this work, we identified a 50 kDa extracellular protease (MP10 from the Antarctic isolate Pseudomonas sp. AU10. The enzyme was produced by recombinant DNA technology, purified using immobilized metal affinity chromatography and partially characterized. MP10 is an alkaline thermosensitive serine-metallo protease with optimal activity at pH 8.0 and 40 ℃, in the presence of 1.5 mM Ca2+. MP10 showed 100% residual activity and stability (up to 60 min when incubated with 7% of non-ionic surfactants (Triton X-100, Tween-80 and Tween-20 and 1.5% of the oxidizing agent hydrogen peroxide. The 3D MP10 structure was predicted and compared with the crystal structure of mesophilic homologous protease produced by Pseudomonas aeruginosa PA01 (reference strain and other proteases, showing similarity in surface area and volume of proteins, but a significantly higher surface pocket area and volume of MP10. The observed differences presumably may explain the enhanced activity of MP10 for substrate binding at low temperatures. These results give insight to the potential use of MP10 in developing new biotechnologically processes active at low to moderate temperatures, probably with focus in the detergent industry.

  12. Metalloprotease-mediated cleavage of PlexinD1 and its sequestration to actin rods in the motoneuron disease spinal muscular atrophy (SMA)

    NARCIS (Netherlands)

    Rademacher, Sebastian; Verheijen, Bert M.; Hensel, Niko; Peters, Miriam; Bora, Gamze; Brandes, Gudrun; de Sá, Renata Vieira; Heidrich, Natascha; Fischer, Silke; Brinkmann, Hella; van der Pol, W. Ludo; Wirth, Brunhilde; Pasterkamp, R. Jeroen; Claus, Peter

    2017-01-01

    Cytoskeletal rearrangement during axon growth is mediated by guidance receptors and their ligands which act either as repellent, attractant or both. Regulation of the actin cytoskeleton is disturbed in Spinal Muscular Atrophy (SMA), a devastating neurodegenerative disease affecting mainly

  13. Matrix metalloproteinase-2 ablation in dystrophin-deficient mdx muscles reduces angiogenesis resulting in impaired growth of regenerated muscle fibers.

    Science.gov (United States)

    Miyazaki, Daigo; Nakamura, Akinori; Fukushima, Kazuhiro; Yoshida, Kunihiro; Takeda, Shin'ichi; Ikeda, Shu-ichi

    2011-05-01

    Matrix metalloproteases (MMPs) are a family of endopeptidases classified into subgroups based on substrate preference in normal physiological processes such as embryonic development and tissue remodeling, as well as in various disease processes via degradation of extracellular matrix components. Among the MMPs, MMP-9 and MMP-2 have been reported to be up-regulated in skeletal muscles in the lethal X-linked muscle disorder Duchenne muscular dystrophy (DMD), which is caused by loss of dystrophin. A recent study showed that deletion of the MMP9 gene in mdx, a mouse model for DMD, improved skeletal muscle pathology and function; however, the role of MMP-2 in the dystrophin-deficient muscle is not well known. In this study, we aimed at verifying the role of MMP-2 in the dystrophin-deficient muscle by using mdx mice with genetic ablation of MMP-2 (mdx/MMP-2(-/-)). We found impairment of regenerated muscle fiber growth with reduction of angiogenesis in mdx/MMP-2(-/-) mice at 3 months of age. Expression of vascular endothelial growth factor-A (VEGF-A), an important angiogenesis-related factor, decreased in mdx/MMP-2(-/-) mice at 3 months of age. MMP-2 had not a critical role in the degradation of dystrophin-glycoprotein complex (DGC) components such as β-dystroglycan and β-sarcoglycan in the regeneration process of the dystrophic muscle. Accordingly, MMP-2 may be essential for growth of regenerated muscle fibers through VEGF-associated angiogenesis in the dystrophin-deficient skeletal muscle.

  14. [Pt(O,O’-acac)(γ-acac)(DMS)] Alters SH-SY5Y Cell Migration and Invasion by the Inhibition of Na+/H+ Exchanger Isoform 1 Occurring through a PKC-ε/ERK/mTOR Pathway

    Science.gov (United States)

    Muscella, Antonella; Vetrugno, Carla; Calabriso, Nadia; Cossa, Luca Giulio; De Pascali, Sandra Angelica; Fanizzi, Francesco Paolo; Marsigliante, Santo

    2014-01-01

    We previously showed that [Pt(O,O’-acac)(γ-acac)(DMS)] ([Pt(acac)2(DMS)]) exerted substantial cytotoxic effects in SH-SY5Y neuroblastoma cells, and decreased metalloproteases (MMPs) production and cells migration in MCF-7 breast cancer cells. The ubiquitously distributed sodium-hydrogen antiporter 1 (NHE1) is involved in motility and invasion of many solid tumours. The present study focuses on the effects of [Pt(acac)2(DMS)] in SH-SY5Y cell migration and also on the possibility that NHE1 may be involved in such effect. After sublethal [Pt(acac)2(DMS)] treatment cell migration was examined by wounding assay and cell invasion by transwell assay. NHE1 activity was measured in BCECF-loaded SH-SY5Y as the rate of Na+-dependent intracellular pH recovery in response to an acute acid pulse. Gelatin zymography for MMP-2/9 activities, Western blottings of MMPs, MAPKs, mTOR, S6 and PKCs and small interfering RNAs to PKC-ε/-δ mRNA were performed. Sublethal concentrations of [Pt(acac)2(DMS)] decreases NHE1 activity, inhibites cell migration and invasion and decreases expression and activity of MMP-2 and -9. [Pt(acac)2(DMS)] administered to SH-SY5Y cells provokes the increment of ROS, generated by NADPH oxidase, responsible for the PKC-ε and PKC-δ activation. Whilst PKC-δ activates p38/MAPK, responsible for the inhibition of MMP-2 and -9 secretion, PKC-ε activates a pathway made of ERK1/2, mTOR and S6K responsible for the inhibition of NHE1 activity and cell migration. In conclusion, we have shown a drastic impairment in tumour cell metastatization in response to inhibition of NHE1 and MMPs activities by [Pt(acac)2(DMS)] occurring through a novel mechanism mediated by PKC-δ/-ε activation. PMID:25372487

  15. Action of hexachlorobenzene on tumor growth and metastasis in different experimental models

    Energy Technology Data Exchange (ETDEWEB)

    Pontillo, Carolina Andrea, E-mail: caroponti@hotmail.com [Laboratorio de Efectos Biológicos de Contaminantes Ambientales, Departamento de Bioquímica Humana, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires (Argentina); Rojas, Paola, E-mail: parojas2010@gmail.com [Laboratorio de Carcinogénesis Hormonal, Instituto de Biología y Medicina Experimental (IBYME-CONICET), Buenos Aires (Argentina); Chiappini, Florencia, E-mail: florenciachiappini@hotmail.com [Laboratorio de Efectos Biológicos de Contaminantes Ambientales, Departamento de Bioquímica Humana, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires (Argentina); Sequeira, Gonzalo, E-mail: chicon27_7@hotmail.com [Laboratorio de Carcinogénesis Hormonal, Instituto de Biología y Medicina Experimental (IBYME-CONICET), Buenos Aires (Argentina); Cocca, Claudia, E-mail: cm_cocca@hotmail.com [Laboratorio de Radioisótopos, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires (Argentina); Crocci, Máximo, E-mail: info@crescenti.com.ar [Instituto de Inmunooncología Crescenti, Buenos Aires (Argentina); Colombo, Lucas, E-mail: lucascol2003@yahoo.com.ar [Instituto de Oncología Angel Roffo, Universidad de Buenos Aires, Buenos Aires,Argentina (Argentina); Lanari, Claudia, E-mail: lanari.claudia@gmail.com [Laboratorio de Carcinogénesis Hormonal, Instituto de Biología y Medicina Experimental (IBYME-CONICET), Buenos Aires (Argentina); and others

    2013-05-01

    Hexachlorobenzene (HCB) is a widespread organochlorine pesticide, considered a possible human carcinogen. It is a dioxin-like compound and a weak ligand of the aryl hydrocarbon receptor (AhR). We have found that HCB activates c-Src/HER1/STAT5b and HER1/ERK1/2 signaling pathways and cell migration, in an AhR-dependent manner in MDA-MB-231 breast cancer cells. The aim of this study was to investigate in vitro the effect of HCB (0.005, 0.05, 0.5, 5 μM) on cell invasion and metalloproteases (MMPs) 2 and 9 activation in MDA-MB-231 cells. Furthermore, we examined in vivo the effect of HCB (0.3, 3, 30 mg/kg b.w.) on tumor growth, MMP2 and MMP9 expression, and metastasis using MDA-MB-231 xenografts and two syngeneic mouse breast cancer models (spontaneous metastasis using C4-HI and lung experimental metastasis using LM3). Our results show that HCB (5 μM) enhances MMP2 expression, as well as cell invasion, through AhR, c-Src/HER1 pathway and MMPs. Moreover, HCB increases MMP9 expression, secretion and activity through a HER1 and AhR-dependent mechanism, in MDA-MB-231 cells. HCB (0.3 and 3 mg/kg b.w.) enhances subcutaneous tumor growth in MDA-MB-231 and C4-HI in vivo models. In vivo, using MDA-MB-231 model, the pesticide (0.3, 3 and 30 mg/kg b.w.) activated c-Src, HER1, STAT5b, and ERK1/2 signaling pathways and increased MMP2 and MMP9 protein levels. Furthermore, we observed that HCB stimulated lung metastasis regardless the tumor hormone-receptor status. Our findings suggest that HCB may be a risk factor for human breast cancer progression. - Highlights: ► HCB enhances MMP2 and MMP9 expression and cell invasion in MDA-MB-231, in vitro. ► HCB-effects are mediated through AhR, HER1 and/or c-Src. ► HCB increases subcutaneous tumor growth in MDA-MB-231 and C4-HI in vivo models. ► HCB activates c-Src/HER1 pathway and increases MMPs levels in MDA-MB-231 tumors. ► HCB stimulates lung metastasis in C4-HI and LM3 in vivo models.

  16. Investigating Mechanisms of Alkalinization for Reducing Primary Breast Tumor Invasion

    Directory of Open Access Journals (Sweden)

    Ian F. Robey

    2013-01-01

    Full Text Available The extracellular pH (pHe of many solid tumors is acidic as a result of glycolytic metabolism and poor perfusion. Acidity promotes invasion and enhances metastatic potential. Tumor acidity can be buffered by systemic administration of an alkaline agent such as sodium bicarbonate. Tumor-bearing mice maintained on sodium bicarbonate drinking water exhibit fewer metastases and survive longer than untreated controls. We predict this effect is due to inhibition of tumor invasion. Reducing tumor invasion should result in fewer circulating tumor cells (CTCs. We report that bicarbonate-treated MDA-MB-231 tumor-bearing mice exhibited significantly lower numbers of CTCs than untreated mice (. Tumor pHe buffering may reduce optimal conditions for enzymes involved in tumor invasion such as cathepsins and matrix metalloproteases (MMPs. To address this, we tested the effect of transient alkalinization on cathepsin and MMP activity using enzyme activatable fluorescence agents in mice bearing MDA-MB-231 mammary xenografts. Transient alkalinization significantly reduced the fluorescent signal of protease-specific activatable agents in vivo (. Alkalinization, however, did not affect expression of carbonic anhydrase IX (CAIX. The findings suggest a possible mechanism in a live model system for breast cancer where systemic alkalinization slows the rate of invasion.

  17. Diagnostic, Predictive, Prognostic, and Therapeutic Molecular Biomarkers in Third Millennium: A Breakthrough in Gastric Cancer

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    Nicola Carlomagno

    2017-01-01

    Full Text Available Introduction. Gastric cancer is the fifth most common cancer and the third cause of cancer death. The clinical outcomes of the patients are still not encouraging with a low rate of 5 years’ survival. Often the disease is diagnosed at advanced stages and this obviously negatively affects patients outcomes. A deep understanding of molecular basis of gastric cancer can lead to the identification of diagnostic, predictive, prognostic, and therapeutic biomarkers. Main Body. This paper aims to give a global view on the molecular classification and mechanisms involved in the development of the tumour and on the biomarkers for gastric cancer. We discuss the role of E-cadherin, HER2, fibroblast growth factor receptor (FGFR, MET, human epidermal growth factor receptor (EGFR, hepatocyte growth factor receptor (HGFR, mammalian target of rapamycin (mTOR, microsatellite instability (MSI, PD-L1, and TP53. We have also considered in this manuscript new emerging biomarkers as matrix metalloproteases (MMPs, microRNAs, and long noncoding RNAs (lncRNAs. Conclusions. Identifying and validating diagnostic, prognostic, predictive, and therapeutic biomarkers will have a huge impact on patients outcomes as they will allow early detection of tumours and also guide the choice of a targeted therapy based on specific molecular features of the cancer.

  18. Curcumin as anti-endometriotic agent: implication of MMP-3 and intrinsic apoptotic pathway.

    Science.gov (United States)

    Jana, Sayantan; Paul, Sumit; Swarnakar, Snehasikta

    2012-03-15

    The disease of reproductive women, endometriosis represents implantation of functional endometrial glands outside uterine cavity. This invasive disorder is associated with dysregulation of matrix metalloproteases (MMP)s and extracellular matrix (ECM) remodeling. In this study, we investigated the role of MMP-3 on apoptosis during endometriosis. We also checked whether curcumin has potency to regress endometriosis by modulating MMP-3 and apoptotic pathway. Mouse model of endometriosis was designed by intraperitoneal inoculation of endometrial tissues to syngeneic female BALB/c. At 15th day, stable endometriotic developments were observed with increased MMP-3 expression. TUNEL positive cells were also found with endometriotic progression, which might resulted from destruction of local immune cells. We speculate that increased MMP-3 activity might be involved in the Fas mediated apoptosis. Curcumin treatment regressed endometriosis by inhibiting NFκB translocation and MMP-3 expression. It also accelerated apoptosis in endometriomas predominantly via cytochrome-c mediated mitochondrial pathway. Involvement of mitochondria in apoptosis was further confirmed by atomic force microscopy (AFM). These results were also supported by our therapeutic study, where curcumin induced apoptosis both by p53 dependent and independent manner, while celecoxib followed only p53 independent pathway. Altogether, our study establishes the novel role of curcumin as a potent anti-endometriotic compound. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Wound healing and protease inhibition activity of Bacoside-A, isolated from Bacopa monnieri wettest.

    Science.gov (United States)

    Sharath, R; Harish, B G; Krishna, V; Sathyanarayana, B N; Swamy, H M Kumara

    2010-08-01

    Bacopa monnieri (L.) Wettest. (Scrophulariaceae) is a well-known medicinal herb. In the Indian system of medicine it is known as Brahmi (Sanskrit) and Indian water hyssop. Methanolic extract of Bacopa monnieri and its isolated constituent Bacoside-A were screened for wound healing activity. Bacoside-A was screened for wound healing activity by excision, incision and dead space wound on Swiss albino rats. Significant wound healing activity was observed in both extract and the Bacoside-A treated groups. The SDS-PAGE caseinolytic zymogram analysis of inhibition of matrix metalloproteases (MMPs) enzyme from the excision wound by Bacoside-A, an isolated constituent, was done with the concentrations 100 and 200 micromg/ml. In Bacoside-A treated groups, epithelialization of the excision wound was faster with a high rate (18.30 +/- 0.01 days) of wound contraction. The tensile strength of the incision wound was increased (538.47 +/- 0.14 g) in the Bacoside-A treated group. In the dead space wound model, the weight of the granuloma was also increased (89.15 +/- 0.08 g). The histological examination of the granuloma tissue of the Bacoside-A treated group showed increased cross-linking of collagen fibers and absence of monocytes. The wound healing activity of Bacoside-A was more effective in various wound models compared to the standard skin ointment Nitrofurazone.

  20. Tetracycline Reduces Kidney Damage Induced by Loxosceles Spider Venom

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    Cinthya Kimori Okamoto

    2017-03-01

    Full Text Available Envenomation by Loxosceles spider can result in two clinical manifestations: cutaneous and systemic loxoscelism, the latter of which includes renal failure. Although incidence of renal failure is low, it is the main cause of death, occurring mainly in children. The sphingomyelinase D (SMase D is the main component in Loxosceles spider venom responsible for local and systemic manifestations. This study aimed to investigate the toxicity of L. intermedia venom and SMase D on kidney cells, using both In vitro and in vivo models, and the possible involvement of endogenous metalloproteinases (MMP. Results demonstrated that venom and SMase D are able to cause death of human kidney cells by apoptosis, concomitant with activation and secretion of extracellular matrix metalloproteases, MMP-2 and MMP-9. Furthermore, cell death and MMP synthesis and secretion can be prevented by tetracycline. In a mouse model of systemic loxoscelism, Loxosceles venom-induced kidney failure was observed, which was abrogated by administration of tetracycline. These results indicate that MMPs may play an important role in Loxosceles venom-induced kidney injury and that tetracycline administration may be useful in the treatment of human systemic loxoscelism.

  1. Proinflammatory Effects of Diesel Exhaust Nanoparticles on Scleroderma Skin Cells

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    A. Mastrofrancesco

    2014-01-01

    Full Text Available Autoimmune diseases are complex disorders of unknown etiology thought to result from interactions between genetic and environmental factors. We aimed to verify whether environmental pollution from diesel engine exhaust nanoparticulate (DEP of actually operating vehicles could play a role in the development of a rare immune-mediated disease, systemic sclerosis (SSc, in which the pathogenetic role of environment has been highlighted. The effects of carbon-based nanoparticulate collected at the exhaust of newer (Euro 5 and older (Euro 4 diesel engines on SSc skin keratinocytes and fibroblasts were evaluated in vitro by assessing the mRNA expression of inflammatory cytokines (IL-1α, IL-6, IL-8, and TNF-α and fibroblast chemical mediators (metalloproteases 2, 3, 7, 9, and 12; collagen types I and III; VEGF. DEP was shown to stimulate cytokine gene expression at a higher extent in SSc keratinocytes versus normal cells. Moreover, the mRNA gene expression of all MMPs, collagen types, and VEGF genes was significantly higher in untreated SSc fibroblasts versus controls. Euro 5 particle exposure increased the mRNA expression of MMP-2, -7, and -9 in SSc fibroblasts in a dose dependent manner and only at the highest concentration in normal cells. We suggest that environmental DEP could trigger the development of SSc acting on genetically hyperreactive cell systems.

  2. Matrix Metalloproteinases 2 and 3 Gene Polymorphisms and the Risk of Target Vessel Revascularization after Percutaneous Coronary Intervention: Is There Still Room for Determining Genetic Variation of MMPs for Assessment of an Increased Risk of Restenosis?

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    J.J.W. Verschuren

    2010-01-01

    Full Text Available Objective: Mixed results have been reported of matrix metalloproteinases (MMP and their association with restenosis after percutaneous coronary intervention (PCI. The current study examines whether multiple single nucleotide polymorphisms (SNPs, covering the full genomic region of MMP2 and MMP3, were associated with restenosis in the GENDER study population.

  3. An Alginate/Cyclodextrin Spray Drying Matrix to Improve Shelf Life and Antioxidant Efficiency of a Blood Orange By-Product Extract Rich in Polyphenols: MMPs Inhibition and Antiglycation Activity in Dysmetabolic Diseases

    Science.gov (United States)

    Giannone, Virgilio; Ballistreri, Gabriele; Fabroni, Simona; Rapisarda, Paolo; Panico, Anna Maria; Puglisi, Giovanni

    2017-01-01

    Alginate and β-cyclodextrin were used to produce easily dosable and spray-dried microsystems of a dried blood orange extract with antidysmetabolic properties, obtained from a by-product fluid extract. The spray-dried applied conditions were able to obtain a concentrate dried extract without the loss of AOA and with TPC and TMA values of 35–40% higher than that of the starting material. They were also effective in producing microparticles with 80–100% of encapsulation efficiency. The 2% sodium alginate was capable of improving the extract shelf life, while the beta-cyclodextrin (1 : 1 molar ratio with dried extract) prolonged the extract antioxidant efficiency by 6 hours. The good inhibition effect of the dried extract on the AGE formation and the MMP-2 and MMP-9 activity is presumably due to a synergic effect exerted by both anthocyanin and bioflavonoid extract compounds and was improved by the use of alginate and cyclodextrin. PMID:29230268

  4. Treinamento de força e o envelhecimento alteram a atividade das metalopeptidases de matriz (MMPs) em músculos esqueléticos e em soro de ratos

    OpenAIRE

    Prado, Gleyce Pires Gonçalves do

    2017-01-01

    O envelhecimento é um processo multifatorial complexo, no qual as propriedades mecânicas do músculo se deterioram o que contribui para perda de função e independência dos idosos, essa deterioração é principalmente devido à perda de massa muscular. É possível que as alterações no músculo esquelético também possam estar relacionadas a matriz extracelular. O objetivo deste estudo foi investigar os efeitos do TF sobre a atividade das MMP-2 e MMP-9 em músculos esqueléticos e soro de ratos jovens e...

  5. InfluÃncia de inibidores de metaloproteinases da matriz (MMPS) na resistÃncia de uniÃo de um sistema adesivo autocondicionante em dentina hÃgida e erosivamente desmineralizada

    OpenAIRE

    CecÃlia Atem GonÃalves de Araujo Costa

    2013-01-01

    Objetivo: analisar a influÃncia do uso de inibidores de metaloproteinases da matriz [epigalocatequina-3-galato (EGCG) e digluconato de clorexidina (CHX)] utilizados como prÃ-tratamento dentinÃrio sobre a resistÃncia de uniÃo de um sistema adesivo autocondicionante em dentina hÃgida (H) e erosivamente desmineralizada (ED). Materiais e MÃtodos: terceiros molares humanos hÃgidos extraÃdos tiveram o terÃo oclusal seccionado, expondo a superfÃcie dentinÃria subjacente ao esmalte. A microdureza de ...

  6. An Alginate/Cyclodextrin Spray Drying Matrix to Improve Shelf Life and Antioxidant Efficiency of a Blood Orange By-Product Extract Rich in Polyphenols: MMPs Inhibition and Antiglycation Activity in Dysmetabolic Diseases

    Directory of Open Access Journals (Sweden)

    Maria Rosaria Lauro

    2017-01-01

    Full Text Available Alginate and β-cyclodextrin were used to produce easily dosable and spray-dried microsystems of a dried blood orange extract with antidysmetabolic properties, obtained from a by-product fluid extract. The spray-dried applied conditions were able to obtain a concentrate dried extract without the loss of AOA and with TPC and TMA values of 35–40% higher than that of the starting material. They were also effective in producing microparticles with 80–100% of encapsulation efficiency. The 2% sodium alginate was capable of improving the extract shelf life, while the beta-cyclodextrin (1 : 1 molar ratio with dried extract prolonged the extract antioxidant efficiency by 6 hours. The good inhibition effect of the dried extract on the AGE formation and the MMP-2 and MMP-9 activity is presumably due to a synergic effect exerted by both anthocyanin and bioflavonoid extract compounds and was improved by the use of alginate and cyclodextrin.

  7. An Alginate/Cyclodextrin Spray Drying Matrix to ImproveShelf Lifeand Antioxidant Efficiency of a Blood Orange By-Product Extract Rich in Polyphenols: MMPs Inhibition and Antiglycation Activity in Dysmetabolic Diseases.

    Science.gov (United States)

    Lauro, Maria Rosaria; Crascì, Lucia; Giannone, Virgilio; Ballistreri, Gabriele; Fabroni, Simona; Sansone, Francesca; Rapisarda, Paolo; Panico, Anna Maria; Puglisi, Giovanni

    2017-01-01

    Alginate and β -cyclodextrin were used to produce easily dosable and spray-dried microsystems of a dried blood orange extract with antidysmetabolic properties, obtained from a by-product fluid extract. The spray-dried applied conditions were able to obtain a concentrate dried extract without the loss of AOA and with TPC and TMA values of 35-40% higher than that of the starting material. They were also effective in producing microparticles with 80-100% of encapsulation efficiency. The 2% sodium alginate was capable of improving the extract shelf life , while the beta-cyclodextrin (1 : 1 molar ratio with dried extract) prolonged the extract antioxidant efficiency by 6 hours. The good inhibition effect of the dried extract on the AGE formation and the MMP-2 and MMP-9 activity is presumably due to a synergic effect exerted by both anthocyanin and bioflavonoid extract compounds and was improved by the use of alginate and cyclodextrin.

  8. Matrix metalloproteinases 2 and 3 gene polymorphisms and the risk of target vessel revascularization after percutaneous coronary intervention: Is there still room for determining genetic variation of MMPs for assessment of an increased risk of restenosis?

    NARCIS (Netherlands)

    Verschuren, J. J. W.; Sampietro, M. L.; Pons, D.; Trompet, S.; Ewing, M. M.; Quax, P. H. A.; de Knijff, P.; Zwinderman, A. H.; de Winter, R. J.; Tio, R. A.; de Maat, M. P.; Doevendans, P. A. F. M.; Jukema, J. W.

    2010-01-01

    Objective: Mixed results have been reported of matrix metalloproteinases (MMP) and their association with restenosis after percutaneous coronary intervention (PCI). The current study examines whether multiple single nucleotide polymorphisms (SNPs), covering the full genomic region of MMP2 and MMP3,

  9. Activation of PPARs α, β/δ, and γ Impairs TGF-β1-Induced Collagens' Production and Modulates the TIMP-1/MMPs Balance in Three-Dimensional Cultured Chondrocytes

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    Paul-Emile Poleni

    2010-01-01

    Full Text Available Background and Purpose. We investigated the potency of Peroxisome Proliferators-Activated Receptors (PPARs α, β/δ, and γ agonists to modulate Transforming Growth Factor-β1 (TGF-β1- induced collagen production or changes in Tissue Inhibitor of Matrix Metalloproteinase- (TIMP- 1/Matrix Metalloproteinase (MMP balance in rat chondrocytes embedded in alginate beads. Experimental Approach. Collagen production was evaluated by quantitative Sirius red staining, while TIMP-1 protein levels and global MMP (-1, -2, -3, -7, and -9 or specific MMP-13 activities were measured by ELISA and fluorigenic assays in culture media, respectively. Levels of mRNA for type II collagen, TIMP-1, and MMP-3 & 13 were quantified by real-time PCR. Key Results. TGF-β1 increased collagen deposition and type II collagen mRNA levels, while inducing TIMP-1 mRNA and protein expression. In contrast, it decreased global MMP or specific MMP-13 activities, while decreasing MMP-3 or MMP-13 mRNA levels. PPAR agonists reduced most of the effects of TGF-β1 on changes in collagen metabolism and TIMP-1/MMP balance in rat in a PPAR-dependent manner, excepted for Wy14643 on MMP activities. Conclusions and Implications. PPAR agonists reduce TGF-β1-modulated ECM turnover and inhibit chondrocyte activities crucial for collagen biosynthesis, and display a different inhibitory profile depending on selectivity for PPAR isotypes.

  10. Análisis de MMPs en fluidos orales en el diagnóstico complementario de las enfermedades periodontales Oral-fluid MMP analysis in the complementary diagnosis of periodontal diseases

    OpenAIRE

    P Hernández; P Mäntylä; T Tervahartiala; T Sorsa; M Hernández

    2012-01-01

    La periodontitis constituye la infección bacteriana más prevalente a nivel mundial y representa un factor de riesgo para diversas patologías sistémicas. El estado de inflamación y destrucción periodontal se manifiestan a través de la presencia de biomarcadores en el suero y fluidos orales, tales como el fluido gingival crevicular (FGC), saliva y enjuague oral. Enzimas como las metaloproteinasas de matriz (MMP) y mieloperoxidasa, constituyen biomarcadores potenciales para ensayos moleculares c...

  11. Physicochemical properties of the modeled structure of astacin metalloprotease moulting enzyme NAS-36 and mapping the druggable allosteric space of Heamonchus contortus, Brugia malayi and Ceanorhabditis elegans via molecular dynamics simulation.

    Science.gov (United States)

    Sharma, Om Prakash; Agrawal, Sonali; Kumar, M Suresh

    2013-12-01

    Nematodes represent the second largest phylum in the animal kingdom. It is the most abundant species (500,000) in the planet. It causes chronic, debilitating infections worldwide such as ascariasis, trichuriasis, hookworm, enterobiasis, strongyloidiasis, filariasis and trichinosis, among others. Molecular modeling tools can play an important role in the identification and structural investigation of molecular targets that can act as a vital candidate against filariasis. In this study, sequence analysis of NAS-36 from H. contortus (Heamonchus contortus), B. malayi (Brugia malayi) and C. elegans (Ceanorhabditis elegans) has been performed, in order to identify the conserved residues. Tertiary structure was developed for an insight into the molecular structure of the enzyme. Molecular Dynamics Simulation (MDS) studies have been carried out to analyze the stability and the physical properties of the proposed enzyme models in the H. contortus, B. malayi and C. elegans. Moreover, the drug binding sites have been mapped for inhibiting the function of NAS-36 enzyme. The molecular identity of this protease could eventually demonstrate how ex-sheathment is regulated, as well as provide a potential target of anthelmintics for the prevention of nematode infections.

  12. Impact of CD68/(CD3+CD20 ratio at the invasive front of primary tumors on distant metastasis development in breast cancer.

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    Noemí Eiró

    Full Text Available Tumors are infiltrated by macrophages, T and B-lymphocytes, which may favor tumor development by promoting angiogenesis, growth and invasion. The aim of this study was to investigate the clinical relevance of the relative amount of macrophages (CD68⁺, T-cells (CD3⁺ and B-cells (CD20⁺ at the invasive front of breast carcinomas, and the expression of matrix metalloproteases (MMPs and their inhibitors (TIMPs either at the invasive front or at the tumor center. We performed an immunohistochemical study counting CD3, CD20 and CD68 positive cells at the invasive front, in 102 breast carcinomas. Also, tissue sections were stained with MMP-2, -9, -11, -14 and TIMP-2 antibodies, and immunoreactivity location, percentage of reactive area and intensity were determined at the invasive front and at the tumor center. The results showed that an increased CD68 count and CD68/(CD3+CD20 ratio were directly associated with both MMP-11 and TIMP-2 expression by mononuclear inflammatory cells at the tumor center (p = 0.041 and p = 0.025 for CD68 count and p = 0.001 and p = 0.045 for ratio, respectively for MMP-11 and TIMP-2. In addition, a high CD68/(CD3+CD20 ratio (>0.05 was directly associated with a higher probability of shortened relapse-free survival. Multivariate analysis revealed that CD68/(CD3+CD20 ratio was an independent factor associated with distant relapse-free survival (RR: 2.54, CI: (1.23-5.24, p<0.01. Therefore, CD68/(CD3+CD20 ratio at the invasive front could be used as an important prognostic marker.

  13. Rational modulation of the innate immune system for neuroprotection in ischemic stroke

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    Diana eAmantea

    2015-04-01

    Full Text Available The innate immune system plays a dualistic role in the evolution of ischemic brain damage and has also been implicated in ischemic tolerance produced by different conditioning stimuli. Early after ischemia, perivascular astrocytes release cytokines and activate metalloproteases (MMPs that contribute to blood–brain barrier (BBB disruption and vasogenic oedema; whereas at later stages, they provide extracellular glutamate uptake, BBB regeneration and neurotrophic factors release. Similarly, early activation of microglia contributes to ischemic brain injury via the production of inflammatory cytokines, including tumor necrosis factor (TNF and interleukin (IL-1, reactive oxygen and nitrogen species and proteases. Nevertheless, microglia also contributes to the resolution of inflammation, by releasing IL-10 and tumor growth factor (TGF-beta, and to the late reparative processes by phagocytic activity and growth factors production. Indeed, after ischemia, microglia/macrophages differentiate towards several phenotypes: the M1 pro-inflammatory phenotype is classically activated via toll-like receptors or interferon-γ, whereas M2 phenotypes are alternatively activated by regulatory mediators, such as ILs 4, 10, 13 or TGF-beta. Thus, immune cells exert a dualistic role on the evolution of ischemic brain damage, since the classic phenotypes promote injury, whereas alternatively activated M2 macrophages or N2 neutrophils prompt tissue remodeling and repair.Moreover, a subdued activation of the immune system has been involved in ischemic tolerance, since different preconditioning stimuli act via modulation of inflammatory mediators, including toll-like receptors and cytokine signaling pathways. This further underscores that the immuno-modulatory approach for the treatment of ischemic stroke should be aimed at blocking the detrimental effects, while promoting the beneficial responses of the immune reaction.

  14. Conditionally replicating adenovirus expressing TIMP2 increases survival in a mouse model of disseminated ovarian cancer.

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    Sherry W Yang

    Full Text Available Ovarian cancer remains difficult to treat mainly due to presentation of the disease at an advanced stage. Conditionally-replicating adenoviruses (CRAds are promising anti-cancer agents that selectively kill the tumor cells. The present study evaluated the efficacy of a novel CRAd (Ad5/3-CXCR4-TIMP2 containing the CXCR4 promoter for selective viral replication in cancer cells together with TIMP2 as a therapeutic transgene, targeting the matrix metalloproteases (MMPs in a murine orthotopic model of disseminated ovarian cancer. An orthotopic model of ovarian cancer was established in athymic nude mice by intraperitonal injection of the human ovarian cancer cell line, SKOV3-Luc, expressing luciferase. Upon confirmation of peritoneal dissemination of the cells by non-invasive imaging, mice were randomly divided into four treatment groups: PBS, Ad-ΔE1-TIMP2, Ad5/3-CXCR4, and Ad5/3-CXCR4-TIMP2. All mice were imaged weekly to monitor tumor growth and were sacrificed upon reaching any of the predefined endpoints, including high tumor burden and significant weight loss along with clinical evidence of pain and distress. Survival analysis was performed using the Log-rank test. The median survival for the PBS cohort was 33 days; for Ad-ΔE1-TIMP2, 39 days; for Ad5/3-CXCR4, 52.5 days; and for Ad5/3-CXCR4-TIMP2, 63 days. The TIMP2-armed CRAd delayed tumor growth and significantly increased survival when compared to the unarmed CRAd. This therapeutic effect was confirmed to be mediated through inhibition of MMP9. Results of the in vivo study support the translational potential of Ad5/3-CXCR4-TIMP2 for treatment of human patients with advanced ovarian cancer.

  15. Cigarette smoke-induced collagen destruction; key to chronic neutrophilic airway inflammation?

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    Saskia A Overbeek

    Full Text Available BACKGROUND: Cigarette smoking induces inflammatory responses in all smokers and is the major risk factor for lung disease such as chronic obstructive pulmonary disease (COPD. In this progressive disease, chronic inflammation in the lung contributes to lung tissue destruction leading to the formation of chemotactic collagen fragments such as N-acetylated Proline-Glycine-Proline (N-ac-PGP. The generation of this tripeptide is mediated by a multistep pathway involving matrix metalloproteases (MMPs 8 and 9 and prolyl endopeptidase (PE. Here we investigated whether cigarette smoke extract (CSE stimulates human PMNs to breakdown whole matrix collagen leading to the generation of the chemotactic collagen fragment N-ac-PGP. METHODOLOGY/PRINCIPAL FINDINGS: Incubating PMNs with CSE led to the release of chemo-attractant CXCL8 and proteases MMP8 and MMP9. PMNs constitutively expressed PE activity as well as PE protein. Incubating CSE-primed PMNs with collagen resulted in collagen breakdown and in N-ac-PGP generation. Incubation of PMNs with the tripeptide N-ac-PGP resulted in the release of CXCL8, MMP8 and MMP9. Moreover, we tested whether PMNs from COPD patients are different from PMNs from healthy donors. Here we show that the intracellular basal PE activity of PMNs from COPD patients increased 25-fold compared to PMNs from healthy donors. Immunohistological staining of human lung tissue for PE showed that besides neutrophils, macrophages and epithelial cells express PE. CONCLUSIONS: This study indicates that neutrophils activated by cigarette smoke extract can breakdown collagen into N-ac-PGP and that this collagen fragment itself can activate neutrophils, which may lead in vivo to a self-propagating cycle of neutrophil infiltration, chronic inflammation and lung emphysema. MMP-, PE- or PGP-inhibitors can serve as an attractive therapeutic target and may open new avenues towards effective treatment of COPD.

  16. EGFR signaling downstream of EGF regulates migration, invasion, and MMP secretion of immortalized cells derived from human ameloblastoma.

    Science.gov (United States)

    da Rosa, Marina Rolo Pinheiro; Falcão, Aline Semblano Carreira; Fuzii, Hellen Thais; da Silva Kataoka, Maria Sueli; Ribeiro, André L R; Boccardo, Enrique; de Siqueira, Adriane Sousa; Jaeger, Ruy G; de Jesus Viana Pinheiro, João; de Melo Alves Júnior, Sérgio

    2014-11-01

    Ameloblastoma is an odontogenic tumor characterized by local invasiveness and frequent recurrence. The surrounding stroma, composed of different cell types and extracellular matrix (ECM), may influence ameloblastoma invasive behavior. Furthermore, tumor and stromal cells secrete matrix metalloproteases (MMPs), which, in turn, can modulate the matrix and promote the release of ECM-bound growth factors. Among these growth factors, epidermal growth factor (EGF) and its receptor, EGFR, have already been shown to stimulate MMP synthesis, suggesting that an interdependent mechanism, involving MMP activity and growth factors release, may contribute to tumor invasiveness. The aim of this study was to evaluate the effects of the EGF/EGFR signaling pathway on migration, invasion, and MMP activity, in a primary cell line derived from human ameloblastoma. We established and characterized a primary cell line (AME-1) from a human ameloblastoma sample. This cell line was transduced with human papillomavirus type 16 (HPV16) E6/E7 oncogenes, generating the AME-HPV continuous cell line. EGF, MMP2, and MMP9 expression in ameloblastoma biopsies and in the AME-HPV cell line was analyzed by immunohistochemistry and immunofluorescence, respectively. Migratory activity of EGF-treated AME-HPV cells was investigated using monolayer wound assays and Transwell chambers. EGF-induced invasion was assessed in Boyden chambers coated with Matrigel. Conditioned medium from EGF-treated cells was subjected to zymography. EGFR expression in AME-HPV cells was silenced by small interfering RNA (siRNA), to verify the relationship between this receptor and MMP secretion. Ameloblastoma samples and AME-HPV cells expressed EGF, EGFR, MMP2, and MMP9. AME-HPV cells treated with EGF showed increased rates of migration and invasion, as well as enhanced MMP2 and MMP9 activity. EGFR knockdown decreased MMP2 and MMP9 levels in AME-HPV cells. EGFR signaling downstream of EGF probably regulates migration, invasion

  17. Interaction of Leptospira interrogans with Human Proteolytic Systems Enhances Dissemination through Endothelial Cells and Protease Levels

    Science.gov (United States)

    Vieira, Monica L.; Alvarez-Flores, Miryam P.; Kirchgatter, Karin; Romero, Eliete C.; Alves, Ivy J.; de Morais, Zenaide M.; Vasconcellos, Silvio A.; Chudzinski-Tavassi, Ana M.

    2013-01-01

    We have recently reported the ability of Leptospira to capture plasminogen (PLG) and generate plasmin (PLA) bound on the microbial surface in the presence of exogenous activators. In this work, we examined the effects of leptospiral PLG binding for active penetration through the endothelial cell barrier and activation. The results indicate that leptospires with PLG association or PLA activation have enhanced migration activity through human umbilical vein endothelial cell (HUVEC) monolayers compared with untreated bacteria. Leptospira cells coated with PLG were capable of stimulating the expression of PLG activators by HUVECs. Moreover, leptospires endowed with PLG or PLA promoted transcriptional upregulation matrix metalloprotease 9 (MMP-9). Serum samples from patients with confirmed leptospirosis showed higher levels of PLG activators and total MMP-9 than serum samples from normal (healthy) subjects. The highest level of PLG activators and total MMP-9 was detected with microscopic agglutination test (MAT)-negative serum samples, suggesting that this proteolytic activity stimulation occurs at the early stage of the disease. Furthermore, a gelatin zymography profile obtained for MMPs with serum samples from patients with leptospirosis appears to be specific to leptospiral infection because serum samples from patients with unrelated infectious diseases produced no similar degradation bands. Altogether, the data suggest that the Leptospira-associated PLG or PLA might represent a mechanism that contributes to bacterial penetration of endothelial cells through an activation cascade of events that enhances the proteolytic capability of the organism. To our knowledge, this is the first proteolytic activity associated with leptospiral pathogenesis described to date. PMID:23478319

  18. Effect of synthetic matrix-metalloproteinase inhibitors on invasive capacity and proliferation of human malignant gliomas in vitro.

    Science.gov (United States)

    Tonn, J C; Kerkau, S; Hanke, A; Bouterfa, H; Mueller, J G; Wagner, S; Vince, G H; Roosen, K

    1999-03-01

    Glioma invasion into the surrounding brain tissue is still a major obstacle for any therapeutical approach. As in other solid tumors, matrix-metalloproteases (MMPs) have been suggested as being involved. The aim of this study was to evaluate whether the use of MMP inhibitors to target the protease-mediated invasion process could be a feasible approach. Two human cell lines (U251 and GaMG) and surgical specimens of 6 patients with malignant gliomas were grown as monolayers and spheroid cultures respectively. MMP- and u-PA-mRNA expression was investigated by semi-quantitative RT-PCR. Invasion was studied in Matrigel-coated Boyden chamber transwell assays for monolayers and in confrontation cultures of tumor spheroids with fetal rat brain aggregates in the presence of the synthetic MMP inhibitors batimastat (BB-94) and marimastat (BB-2516). Cytotoxicity/cytostatic effects of high concentrations of both compounds were assessed by growth curves, MTT assays and flow cytometry in human glioma cell lines. Batimastat and marimastat revealed a cytostatic effect at high concentrations (above 1 microM) without cytotoxicity. Both MMP inhibitors effectively reduced glioma invasion in Boyden-chamber assays at low concentrations of 0.3 microM. In confrontation cultures, concentrations of 10 microM and above were necessary to reduce invasion. This effect was observable with inter-individual heterogeneity in the patient's tumor material. MMP inhibitors effectively reduce glioma invasion, although high concentrations were required in 3-dimensional culture systems. At these concentrations, both compounds revealed a cytostatic, but no cytotoxic effect. Thus, high local concentrations of MMP inhibitors could offer a new therapeutic strategy for the treatment of gliomas.

  19. Genetic polymorphisms in MMP 2, 9 and 3 genes modify lung cancer risk and survival

    Directory of Open Access Journals (Sweden)

    González-Arriaga Patricia

    2012-03-01

    Full Text Available Abstract Background Matrix metalloproteases (MMPs are proteolytic enzymes that contribute to all stages of tumour progression, including the later stages of invasion and metastasis. Genetic variants in the MMP genes may influence the biological function of these enzymes and change their role in carcinogenesis and progression. We have investigated the association between the -735 C/T, the -1171 5A/6A, and the -1562 C/T polymorphisms in the MMP2, MMP3 and MMP9 genes, respectively, and the risk and survival of lung cancer. Methods The case-control study includes 879 lung cancer patients and 803 controls from a Caucasian population in Spain (CAPUA study. Genotypes were determined by PCR-RFLP. Odds ratios (OR and 95% confidence intervals (CI were calculated using unconditional logistic regression. The Kaplan-Meier method, long-rank test and Cox's were used for the survival analysis. Results The MMP9 -1562 T/T genotype was associated with a statistically significant decreased risk of developing lung cancer (OR = 0.23; 95% CI: 0.06-0.85, whereas no association was found for the MMP2 -735 C/T and MMP3 -1171 5A/6A polymorphisms. The MMP2 -735 T/T genotype was statistically significantly associated with a decreased survival in non-small cell lung cancer (NSCLC patients, identified as an independent prognosis factor of survival (hazard ratio (HR = 1.79; 95% CI: 1.00-3.20. In contrast, no association was found between the MMP3 -1171 5A/6A and the MMP9 -1562 C/T polymorphisms and survival. Conclusions These findings support the hypothesis that the MMP9 -1562 C/T polymorphism is associated with a protective effect against the development of lung cancer and suggest that the MMP2 -735 C/T polymorphism modify the length of survival in NSCLC patients.

  20. Molecular signature and in vivo behavior of bone marrow endosteal and subendosteal stromal cell populations and their relevance to hematopoiesis

    International Nuclear Information System (INIS)

    Balduino, Alex; Mello-Coelho, Valeria; Wang, Zhou; Taichman, Russell S.; Krebsbach, Paul H.; Weeraratna, Ashani T.; Becker, Kevin G.; Mello, Wallace de; Taub, Dennis D.; Borojevic, Radovan

    2012-01-01

    In the bone marrow cavity, hematopoietic stem cells (HSC) have been shown to reside in the endosteal and subendosteal perivascular niches, which play specific roles on HSC maintenance. Although cells with long-term ability to reconstitute full hematopoietic system can be isolated from both niches, several data support a heterogenous distribution regarding the cycling behavior of HSC. Whether this distinct behavior depends upon the role played by the stromal populations which distinctly create these two niches is a question that remains open. In the present report, we used our previously described in vivo assay to demonstrate that endosteal and subendosteal stromal populations are very distinct regarding skeletal lineage differentiation potential. This was further supported by a microarray-based analysis, which also demonstrated that these two stromal populations play distinct, albeit complementary, roles in HSC niche. Both stromal populations were preferentially isolated from the trabecular region and behave distinctly in vitro, as previously reported. Even though these two niches are organized in a very close range, in vivo assays and molecular analyses allowed us to identify endosteal stroma (F-OST) cells as fully committed osteoblasts and subendosteal stroma (F-RET) cells as uncommitted mesenchymal cells mainly represented by perivascular reticular cells expressing high levels of chemokine ligand, CXCL12. Interestingly, a number of cytokines and growth factors including interleukin-6 (IL-6), IL-7, IL-15, Hepatocyte growth factor (HGF) and stem cell factor (SCF) matrix metalloproteases (MMPs) were also found to be differentially expressed by F-OST and F-RET cells. Further microarray analyses indicated important mechanisms used by the two stromal compartments in order to create and coordinate the “quiescent” and “proliferative” niches in which hematopoietic stem cells and progenitors reside.

  1. Molecular signature and in vivo behavior of bone marrow endosteal and subendosteal stromal cell populations and their relevance to hematopoiesis

    Energy Technology Data Exchange (ETDEWEB)

    Balduino, Alex, E-mail: balduino@uva.edu.br [School of Dentistry, Veiga de Almeida University, Rio de Janeiro, RJ (Brazil); Mello-Coelho, Valeria [Biomedical Science Institute, Federal University of Rio de Janeiro, Rio de Janeiro, RJ (Brazil); National Institute on Aging, National Institute of Health, Baltimore, MD (United States); Wang, Zhou; Taichman, Russell S.; Krebsbach, Paul H. [Department of Periodontics, Prevention and Geriatrics, University of Michigan School of Dentistry, Ann Arbor, MI (United States); Weeraratna, Ashani T.; Becker, Kevin G. [National Institute on Aging, National Institute of Health, Baltimore, MD (United States); Mello, Wallace de [Instituto Oswaldo Cruz, Rio de Janeiro, RJ (Brazil); Taub, Dennis D. [National Institute on Aging, National Institute of Health, Baltimore, MD (United States); Borojevic, Radovan [Biomedical Science Institute, Federal University of Rio de Janeiro, Rio de Janeiro, RJ (Brazil)

    2012-11-15

    In the bone marrow cavity, hematopoietic stem cells (HSC) have been shown to reside in the endosteal and subendosteal perivascular niches, which play specific roles on HSC maintenance. Although cells with long-term ability to reconstitute full hematopoietic system can be isolated from both niches, several data support a heterogenous distribution regarding the cycling behavior of HSC. Whether this distinct behavior depends upon the role played by the stromal populations which distinctly create these two niches is a question that remains open. In the present report, we used our previously described in vivo assay to demonstrate that endosteal and subendosteal stromal populations are very distinct regarding skeletal lineage differentiation potential. This was further supported by a microarray-based analysis, which also demonstrated that these two stromal populations play distinct, albeit complementary, roles in HSC niche. Both stromal populations were preferentially isolated from the trabecular region and behave distinctly in vitro, as previously reported. Even though these two niches are organized in a very close range, in vivo assays and molecular analyses allowed us to identify endosteal stroma (F-OST) cells as fully committed osteoblasts and subendosteal stroma (F-RET) cells as uncommitted mesenchymal cells mainly represented by perivascular reticular cells expressing high levels of chemokine ligand, CXCL12. Interestingly, a number of cytokines and growth factors including interleukin-6 (IL-6), IL-7, IL-15, Hepatocyte growth factor (HGF) and stem cell factor (SCF) matrix metalloproteases (MMPs) were also found to be differentially expressed by F-OST and F-RET cells. Further microarray analyses indicated important mechanisms used by the two stromal compartments in order to create and coordinate the 'quiescent' and 'proliferative' niches in which hematopoietic stem cells and progenitors reside.

  2. Regulatory effects of zinc on cadmium-induced cytotoxicity in chronic inflammation.

    Directory of Open Access Journals (Sweden)

    Paola Bonaventura

    Full Text Available Zinc (Zn has major effects on immune system activation while Cadmium (Cd has anti-inflammatory and anti-proliferative effects in several chronic inflammatory contexts. The aim of this work was to investigate by which mechanisms Zn could compete with Cd and eventually counteract its deleterious effects. Rheumatoid arthritis (RA synoviocytes exposed to cytokines were used as a model of chronic inflammation; osteoarthritis (OA synoviocytes were used as control.Cell/medium fractionation constants were analyzed for different metals by inductively-coupled-plasma mass-spectrometry by comparison to the 70Zn spike. Interleukin-17 (IL-17 and tumor necrosis factor-alpha (TNF-α were used to mimic inflammation. Gene expression of ZIP-8 importer, metallothioneins-1 (MT-1s and the ratio between metalloprotease-3 and the tissue inhibitor of metalloproteinases (MMP-3/TIMP-1 were evaluated after pre-exposure to cytokines and Cd, with or without the addition of exogenous Zn (0.9 ppm. Cell viability was measured by neutral red assay and IL-6 production by ELISA.Synoviocytes selectively absorbed and retained Cd in comparison to Zn. Metal import increased with IL-17/TNF-α exposure, through the enhanced ZIP-8 expression. Zn did not modify ZIP-8 expression, while Cd reduced it (p<0.05. Zn induced a reduction of Cd-induced MT-1s expression, in particular of MT-1X (3-fold, and subsequently the final intra-cellular content of Cd. By reducing Cd accumulation in cells, Zn reversed Cd anti-proliferative and anti-inflammatory effects but preserved the low MMP-3/TIMP-1 ratio induced by Cd, which was enhanced by inflammatory conditions.Zinc counteracts the deleterious effect of Cd by reducing its import and accumulation in the cell, without the reactivation of destructive pathways such as MMPs.

  3. Matrix metalloproteinase inhibition after myocardial infarction: a new approach to prevent heart failure?

    NARCIS (Netherlands)

    Creemers, E. E.; Cleutjens, J. P.; Smits, J. F.; Daemen, M. J.

    2001-01-01

    Increased activity of matrix metalloproteinases (MMPs) has been implicated in numerous disease processes, including tumor growth and metastasis, arthritis, and periodontal disease. It is now becoming increasingly clear that extracellular matrix degradation by MMPs is also involved in the

  4. Collagenolytic fragments and active gelatinase complexes in periodontitis.

    NARCIS (Netherlands)

    Bildt, M.M.; Bloemen, M.; Kuijpers-Jagtman, A.M.; Hoff, J.W. Von den

    2008-01-01

    BACKGROUND: Periodontal tissues remodel rapidly, which enables quick adaptation to mechanical changes. Matrix metalloproteinases (MMPs) are involved in these remodeling processes under control of tissue inhibitor of metalloproteinases (TIMPs). In periodontitis, overactivity of MMPs results in

  5. Matrix metalloproteinase 2 and 9 activity in patients with colorectal cancer liver metastasis.

    NARCIS (Netherlands)

    Waas, E.T.; Wobbes, Th.; Lomme, R.M.L.M.; Groot, J.H. de; Ruers, T.J.M.; Hendriks, T.

    2003-01-01

    BACKGROUND: Matrix metalloproteinases (MMPs) have been reported to play an important role in tumour cell invasion and metastasis. The bioactivity of MMPs in liver metastasis from colorectal cancer was investigated and correlated with clinicopathological variables. METHOD: Thirty-two patients

  6. Increased expression of matrix metalloproteinases in the murine zymosan-induced multiple organ dysfunction syndrome.

    NARCIS (Netherlands)

    Volman, T.J.H.; Goris, R.J.A.; Lomme, R.M.L.M.; Groot, J. de; Verhofstad, A.A.J.; Hendriks, T.

    2004-01-01

    Matrix metalloproteinases (MMPs) have been implicated as mediators of tissue damage in several inflammatory diseases. Since the multiple organ dysfunction syndrome (MODS) is thought to result from systemic inflammation, overactivation of MMPs could contribute to the organ damage observed. The

  7. Comprehensive profiling and localisation of the matrix metalloproteinases in urothelial carcinoma

    OpenAIRE

    Wallard, M J; Pennington, C J; Veerakumarasivam, A; Burtt, G; Mills, I G; Warren, A; Leung, H Y; Murphy, G; Edwards, D R; Neal, D E; Kelly, J D

    2006-01-01

    The matrix metalloproteinases (MMPs) are endopeptidases which break down the extracellular matrix and regulate cytokine and growth factor activity. Several MMPs have been implicated in the promotion of invasion and metastasis in a broad range of tumours including urothelial carcinoma. In this study, RNA from 132 normal bladder and urothelial carcinoma specimens was profiled for each of the 24 human MMPs, the four endogenous tissue inhibitors of MMPs (TIMPs) and several key growth factors and ...

  8. Localization of mRNAs coding for mitochondrial proteins in the yeast Saccharomyces cerevisiae

    OpenAIRE

    Gadir, Noga; Haim-Vilmovsky, Liora; Kraut-Cohen, Judith; Gerst, Jeffrey E.

    2011-01-01

    Targeted mRNA localization is a likely determinant of localized protein synthesis. To investigate whether mRNAs encoding mitochondrial proteins (mMPs) localize to mitochondria and, thus, might confer localized protein synthesis and import, we visualized endogenously expressed mMPs in vivo for the first time. We determined the localization of 24 yeast mMPs encoding proteins of the mitochondrial matrix, outer and inner membrane, and intermembrane space and found that many mMPs colocalize with m...

  9. Urinary high molecular weight matrix metalloproteinases as non-invasive biomarker for detection of bladder cancer

    OpenAIRE

    Mohammed, Mohammed A; Seleim, Manar F; Abdalla, Mohga S; Sharada, Hayat M; Abdel Wahab, Abdel Hady A

    2013-01-01

    Background Matrix Metalloproteinases (MMPs) are key molecules for tumor growth, invasion and metastasis. Over-expression of different MMPs in tumor tissues can disturb the homeostasis and increase the level of various body fluids. Many MMPs including high molecular weights (HMWs) were detected in the urine of prostate and bladder cancer patients. Our aim here is to assess the usefulness of HMW MMPs as non invasive biomarkers in bilharzial bladder cancer in Egyptian patients. Methods The activ...

  10. Downregulation of membrane type-matrix metalloproteinases in the inflamed or injured central nervous system

    DEFF Research Database (Denmark)

    Toft-Hansen, Henrik; Babcock, Alicia A; Millward, Jason M

    2007-01-01

    BACKGROUND: Matrix metalloproteinases (MMPs) are thought to mediate cellular infiltration in central nervous system (CNS) inflammation by cleaving extracellular matrix proteins associated with the blood-brain barrier. The family of MMPs includes 23 proteinases, including six membrane type-MMPs (MT...

  11. An Examination of Preservice Teachers' Capacity to Create Mathematical Modeling Problems for Children

    Science.gov (United States)

    Paolucci, Catherine; Wessels, Helena

    2017-01-01

    This study examined preservice teachers' (PSTs) capacity to create mathematical modeling problems (MMPs) for grades 1 to 3. PSTs created MMPs for their choice of grade level and aligned the mathematical content of their MMPs with the relevant mathematics curriculum. PSTs were given criteria adapted from Galbraith's MMP design principles to guide…

  12. Estrogen and progesterone receptors have distinct roles in the establishment of the hyperplastic phenotype in PR-A transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Simian, Marina; Bissell, Mina J.; Barcellos-Hoff, Mary Helen; Shyamala, Gopalan

    2009-05-11

    Expression of the A and B forms of progesterone receptor (PR) in an appropriate ratio is critical for mammary development. Mammary glands of PR-A transgenic mice, carrying an additional A form of PR as a transgene, exhibit morphological features associated with the development of mammary tumors. Our objective was to determine the roles of estrogen (E) and progesterone (P) in the genesis of mammary hyperplasias/preneoplasias in PR-A transgenics. We subjected PR-A mice to hormonal treatments and analyzed mammary glands for the presence of hyperplasias and used BrdU incorporation to measure proliferation. Quantitative image analysis was carried out to compare levels of latency-associated peptide and transforming growth factor beta 1 (TGF{beta}1) between PR-A and PR-B transgenics. Basement membrane disruption was examined by immunofluorescence and proteolytic activity by zymography. The hyperplastic phenotype of PR-A transgenics is inhibited by ovariectomy, and is reversed by treatment with E + P. Studies using the antiestrogen ICI 182,780 or antiprogestins RU486 or ZK 98,299 show that the increase in proliferation requires signaling through E/estrogen receptor alpha but is not sufficient to give rise to hyperplasias, whereas signaling through P/PR has little impact on proliferation but is essential for the manifestation of hyperplasias. Increased proliferation is correlated with decreased TGF{beta}1 activation in the PR-A transgenics. Analysis of basement membrane integrity showed loss of laminin-5, collagen III and collagen IV in mammary glands of PR-A mice, which is restored by ovariectomy. Examination of matrix metalloproteases (MMPs) showed that total levels of MMP-2 correlate with the steady-state levels of PR, and that areas of laminin-5 loss coincide with those of activation of MMP-2 in PR-A transgenics. Activation of MMP-2 is dependent on treatment with E and P in ovariectomized wild-type mice, but is achieved only by treatment with P in PR-A mice. These data

  13. T117

    Directory of Open Access Journals (Sweden)

    A. Alexandrova

    2015-11-01

    Full Text Available During cancer development, tumor cells gain the ability to invade and metastasize. Individual cells use alternative migration modes based on different cellular mechanisms. One of them is mesenchymal motility mode which is driven by leading edge protrusion in the form of filopodia or/and lamellipodia based on Arp2/3 dependent actin polymerization. Mesenchymal motility depends on formation of cell-substrate adhesions, activity of matrix metalloproteases (MMPs and on activity of small GTPaseRac. Another mode is amoeboid motility, which involves formation of blebs – hollow membrane protrusions extruded from the cell surface by actin-myosin contraction. Amoeboid motility does not need both pronounced cell-substrate adhesions and MMPs activity and required increase of activity of small GTPase Rho. Fibroblasts and scattered epithelial cells migrate by mesenchymal mode, while blood cells – lymphocytes or macrophages mainly use amoeboid mode for migration. It was shown that some treatments cause transition from one motility mode to another. Switches from mesenchymal to amoeboid motility and opposite are called mesenchymal-amoeboid transition (MAT and amoeboid -mesenchymal transition (AMT respectively. The ability of cells for such transitions was named as plasticity of migration. We compared the plasticity of migration of normal and tumor cells. To study plasticity of mesenchymally migrated cells (MAT we choose fibrosarcoma cells HT1080 as tumor and non-transformed subcutaneous fibroblasts 1036 as normal counterpart. To study AMT we choose a few lines of myeloid leukemia THP1, K562, KG1 in contrast to normal leukocytes obtained from healthy donors. We showed that fibrosarcoma cells in opposite to non-transformed fibroblasts could undergo MAT under treatments, which limited mesenchymal migration. Two approaches to limit mesenchymal motility of cells were used. One was decrease of substrate adhesiveness by treatment of coverslips with Poly

  14. Acquired thrombotic thrombocytopenic purpura. Development of an autoimmune response

    NARCIS (Netherlands)

    Schaller, M.; Studt, J.-D.; Voorberg, J.; Kremer Hovinga, J. A.

    2013-01-01

    The von Willebrand factor (VWF)-cleaving metalloprotease, ADAMTS13 (adisintegrin and metalloprotease with thrombospondin type 1 motifs-13) is the only known target of the dysregulated immune response in acquired TTP. Autoantibodies to ADAMTS13 either neutralize its activity or accelerate its

  15. Analysis of Enzymatic Activity of Matrix Metalloproteinase (MMP) by Collagen Zymography in Melanoma.

    Science.gov (United States)

    Walia, Vijay; Samuels, Yardena

    2018-01-01

    Protein zymography is the most commonly used technique to study the enzymatic activity of matrix metalloproteinases (MMPs) and their inhibitors. MMPs are proteolytic enzymes that promote extracellular matrix degradation. MMPs are frequently mutated in malignant melanomas as well as other cancers and are linked to increasing incidence of tumor metastasis. Substrate zymography characterizes MMP activity by their ability to degrade preferred substrates. Here we describe the collagen zymography technique to measure the active or latent form of MMPs using MMP-8 as an example, which is a frequently mutated MMP family member in malignant melanomas. The same technique can be used with the modification of substrate to detect metalloproteinase activity of other MMPs. Both wild-type and mutated forms of MMPs can be analyzed using a single gel using this method.

  16. Beneficial Regulation of Matrix Metalloproteinases for Skin Health

    Directory of Open Access Journals (Sweden)

    Neena Philips

    2011-01-01

    Full Text Available Matrix metalloproteinases (MMPs are essential to the remodeling of the extracellular matrix. While their upregulation facilitates aging and cancer, they are essential to epidermal differentiation and the prevention of wound scars. The pharmaceutical industry is active in identifying products that inhibit MMPs to prevent or treat aging and cancer and products that stimulate MMPs to prevent epidermal hyperproliferative diseases and wound scars.

  17. Periodontal Disease, Matrix Metalloproteinases and Chemically Modified Tetracyclines

    OpenAIRE

    Steinsvoll, Svein

    2011-01-01

    Matrix metalloproteinases (MMPs) are crucial in the degradation of the main components in the extracellular matrix and thereby play important roles in cell migration, wound healing and tissue remodelling. MMPs have pathogenic roles in arthritis, periodontitis, hepatitis, glomerulonephritis, atherosclerosis and cancer cell invasion. MMPs are activators of pro-inflammatory mediators that occur in latent forms, such as interleukin (IL)-1β, membrane-bound tumour necrosis factor (TNF) and dif...

  18. EMMPRIN-Mediated Induction of Uterine and Vascular Matrix Metalloproteinases during Pregnancy and in Response to Estrogen and Progesterone

    OpenAIRE

    Dang, Yiping; Li, Wei; Tran, Victoria; Khalil, Raouf A.

    2013-01-01

    Pregnancy is associated with uteroplacental and vascular remodeling in order to adapt for the growing fetus and the hemodynamic changes in the maternal circulation. We have previously shown upregulation of uterine matrix metalloproteinases (MMPs) during pregnancy. Whether pregnancy-associated changes in MMPs are localized to the uterus or are generalized in feto-placental and maternal circulation is unclear. Also, the mechanisms causing the changes in uteroplacental and vascular MMPs during p...

  19. Matrix Metalloproteinases as Therapeutic Targets for Idiopathic Pulmonary Fibrosis

    OpenAIRE

    Craig, Vanessa J.; Zhang, Li; Hagood, James S.; Owen, Caroline A.

    2015-01-01

    Idiopathic pulmonary fibrosis (IPF) is a restrictive lung disease that is associated with high morbidity and mortality. Current medical therapies are not fully effective at limiting mortality in patients with IPF, and new therapies are urgently needed. Matrix metalloproteinases (MMPs) are proteinases that, together, can degrade all components of the extracellular matrix and numerous nonmatrix proteins. MMPs and their inhibitors, tissue inhibitors of MMPs (TIMPs), have been implicated in the p...

  20. Matrix metalloproteinases in stem cell regulation and cancer

    OpenAIRE

    Kessenbrock, K; Wang, CY; Wang, CY; Werb, Z

    2014-01-01

    © 2015. Since Gross and Lapiere firstly discovered matrix metalloproteinases (MMPs) as important collagenolytic enzymes during amphibian tadpole morphogenesis in 1962, this intriguing family of extracellular proteinases has been implicated in various processes of developmental biology. However, the pathogenic roles of MMPs in human diseases such as cancer have also garnered widespread attention. The most straightforward explanation for their role in cancer is that MMPs, through extracellular ...

  1. MMP-1 polymorphism and its relationship to pathological processes

    Indian Academy of Sciences (India)

    Matrix metalloproteinases (MMPs) are a family of zinc (Zn)-dependent endopeptidases that are collectively capable of cleaving virtually all extracellular matrix (ECM) substrates and play an important role in diverse physiological and pathological processes. The activity of MMPs is regulated at multiple levels.

  2. Expression and response to angiotensin-converting enzyme inhibition of matrix metalloproteinases 2 and 9 in renal glomerular damage in young transgenic rats with renin-dependent hypertension

    NARCIS (Netherlands)

    Bolbrinker, J; Markovic, S; Wehland, M; Melenhorst, WBWH; van Goor, H; Kreutz, R

    Extracellular matrix expansion in the glomerular mesangium contributes to the development of glomerulosclerosis and chronic renal disease in arterial hypertension. Transforming growth factor-beta 1 (TGF-beta 1), matrix metalloproteinases (MMPs), and tissue inhibitors of MMPs (TIMPs) are involved in

  3. The possible role of matrix metalloproteinase (MMP)-2 and MMP-9 in cancer, e.g. acute leukemia

    NARCIS (Netherlands)

    Klein, G.; Vellenga, E.; Fraaije, M.W.; Kamps, W.A.; Bont, E.S.J.M. de

    2004-01-01

    In the past decades, a lot of effort has been put in identifying the role of matrix metalloproteinases (MMPs) in cancer. The main role of MMPs in angiogenesis, tumor growth and metastasis is degradation of extracellular matrix (ECM) and release and/or activation of growth factors through their

  4. Matrix metalloproteinases in premature coronary atherosclerosis: influence of inhibitors, inflammation, and genetic polymorphisms

    NARCIS (Netherlands)

    Nanni, S.; Melandri, G.; Hanemaaijer, R.; Cervi, V.; Tomasi, L.; Altimari, A.; Lent, N. van; Tricoci, P.; Bacchi, L.; Branzi, A.

    2007-01-01

    Matrix metalloproteinases (MMPs) are thought to participate in the pathogenesis of coronary artery disease (CAD), particularly in the occurrence of acute coronary syndrome (ACS). Little is known about human in vivo MMP regulation in CAD. The expression and regulation of MMPs and their tissue

  5. A study on the expression levels of matrix metalloproteinases and ...

    African Journals Online (AJOL)

    Purpose: To investigate the expression matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in ulcerative colitis (UC) patients admitted to The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China. Methods: The expression of MMPs and TIMPs) was evaluated in 50 ...

  6. Downregulation of membrane type-matrix metalloproteinases in the inflamed or injured central nervous system

    Directory of Open Access Journals (Sweden)

    Millward Jason M

    2007-09-01

    Full Text Available Abstract Background Matrix metalloproteinases (MMPs are thought to mediate cellular infiltration in central nervous system (CNS inflammation by cleaving extracellular matrix proteins associated with the blood-brain barrier. The family of MMPs includes 23 proteinases, including six membrane type-MMPs (MT-MMPs. Leukocyte infiltration is an integral part of the pathogenesis of autoimmune inflammation in the CNS, as occurs in multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE, as well as in the response to brain trauma and injury. We have previously shown that gene expression of the majority of MMPs was upregulated in the spinal cord of SJL mice with severe EAE induced by adoptive transfer of myelin basic protein-reactive T cells, whereas four of the six MT-MMPs (MMP-15, 16, 17 and 24 were downregulated. The two remaining MT-MMPs (MMP-14 and 25 were upregulated in whole tissue. Methods We used in vivo models of CNS inflammation and injury to study expression of MT-MMP and cytokine mRNA by real-time RT-PCR. Expression was also assessed in microglia sorted from CNS by flow cytometry, and in primary microglia cultures following treatment with IFNγ. Results We now confirm the expression pattern of MT-MMPs in the B6 mouse, independent of effects of adjuvant. We further show expression of all the MT-MMPs, except MMP-24, in microglia. Microglia isolated from mice with severe EAE showed statistically significant downregulation of MMP-15, 17 and 25 and lack of increase in levels of other MT-MMPs. Downregulation of MT-MMPs was also apparent following CNS injury. The pattern of regulation of MT-MMPs in neuroinflammation showed no association with expression of the proinflammatory cytokines TNFα, IL-1β, or IFNγ. Conclusion CNS inflammation and injury leads to downregulation in expression of the majority of MT-MMPs. Microglia in EAE showed a general downregulation of MT-MMPs, and our findings suggest that MT-MMP levels may

  7. A new generation of topical chronic wound treatments containing specific MMP inhibitors

    Directory of Open Access Journals (Sweden)

    Shrivastava R

    2014-09-01

    Full Text Available Ravi Shrivastava, Nathalie Cucuat, Monika Rousse, Thomas Weigand, Pedro Neto, Claire Janicot, Christiane Shrivastava VITROBIO Research Institute, Issoire, France Purpose: Incidence of chronic wounds is constantly rising worldwide, but all currently available treatments are intended either to provide symptomatic relief or to assist cicatrization to some extent, but not to directly stimulate cellular growth. Physiologically, chronic wound healing simply requires cell growth to fill the injured cavity. To grow, our cells need to attach onto a cushion, called extracellular matrix (ECM, secreted by the mother cells and composed of multiple proteins. Recent scientific works prove that the concentration of certain matrix metalloproteinases (MMPs is extremely high in all chronic wounds and, because of their proteolytic nature, some MMPs completely degrade the ECM, hindering cell attachment and cell growth. The aim of this study was to identify, neutralize, and eliminate these MMPs from the wound surface so as to design an effective treatment for chronic wounds. Methods: Acute and chronic models of human epithelial and fibroblast cells were prepared on a defined ECM cushion in vitro and MMPs were added in the culture medium to identify the MMPs causing ECM disintegration for each cell type. ECM-degrading MMPs were then incubated with selected procyanidin-rich plant extracts (PCDs and cell growth was reanalyzed. Results: It was observed that: 1 multiple MMPs are involved in cellular matrix destruction; 2 ECM-destroying MMPs are specific with respect to cell type; and 3 specific PCDs may bind and neutralize selected MMPs. Conclusion: Topical application of specific plant PCDs to selectively neutralize ECM-destroying MMPs in acute and chronic wounds represents a novel approach for the treatment of superficial and deep skin wounds. Keywords: extra cellular matrix (ECM, matrix metalloproteinases, procyanidins (PCDs, tannins, ulcers

  8. Analysis and protease-catalysed synthesis of sucrose alkanoate regioisomers

    DEFF Research Database (Denmark)

    Lie, Aleksander

    2014-01-01

    laurate in DMF using serine proteases and a metalloprotease. A broad range of elution strategies for the chromatographic analysis of sucrose alkanoate regioisomers was systematically investigated using design of experiments strategies and statistical and multivariate analysis and modelling. Efficiency...

  9. New Approaches towards the Elucidation of Epidermal-Dermal Separation in Sulfur Mustard-Exposed Human Skin and Directions for Therapy

    National Research Council Canada - National Science Library

    Mol, Marijke

    2005-01-01

    .... Results of experiments with a human skin ex vivo model show that apoptosis and metalloprotease activity are key elements in HD-induced skin pathogenesis, and that intervention in these two processes...

  10. AcEST: DK953938 [AcEST

    Lifescience Database Archive (English)

    Full Text Available on tr|A3TZ18|A3TZ18_9RHOB ATP-dependent metalloprotease FtsH OS=Oceanicola batsensis HTCC2597 Align length 7...0E06711p OS=Kluyveromyces lactis GN=K... 33 9.8 >tr|A3TZ18|A3TZ18_9RHOB ATP-dependent metalloprotease FtsH OS=Oceanicola bats

  11. AcEST: BP919546 [AcEST

    Lifescience Database Archive (English)

    Full Text Available Definition tr|A3TZ18|A3TZ18_9RHOB ATP-dependent metalloprotease FtsH OS=Oceanicola batsensis HTCC2597 Align ...r... 33 9.2 >tr|A3TZ18|A3TZ18_9RHOB ATP-dependent metalloprotease FtsH OS=Oceanicola batsensis HTCC2597 GN=O

  12. The interplay of matrix metalloproteinases, morphogens and growth factors is necessary for branching of mammary epithelial cells

    International Nuclear Information System (INIS)

    Simian, Marina; Hirai, Yohei; Navre, Marc; Werb, Zena; Lochter, Andre; Bissell, Mina J.

    2002-01-01

    The mammary gland develops its adult form by a process referred to as branching morphogenesis. Many factors have been reported to affect this process. We have used cultured primary mammary epithelial organoids and mammary epithelial cell lines in three-dimensional collagen gels to elucidate which growth factors, matrix metalloproteinases (MMPs) and mammary morphogens interact in branching morphogenesis. Branching stimulated by stromal fibroblasts, epidermal growth factor, fibroblast growth factor 7, fibroblast growth factor 2 and hepatocyte growth factor was strongly reduced by inhibitors of MMPs, indicating the requirement of MMPs for three-dimensional growth involved in morphogenesis. Recombinant stromelysin 1/MMP-3 alone was sufficient to drive branching in the absence of growth factors in the organoids. Plasmin also stimulated branching; however, plasmin-dependent branching was abolished by both inhibitors of plasmin and MMPs, suggesting that plasmin activates MMPs. To differentiate between signals for proliferation and morphogenesis, we used a cloned mammary epithelial cell line that lacks epimorphin, an essential mammary morphogen. Both epimorphin and MMPs were required for morphogenesis, but neither was required for epithelial cell proliferation. These results provide direct evidence for a critical role of MMPs in branching in mammary epithelium and suggest that, in addition to epimorphin, MMP activity is a minimum requirement for branching morphogenesis in the mammary gland

  13. The interplay of matrix metalloproteinases, morphogens and growth factors is necessary for branching of mammary epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Simian, M.; Harail, Y.; Navre, M.; Werb, Z.; Lochter, A.; Bissell, M.J.

    2002-03-06

    The mammary gland develops its adult form by a process referred to as branching morphogenesis. Many factors have been reported to affect this process. We have used cultured primary mammary epithelial organoids and mammary epithelial cell lines in three-dimensional collagen gels to elucidate which growth factors, matrix metalloproteinases (MMPs) and mammary morphogens interact in branching morphogenesis. Branching stimulated by stromal fibroblasts, epidermal growth factor, fibroblast growth factor 7, fibroblast growth factor 2 and hepatocyte growth factor was strongly reduced by inhibitors of MMPs, indicating the requirement of MMPs for three-dimensional growth involved in morphogenesis. Recombinant stromelysin 1/MMP-3 alone was sufficient to drive branching in the absence of growth factors in the organoids. Plasmin also stimulated branching; however, plasmin-dependent branching was abolished by both inhibitors of plasmin and MMPs, suggesting that plasmin activates MMPs. To differentiate between signals for proliferation and morphogenesis, we used a cloned mammary epithelial cell line that lacks epimorphin, an essential mammary morphogen. Both epimorphin and MMPs were required for morphogenesis, but neither was required for epithelial cell proliferation. These results provide direct evidence for a critical role of MMPs in branching in mammary epithelium and suggest that, in addition to epimorphin, MMP activity is a minimum requirement for branching morphogenesis in the mammary gland.

  14. Matrix metalloproteinase activity assays: Importance of zymography.

    Science.gov (United States)

    Kupai, K; Szucs, G; Cseh, S; Hajdu, I; Csonka, C; Csont, T; Ferdinandy, P

    2010-01-01

    Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases capable of degrading extracellular matrix, including the basement membrane. MMPs are associated with various physiological processes such as morphogenesis, angiogenesis, and tissue repair. Moreover, due to the novel non-matrix related intra- and extracellular targets of MMPs, dysregulation of MMP activity has been implicated in a number of acute and chronic pathological processes, such as arthritis, acute myocardial infarction, chronic heart failure, chronic obstructive pulmonary disease, inflammation, and cancer metastasis. MMPs are considered as viable drug targets in the therapy of the above diseases. For the development of selective MMP inhibitor molecules, reliable methods are necessary for target validation and lead development. Here, we discuss the major methods used for MMP assays, focusing on substrate zymography. We highlight some problems frequently encountered during sample preparations, electrophoresis, and data analysis of zymograms. Zymography is a widely used technique to study extracellular matrix-degrading enzymes, such as MMPs, from tissue extracts, cell cultures, serum or urine. This simple and sensitive technique identifies MMPs by the degradation of their substrate and by their molecular weight and therefore helps to understand the widespread role of MMPs in different pathologies and cellular pathways. Copyright 2010 Elsevier Inc. All rights reserved.

  15. Differential inhibitory effect of fondaparinux on the procoagulant potential of intact monocytes and monocyte-derived microparticles.

    Science.gov (United States)

    Ben-Hadj-Khalifa-Kechiche, Sonia; Hezard, Nathalie; Poitevin, Stephane; Remy, Marie-Geneviève; Florent, Bernadette; Mahjoub, Touhami; Nguyen, Philippe

    2010-11-01

    Monocytes and monocyte-derived microparticles (MMPs) play a major role in acute coronary syndrome (ASC). Activated monocytes (ac-M) and MMPs support thrombin generation via tissue factor (TF). The aim of this study was to evaluate the inhibitory effect of fondaparinux, a selective Xa inhibitor, on thrombin generation supported by activated monocytes and MMPs. Monocytes were purified by elutriation. They were activated by LPS, allowing to obtain both ac-M and MMPs. Thrombin generation was performed using Fluoroscan(®) in these two cell models, in comparison with a cell-free model (TF 5 pM final). Two concentrations of ac-M (0.2 × 10⁶ and 1 × 10⁶/well) and four concentrations of MMPs (40,000; 80,000; 120,000 and 160,000/well) were tested. TGT was evaluated for increasing fondaparinux concentrations (0, 0.1, 0.4, 0.7 and 1.2 μg/ml). Without fondaparinux, 0.2 × 10⁶ ac-M and 160,000 MMPs induced comparable results. Fondaparinux inhibited thrombin generation in the three models. Inhibition was fondaparinux concentration dependent. Rate index was the most sensitive parameter, compared to lag-time, peak and endogenous thrombin potential. The rate index IC(50) were 0.69 ± 0.03 μg/ml for ac-M, 0.20 ± 0.03 μg/ml for MMPs, and 0.22 ± 0.02 μg/ml for cell-free model. Fondaparinux exerted an inhibitory effect at all concentrations, including the lowest (0.1 μg/ml). The extend of inhibition was similar between MMPs and cell-free models, and stronger than ac-M model. We assume that the efficacy of fondaparinux 2.5 mg once daily in ACS patients may be in part attributed to its inhibitory effect on MMPs.

  16. Matrix metalloproteinases as therapeutic targets for idiopathic pulmonary fibrosis.

    Science.gov (United States)

    Craig, Vanessa J; Zhang, Li; Hagood, James S; Owen, Caroline A

    2015-11-01

    Idiopathic pulmonary fibrosis (IPF) is a restrictive lung disease that is associated with high morbidity and mortality. Current medical therapies are not fully effective at limiting mortality in patients with IPF, and new therapies are urgently needed. Matrix metalloproteinases (MMPs) are proteinases that, together, can degrade all components of the extracellular matrix and numerous nonmatrix proteins. MMPs and their inhibitors, tissue inhibitors of MMPs (TIMPs), have been implicated in the pathogenesis of IPF based upon the results of clinical studies reporting elevated levels of MMPs (including MMP-1, MMP-7, MMP-8, and MMP-9) in IPF blood and/or lung samples. Surprisingly, studies of gene-targeted mice in murine models of pulmonary fibrosis (PF) have demonstrated that most MMPs promote (rather than inhibit) the development of PF and have identified diverse mechanisms involved. These mechanisms include MMPs: (1) promoting epithelial-to-mesenchymal transition (MMP-3 and MMP-7); (2) increasing lung levels or activity of profibrotic mediators or reducing lung levels of antifibrotic mediators (MMP-3, MMP-7, and MMP-8); (3) promoting abnormal epithelial cell migration and other aberrant repair processes (MMP-3 and MMP-9); (4) inducing the switching of lung macrophage phenotypes from M1 to M2 types (MMP-10 and MMP-28); and (5) promoting fibrocyte migration (MMP-8). Two MMPs, MMP-13 and MMP-19, have antifibrotic activities in murine models of PF, and two MMPs, MMP-1 and MMP-10, have the potential to limit fibrotic responses to injury. Herein, we review what is known about the contributions of MMPs and TIMPs to the pathogenesis of IPF and discuss their potential as therapeutic targets for IPF.

  17. Magnetically-responsive, multifunctional drug delivery nanoparticles for elastic matrix regenerative repair.

    Science.gov (United States)

    Sivaraman, Balakrishnan; Swaminathan, Ganesh; Moore, Lee; Fox, Jonathan; Seshadri, Dhruv; Dahal, Shataakshi; Stoilov, Ivan; Zborowski, Maciej; Mecham, Robert; Ramamurthi, Anand

    2017-04-01

    Arresting or regressing growth of abdominal aortic aneurysms (AAAs), localized expansions of the abdominal aorta are contingent on inhibiting chronically overexpressed matrix metalloproteases (MMPs)-2 and -9 that disrupt elastic matrix within the aortic wall, concurrent with providing a stimulus to augmenting inherently poor auto-regeneration of these matrix structures. In a recent study we demonstrated that localized, controlled and sustained delivery of doxycycline (DOX; a tetracycline-based antibiotic) from poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs), enhances elastic matrix deposition and MMP-inhibition at a fraction of the therapeutically effective oral dose. The surface functionalization of these NPs with cationic amphiphiles, which enhances their arterial uptake, was also shown to have pro-matrix regenerative and anti-MMP effects independent of the DOX. Based on the hypothesis that the incorporation of superparamagnetic iron oxide NPs (SPIONs) within these PLGA NPs would enhance their targetability to the AAA site under an applied external magnetic field, we sought to evaluate the functional effects of NPs co-encapsulating DOX and SPIONs (DOX-SPION NPs) on elastic matrix regeneration and MMP synthesis/activity in vitro within aneurysmal smooth muscle cell (EaRASMC) cultures. The DOX-SPION NPs were mobile under an applied external magnetic field, while enhancing elastic matrix deposition 1.5-2-fold and significantly inhibiting MMP-2 synthesis and MMP-2 and -9 activities, compared to NP-untreated control cultures. These results illustrate that the multifunctional benefits of NPs are maintained following SPION co-incorporation. Additionally, preliminary studies carried out demonstrated enhanced targetability of SPION-loaded NPs within proteolytically-disrupted porcine carotid arteries ex vivo, under the influence of an applied external magnetic field. Thus, this dual-agent loaded NP system proffers a potential non-surgical option for treating small

  18. Accumbens nNOS Interneurons Regulate Cocaine Relapse.

    Science.gov (United States)

    Smith, Alexander C W; Scofield, Michael D; Heinsbroek, Jasper A; Gipson, Cassandra D; Neuhofer, Daniela; Roberts-Wolfe, Doug J; Spencer, Sade; Garcia-Keller, Constanza; Stankeviciute, Neringa M; Smith, Rachel J; Allen, Nicholas P; Lorang, Melissa R; Griffin, William C; Boger, Heather A; Kalivas, Peter W

    2017-01-25

    Relapse to drug use can be initiated by drug-associated cues. The intensity of cue-induced relapse is correlated with the induction of transient synaptic potentiation (t-SP) at glutamatergic synapses on medium spiny neurons (MSNs) in the nucleus accumbens core (NAcore) and requires spillover of glutamate from prefrontal cortical afferents. We used a rodent self-administration/reinstatement model of relapse to show that cue-induced t-SP and reinstated cocaine seeking result from glutamate spillover, initiating a metabotropic glutamate receptor 5 (mGluR5)-dependent increase in nitric oxide (NO) production. Pharmacological stimulation of mGluR5 in NAcore recapitulated cue-induced reinstatement in the absence of drug-associated cues. Using NO-sensitive electrodes, mGluR5 activation by glutamate was shown to stimulate NO production that depended on activation of neuronal nitric oxide synthase (nNOS). nNOS is expressed in ∼1% of NAcore neurons. Using a transgene strategy to express and stimulate designer receptors that mimicked mGluR5 signaling through Gq in nNOS interneurons, we recapitulated cue-induced reinstatement in the absence of cues. Conversely, using a transgenic caspase strategy, the intensity of cue-induced reinstatement was correlated with the extent of selective elimination of nNOS interneurons. The induction of t-SP during cued reinstatement depends on activating matrix metalloproteinases (MMPs) and selective chemogenetic stimulation of nNOS interneurons recapitulated MMP activation and t-SP induction (increase in AMPA currents in MSNs). These data demonstrate critical involvement of a sparse population of nNOS-expressing interneurons in cue-induced cocaine seeking, revealing a bottleneck in brain processing of drug-associated cues where therapeutic interventions could be effective in treating drug addiction. Relapse to cocaine use in a rat model is associated with transient increases in synaptic strength at prefrontal cortex synapses in the nucleus

  19. Novel serological neo-epitope markers of extracellular matrix proteins for the detection of portal hypertension

    DEFF Research Database (Denmark)

    Leeming, Diana Julie; Karsdal, M A; Byrjalsen, I

    2013-01-01

    The hepatic venous pressure gradient (HVPG) is an invasive, but important diagnostic and prognostic marker in cirrhosis with portal hypertension (PHT). During cirrhosis, remodelling of fibrotic tissue by matrix metalloproteinases (MMPs) is a permanent process generating small fragments of degrade...

  20. Distribution of Matrix Metalloproteinases in Human Atherosclerotic Carotid Plaques and Their Production by Smooth Muscle Cells and Macrophage Subsets

    NARCIS (Netherlands)

    Jager, Nynke A.; de Vries, Bastiaan M. Wallis; Hillebrands, Jan-Luuk; Harlaar, Niels J.; Tio, Rene A.; Slart, Riemer H. J. A.; van Dam, Gooitzen M.; Boersma, Hendrikus H.; Zeebregts, Clark J.; Westra, Johanna

    In this study, the potential of matrix metalloproteinase (MMP) sense for detection of atherosclerotic plaque instability was explored. Secondly, expression of MMPs by macrophage subtypes and smooth muscle cells (SMCs) was investigated. Twenty-three consecutive plaques removed during carotid

  1. Genetic variations in matrix metalloproteinases may be associated with increased risk of ulcerative colitis

    NARCIS (Netherlands)

    Morgan, Angharad R.; Han, Dug-Yeo; Lam, Wen-Jiun; Triggs, Christopher M.; Fraser, Alan G.; Barclay, Murray; Gearry, Richard B.; Meisner, Sander; Stokkers, Pieter; Boeckxstaens, Guy E.; Ferguson, Lynnette R.

    2011-01-01

    Increased production of matrix metalloproteinases (MMPs) plays an important role in tissue damage in inflammatory bowel disease (IBD). Genetically encoded variation between individuals in MMP production may therefore contribute to disease onset, type, or severity. We undertook an extensive candidate

  2. Collagenase matrix metalloproteinase-8 expressed in atherosclerotic carotid plaques is associated with systemic cardiovascular outcome

    NARCIS (Netherlands)

    Peeters, W.; Moll, F.L.; Vink, A.; Spek, P.J. van der; Kleijn, D.P.V. de; Vries, J.-P.P.M. de; Verheijen, J.H.; Newby, A.C.; Pasterkamp, G.

    2011-01-01

    Aims Atherosclerotic plaque rupture and subsequent thrombus formation are the major cause of acute cardiovascular events. Local plaque markers may facilitate detection of the vulnerable plaque and help identify the patient at risk for cardiovascular events. Matrix metalloproteinases (MMPs) are

  3. A MMP derived versican neo-epitope is elevated in plasma from patients with atherosclerotic heart disease

    DEFF Research Database (Denmark)

    Barascuk, Natasha; Genovese, Federica; Larsen, Lise Korsager

    2013-01-01

    Extracellular matrix remodelling is a prerequisite for plaque rupture in atherosclerotic lesion. Versican, an extracellular matrix proteoglycan present in normal and atherosclerotic arteries is a substrate for matrix metalloproteinases (MMPs) present in macrophage rich areas. The aim of the curre...

  4. Role of matrix metalloproteinases in the pathophysiology of idiopathic pulmonary fibrosis

    Directory of Open Access Journals (Sweden)

    Bhattacharyya P

    2007-01-01

    Full Text Available Idiopathic pulmonary fibrosis (IPF, a progressive fibrosing lung condition is a ther-apeutic medical challenge. The etiopathogenesis of IPF is complicated and hitherto not adequately understood. However, the concept, of late, is shifting from fibrosis as a result of inflammation to a mechanism of primarily dysregulated fibrogenesis. A class of enzymes called matrix metallo proteinases (MMPs appear important in the pathogenesis of IPF. The heightened activity of MMPs are derived out of an imbalance between them (MMPs and their tissue inhibitors (TIMPs. This leads to breakdown of interstitial matrix and triggering of certain growth factors which play an important mechanistic role in the pathogenesis of IPF. Understanding of the role of MMPs in pathogenesis of IPF may open up a new horizon of therapeutic intervention of the desease.

  5. Matrix metalloproteinase 2 genotype is associated with nonanastomotic biliary strictures after orthotopic liver transplantation

    NARCIS (Netherlands)

    Ten Hove, W. Rogier; Korkmaz, Kerem S.; den Dries, Sanna Op; de Rooij, Bert-Jan F.; van Hoek, Bart; Porte, Robert J.; van der Reijden, Johan J.; Coenraad, Minneke J.; Dubbeld, Jeroen; Hommes, Daniel W.; Verspaget, Hein W.

    Background: Nonanastomotic biliary strictures (NAS) are a serious complication after orthotopic liver transplantation (OLT). Matrix metalloproteinases (MMPs) are involved in connective tissue remodelling in chronic liver disease and complications after OLT. Aim: To evaluate the relationship between

  6. Metalloproteinase Profiling in Lung Transplant Recipients With Good Outcome and Bronchiolitis Obliterans Syndrome

    NARCIS (Netherlands)

    Heijink, Irene H.; Rozeveld, Dennie; van der Heide, Sicco; Bij, van der Wim; Bischoff, Rainer; Oosterhout, van Antoon J,; van der Toorn, Marco

    Background. Bronchiolitis obliterans syndrome (BOS), the major cause of death on lung transplantation, is characterized by bronchiolar inflammation and tissue remodeling. Matrix metalloproteinases (MMPs) have been implicated in these processes, although it is still unclear whether MMP activity and

  7. Matrix metalloproteinases (MMP) and cathepsin K contribute differently to osteoclastic activities

    DEFF Research Database (Denmark)

    Delaissé, Jean-Marie; Andersen, Thomas L; Engsig, Michael T

    2003-01-01

    The best established proteolytic event of osteoclasts is bone matrix solubilization by the cysteine proteinase cathepsin K. Here, however, we draw the attention on osteoclastic activities depending on matrix metalloproteinases (MMPs). We discuss the observations supporting that MMPs contribute...... significantly to bone matrix solubilization in specific areas of the skeleton and in some developmental and pathological situations. Our discussion takes into account (1) the characteristics of the bone remodeling persisting in the absence of cathepsin K, (2) the ultrastructure of the resorption zone...... in response to inactivation of MMPs and of cathepsin K in different bone types, (3) bone resorption levels in MMP knockout mice compared to wild-type mice, (4) the identification of MMPs in osteoclasts and surrounding cells, and (5) the effect of different bone pathologies on the serum concentrations...

  8. Matrix metalloproteinases in inflammatory bowel disease : expression, regulation and clinical relevance

    NARCIS (Netherlands)

    Meijer, Martin Jan-Willem

    2009-01-01

    Crohn’s disease (CD) is characterized by chronic, patchy, transmural inflammation of the entire gastrointestinal tract, while ulcerative colitis (UC) is manifested by chronic, continuous, superficial inflammation of the colon. Matrix metalloproteinases (MMPs) constitute a family of matrix degrading

  9. Effects of matrix metallproteinases on dentin bonding and strategies to increase durability of dentin adhesion

    Directory of Open Access Journals (Sweden)

    Jung-Hyun Lee

    2012-02-01

    Full Text Available The limited durability of resin-dentin bonds severely compromises the longevity of composite resin restorations. Resin-dentin bond degradation might occur via degradation of water-rich and resin sparse collagen matrices by host-derived matrix metalloproteinases (MMPs. This review article provides overview of current knowledge of the role of MMPs in dentin matrix degradation and four experimental strategies for extending the longevity of resin-dentin bonds. They include: (1 the use of broad-spectrum inhibitors of MMPs, (2 the use of cross-linking agents for silencing the activities of MMPs, (3 ethanol wet-bonding with hydrophobic resin, (4 biomimetic remineralization of water-filled collagen matrix. A combination of these strategies will be able to overcome the limitations in resin-dentin adhesion.

  10. Zymographic techniques for the analysis of matrix metalloproteinases and their inhibitors.

    NARCIS (Netherlands)

    Snoek, P.A.; Hoff, J.W. Von den

    2005-01-01

    The balance between matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), is largely responsible for the remodeling of tissues. Deregulation of this balance is a characteristic of extensive tissue degradation in certain degenerative diseases. To

  11. Nonselective matrix metalloproteinase but not tumor necrosis factor-a inhibition effectively preserves the early critical colon anastomotic integrity

    DEFF Research Database (Denmark)

    Ågren, Magnus S.; Andersen, Thomas L.; Andersen, Line

    2011-01-01

    Increased matrix metalloproteinase (MMP) activity has been implicated in the pathogenesis of colorectal anastomotic leakage. Tumor necrosis factor-a (TNF-a) induces MMPs and may influence anastomosis repair....

  12. Investigation of enzyme-sensitive lipid nanoparticles for delivery of siRNA to blood–brain barrier and glioma cells

    DEFF Research Database (Denmark)

    Bruun, Jonas; Larsen, Trine Bjørnbo; Jølck, Rasmus Irming

    2015-01-01

    charged PEGylated (poly(ethylene glycol)) cleavable lipopeptide, which contains a recognition sequence for matrix metalloproteinases (MMPs), a class of enzymes often expressed in the tumor microenvironment and inflammatory BBB conditions. Proteolytic cleavage induces PEG release, including the release...

  13. Angiogenesis in vestibular schwannomas

    DEFF Research Database (Denmark)

    Møller, Martin Nue; Werther, Kim; Nalla, Amarnadh

    2010-01-01

    targets the angiogenic process by investigation of tumor expression of MMP-2, MMP-9, and tissue inhibitors of metalloproteinase (TIMP)-1. A possible correlation with gender, patient age, symptom duration, tumor size, and the absolute and relative growth rate is explored.......Vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) are potent mediators of tumor angiogenesis. It has been demonstrated that vestibular schwannoma VEGF expression correlates with tumor growth pattern, whereas knowledge on the expression of MMPs is lacking. This study...

  14. Angiogenesis in vestibular schwannomas: expression of extracellular matrix factors MMP-2, MMP-9, and TIMP-1

    DEFF Research Database (Denmark)

    Møller, Martin Nue; Werther, Kim; Nalla, Amarnadh

    2010-01-01

    targets the angiogenic process by investigation of tumor expression of MMP-2, MMP-9, and tissue inhibitors of metalloproteinase (TIMP)-1. A possible correlation with gender, patient age, symptom duration, tumor size, and the absolute and relative growth rate is explored.......Vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) are potent mediators of tumor angiogenesis. It has been demonstrated that vestibular schwannoma VEGF expression correlates with tumor growth pattern, whereas knowledge on the expression of MMPs is lacking. This study...

  15. Detection of Matrix Metalloproteinases by Zymography.

    Science.gov (United States)

    Tajhya, Rajeev B; Patel, Rutvik S; Beeton, Christine

    2017-01-01

    Matrix metalloproteinases (MMPs) represent more than 20 zinc-containing endopeptidases that cleave internal peptide bonds, leading to protein degradation. They play a critical role in many physiological cell functions, including tissue remodeling, embryogenesis, and angiogenesis. They are also involved in the pathogenesis of a vast array of diseases, including but not limited to systemic inflammation, various cancers, and cardiovascular, neurological, and autoimmune diseases. Here, we describe gel zymography to detect MMPs in cell and tissue samples and in cell culture supernatants.

  16. Matrix Metalloproteinases and Neurotrauma: Evolving Roles in Injury and Reparative Processes

    OpenAIRE

    Zhang, Haoqian; Adwanikar, Hita; Werb, Zena; Noble-Haeusslein, Linda J.

    2010-01-01

    Matrix metalloproteinases (MMPs) are involved in a wide range of proteolytic events in fetal development and normal tissue remodeling as well as wound healing and inflammation. In the CNS, they have been implicated in a variety of neurodegenerative diseases ranging from multiple sclerosis to Alzheimer disease and are integral to stroke-related cell damage. Although studies implicate increased activity of MMPs in pathogenesis in the CNS, there is also a growing literature to support their part...

  17. Overexpression of cathepsin f, matrix metalloproteinases 11 and 12 in cervical cancer

    OpenAIRE

    Vazquez-Ortiz, Guelaguetza; Pina-Sanchez, Patricia; Vazquez, Karla; Duenas, Alfonso; Taja, Lucia; Mendoza, Patricia; Garcia, José A; Salcedo, Mauricio

    2005-01-01

    Abstract Background Cervical carcinoma (CC) is one of the most common cancers among women worldwide and the first cause of death among the Mexican female population. CC progression shows a continuum of neoplastic transitions until invasion. Matrix metalloproteinases (MMPs) and cathepsins play a central role on the enhancement of tumor-induced angiogenesis, cell migration, proliferation, apoptosis and connective tissue degradation. MMPs -2 and -9 expression has been widely studied in cervical ...

  18. From Sensors to Silencers: Quinoline- and Benzimidazole-Sulfonamides as Inhibitors for Zinc Proteases

    Science.gov (United States)

    2010-01-01

    Derived from the extensive work in the area of small molecule zinc(II) ion sensors, chelating fragment libraries of quinoline- and benzimidazole-sulfonamides have been prepared and screened against several different zinc(II)-dependent matrix metalloproteinases (MMPs). The fragments show impressive inhibition of these metalloenzymes and preferences for different MMPs based on the nature of the chelating group. The findings show that focused chelator libraries are a powerful strategy for the discovery of lead fragments for metalloprotein inhibition. PMID:20507095

  19. New Advances in Molecular Therapy for Muscle Repair after Diseases and Injuries

    Science.gov (United States)

    2008-04-01

    AAV serotype because the transduction efficiency of AAV vectors in different cell and tissue types varies widely, as a result of the unique tropism of...300,000 LTP cells obtained fromMSTN/micewere trans- planted in the GMs of 3 8-week-old mdx/scid mice using a protocol previously described (41). The...metalloproteinases (MMPs). MMPs are widely found in plants and animals and are intimately involved in the mainte- nance of the extracellular matrix (ECM

  20. MMP9 is protective against lethal inflammatory mass lesions in the mouse colon

    DEFF Research Database (Denmark)

    Hald, Andreas; Rønø, Birgitte; Melander, Maria C

    2011-01-01

    that concomitant ablation of MMP9 (gelatinase B) and the serine protease plasmin results in lethal inflammatory mass lesions in the colon. These lesions possessed several histological attributes that are characteristic of mucosal prolapse seen in humans, and they were found to be associated with splenomegaly......The family of matrix metalloproteinases (MMPs) is responsible for extracellular matrix degradation during physiological and pathophysiological tissue remodeling processes such as embryogenesis, tissue repair and cancer progression. Despite these important roles of MMPs, inhibition or ablation...

  1. Methadone-maintained patients in primary care have higher rates of chronic disease and multimorbidity, and use health services more intensively than matched controls.

    Science.gov (United States)

    O'Toole, John; Hambly, Róisín; Cox, Anne-Marie; O'Shea, Brendan; Darker, Catherine

    2014-12-01

    Methadone maintenance treatment in primary care is cost-effective and improves outcomes for opiate-dependent patients. A more developed understanding of the evolving needs of this important cohort will facilitate further improvements in their integrated care within the community. The aim of this study was to compare the burden of chronic disease, multi-morbidity and intensity of health-service use between methadone-maintained patients (MMPs) and matched controls in primary care. This is a retrospective matched case-control design. Data on chronic disease and health service use was collected in 13 computerized GP surgeries on 414 patients (207 MMPs and 207 controls). Twelve months of records were examined. MMPs were compared with controls matched by gender, age, socio-economic status (SES) and GP surgery. MMPs suffered more chronic disease (OR = 9.1, 95% CI: 5.4-15.1, P disease. MMPs of lower SES had more chronic disease than their peers (OR = 7.2, 95% CI: 2.4-22.0, P care generated more telephone calls (OR = 4.4, 95% CI: 2.8-6.8, P care and drug-related illnesses, MMPs had a higher burden of chronic disease and used both primary and secondary health services more intensively than matched controls.

  2. Therapeutic potential of matrix metalloproteinases in Duchenne muscular dystrophy

    Directory of Open Access Journals (Sweden)

    Yuji eOgura

    2014-04-01

    Full Text Available Matrix metalloproteinases (MMPs are secreted proteinases that have physiologic roles in degradation and remodeling of extracellular matrix (ECM in almost all tissues. However, their excessive production in disease conditions leads to many pathological features including tissue breakdown, inflammation, cell death, and fibrosis. Duchenne Muscular dystrophy (DMD is a devastating genetic muscle disorder caused by partial or complete loss of cytoskeletal protein dystrophin. Progressive muscle wasting in DMD is accompanied by myofiber necrosis followed by cycles of regeneration and degeneration and inflammation that eventually result in replacement of myofiber by connective and adipose tissues. Emerging evidence suggests that gene expression and the activity of various MMPs are aberrantly regulated in muscle biopsies from DMD patients and in skeletal muscle of animal models of DMD. Moreover, a few studies employing genetic mouse models have revealed that different MMPs play distinct roles in disease progression in DMD. Modulation of the activity of MMPs improves myofiber regeneration and enhances the efficacy of transplantation and engraftment of muscle progenitor cells in dystrophic muscle in mouse models of DMD. Furthermore, recent reports also suggest that some MMPs especially MMP-9 can serve as a biomarker for diagnosis and prognosis of DMD. In this article, we provide a succinct overview of the regulation of various MMPs and their therapeutic importance in DMD.

  3. Peptide inhibitors of botulinum neurotoxin by mRNA display

    International Nuclear Information System (INIS)

    Yiadom, Kwabena P.A.B.; Muhie, Seid; Yang, David C.H.

    2005-01-01

    Botulinum neurotoxins (BoNTs) are extremely toxic. The metalloproteases associated with the toxins cleave proteins essential for neurotransmitter secretion. Inhibitors of the metalloprotease are currently sought to control the toxicity of BoNTs. Toward that goal, we produced a synthetic cDNA for the expression and purification of the metalloprotease of BoNT/A in Escherichia coli as a biotin-ubiquitin fusion protein, and constructed a combinatorial peptide library to screen for BoNT/A light chain inhibitors using mRNA display. A protease assay was developed using immobilized intact SNAP-25 as the substrate. The new peptide inhibitors showed a 10-fold increase in affinity to BoNT/A light chain than the parent peptide. Interestingly, the sequences of the new peptide inhibitors showed abundant hydrophobic residues but few hydrophilic residues. The results suggest that mRNA display may provide a general approach in developing peptide inhibitors of BoNTs

  4. Dimerization of endogenous MT1-MMP is a regulatory step in the activation of the 72-kDa gelatinase MMP-2 on fibroblasts and fibrosarcoma cells

    DEFF Research Database (Denmark)

    Ingvarsen, Signe; Madsen, Daniel H.; Hillig, Thore

    2008-01-01

    The secreted gelatinase matrix metalloprotease-2 (MMP-2) and the membrane-anchored matrix metalloprotease MT1-MMP (MMP-14), are central players in pericellular proteolysis in extracellular matrix degradation. In addition to possessing a direct collagenolytic and gelatinolytic activity......, these enzymes take part in a cascade pathway in which MT1-MMP activates the MMP-2 proenzyme. This reaction occurs in an interplay with the matrix metalloprotease inhibitor, TIMP-2, and the proposed mechanism involves two molecules of MT1-MMP in complex with one TIMP-2 molecule. We provide positive evidence...... that proMMP-2 activation is governed by dimerization of MT1-MMP on the surface of fibroblasts and fibrosarcoma cells. Even in the absence of transfection and overexpression, dimerization of MT1-MMP markedly stimulated the formation of active MMP-2 products. The effect demonstrated here was brought about...

  5. Regulation of membrane fusion and secretory events in the sea urchin embryo

    Energy Technology Data Exchange (ETDEWEB)

    Roe, J.L.

    1990-01-01

    Membrane fusion and secretory events play a key role in fertilization and early development in the sea urchin embryo. To investigate the mechanism of membrane fusion, the effect of inhibitors of metalloendoprotease activity was studied on two model systems of cell fusion; fertilization and spiculogenesis by primary mesenchyme cells in the embryo. Both the zinc chelator, 1,10-phenanthroline, and peptide metalloprotease substrates were found to inhibit both fertilization and gamete fusion, while peptides that are not substrates of metalloproteases did not affect either process. Primary mesenchyme cells form the larval skeleton in the embryo by deposition of mineral and an organic matrix into a syncytial cavity formed by fusion of filopodia of these cells. Metalloprotease inhibitors were found to inhibit spiculogenesis both in vivo and in cultures of isolated primary mesenchyme cells, and the activity of a metalloprotease of the appropriate specificity was found in the primary mesenchyme cells. These two studies implicate the activity of a metalloprotease in a necessary step in membrane fusion. Following fertilization, exocytosis of the cortical granules results in the formation of the fertilization envelope and the hyaline layer, that surround the developing embryo. The hatching enzyme is secreted by the blastula stage sea urchin embryo, which proteolyzes the fertilization envelope surrounding the embryo, allowing the embryo to hatch. Using an assay that measures {sup 125}I-fertilization envelope degradation, the hatching enzyme was identified as a 33 kDa metalloprotease, and was purified by ion-exchange and affinity chromatography from the hatching media of Strongylocentrotus purpuratus embryos. The hatching enzyme showed a substrate preference for only a minor subset of fertilization envelope proteins.

  6. Expression pattern of matrix metalloproteinases in human gynecological cancer cell lines

    International Nuclear Information System (INIS)

    Schröpfer, Andrea; Kammerer, Ulrike; Kapp, Michaela; Dietl, Johannes; Feix, Sonja; Anacker, Jelena

    2010-01-01

    Matrix metalloproteinases (MMPs) are involved in the degradation of protein components of the extracellular matrix and thus play an important role in tumor invasion and metastasis. Their expression is related to the progression of gynecological cancers (e.g. endometrial, cervical or ovarian carcinoma). In this study we investigated the expression pattern of the 23 MMPs, currently known in humans, in different gynecological cancer cell lines. In total, cell lines from three endometrium carcinomas (Ishikawa, HEC-1-A, AN3 CA), three cervical carcinomas (HeLa, Caski, SiHa), three chorioncarcinomas (JEG, JAR, BeWo), two ovarian cancers (BG-1, OAW-42) and one teratocarcinoma (PA-1) were examined. The expression of MMPs was analyzed by RT-PCR, Western blot and gelatin zymography. We demonstrated that the cell lines examined can constitutively express a wide variety of MMPs on mRNA and protein level. While MMP-2, -11, -14 and -24 were widely expressed, no expression was seen for MMP-12, -16, -20, -25, -26, -27 in any of the cell lines. A broad range of 16 MMPs could be found in the PA1 cells and thus this cell line could be used as a positive control for general MMP experiments. While the three cervical cancer cell lines expressed 10-14 different MMPs, the median expression in endometrial and choriocarcinoma cells was 7 different enzymes. The two investigated ovarian cancer cell lines showed a distinctive difference in the number of expressed MMPs (2 vs. 10). Ishikawa, Caski, OAW-42 and BeWo cell lines could be the best choice for all future experiments on MMP regulation and their role in endometrial, cervical, ovarian or choriocarcinoma development, whereas the teratocarcinoma cell line PA1 could be used as a positive control for general MMP experiments

  7. Two-Dimensional Zymography of Proteases from Steatotic Duck Liver.

    Science.gov (United States)

    Wilkesman, Jeff; Padrón, María Fernanda; Kurz, Liliana; Rémignon, Hervé

    2017-01-01

    Protease activity present in liver cells with steatosis can be electrophoretically characterized. Zymographic techniques allow semi-quantitative results, successfully detecting cathepsin and metalloprotease activity using polyacrylamide gels copolymerized with gelatin and quantified by densitometry. By using specific inhibitors, the identity of the proteases can be confirmed. 2D zymography allows the determination of both M r. and pI of the metalloprotease and cathepsin activity present in the homogenates. The analysis of liver proteases activities in force fed ducks may elucidate the mechanisms behind steatosis development.

  8. Neutralization of MMP-2 protects Staphylococcus aureus infection induced septic arthritis in mice and regulates the levels of cytokines.

    Science.gov (United States)

    Sultana, Sahin; Adhikary, Rana; Nandi, Ajeya; Bishayi, Biswadev

    2016-10-01

    Matrix metalloproteinases (MMPs) are crucial players in Staphylococcus aureus mediated synovial tissue destruction in the pathogenesis of septic arthritis. Bacterial insult increases proteolytic matrix fragments by activated chondrocytes and synovial fibroblasts leading to induction of matrix metalloproteinases. Tissue destruction via MMPs induced by bacterial products, necrotic tissues and proinflammatory cytokines have been reported. Cytokines like TNF-α, IL-1β released from host cells in response to S. aureus infection promote cartilage degradation by stimulating the production of MMPs. Antibiotic treatment can eradicate invading bacteria but elevated levels of cytokines and cytokines induced MMPs activation lead to progressive and devastating bone and cartilage destruction even after bacterial clearance. Like other MMPs, MMP-2 also contributes to extracellular matrix degradation in different types of arthritis. Release of certain pro inflammatory cytokines can also be regulated by MMP-2 activation leading to further tissue destruction. The role of MMP-2 in the pathogenesis of S. aureus infection induced septic arthritis and its influence on cytokines regulation needs further investigation. Whether neutralization of MMP-2 provides protection against Staphylococcus aureus infection induced septic arthritis in mice is an obvious question. Here we reported that neutralization of MMP-2 during S. aureus infection induced septic arthritis might be beneficial for preventing infection induced extracellular matrix destruction thereby decreasing bacterial burden in synovial tissues and regulating inflammatory cytokines in arthritic mice. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. The potential role of metalloproteinases in neurogenesis in the gerbil hippocampus following global forebrain ischemia.

    Directory of Open Access Journals (Sweden)

    Luiza Wójcik-Stanaszek

    Full Text Available BACKGROUND: Matrix metalloproteinases (MMPs have recently been considered to be involved in the neurogenic response of adult neural stem/progenitor cells. However, there is a lack of information showing direct association between the activation of MMPs and the development of neuronal progenitor cells involving proliferation and/or further differentiation in vulnerable (Cornus Ammoni-CA1 and resistant (dentate gyrus-DG to ischemic injury areas of the brain hippocampus. PRINCIPAL FINDINGS: We showed that dynamics of MMPs activation in the dentate gyrus correlated closely with the rate of proliferation and differentiation of progenitor cells into mature neurons. In contrast, in the damaged CA1 pyramidal cells layer, despite the fact that some proliferating cells exhibited antigen specific characteristic of newborn neuronal cells, these did not attain maturity. This coincides with the low, near control-level, activity of MMPs. The above results are supported by our in vitro study showing that MMP inhibitors interfered with both the proliferation and differentiation of the human neural stem cell line derived from umbilical cord blood (HUCB-NSCs toward the neuronal lineage. CONCLUSION: Taken together, the spatial and temporal profiles of MMPs activity suggest that these proteinases could be an important component in neurogenesis-associated processes in post-ischemic brain hippocampus.

  10. Matrix Metalloproteinases and Tissue Inhibitor of Metalloproteinases in Inflammation and Fibrosis of Skeletal Muscles.

    Science.gov (United States)

    Alameddine, Hala S; Morgan, Jennifer E

    2016-11-29

    In skeletal muscles, levels and activity of Matrix MetalloProteinases (MMPs) and Tissue Inhibitors of MetalloProteinases (TIMPs) have been involved in myoblast migration, fusion and various physiological and pathological remodeling situations including neuromuscular diseases. This has opened perspectives for the use of MMPs' overexpression to improve the efficiency of cell therapy in muscular dystrophies and resolve fibrosis. Alternatively, inhibition of individual MMPs in animal models of muscular dystrophies has provided evidence of beneficial, dual or adverse effects on muscle morphology or function. We review here the role played by MMPs/TIMPs in skeletal muscle inflammation and fibrosis, two major hurdles that limit the success of cell and gene therapy. We report and analyze the consequences of genetic or pharmacological modulation of MMP levels on the inflammation of skeletal muscles and their repair in light of experimental findings. We further discuss how the interplay between MMPs/TIMPs levels, cytokines/chemokines, growth factors and permanent low-grade inflammation favor cellular and molecular modifications resulting in fibrosis.

  11. Expression of matrix metalloproteinases in Naegleria fowleri and their role in invasion of the central nervous system.

    Science.gov (United States)

    Lam, Charlton; Jamerson, Melissa; Cabral, Guy; Carlesso, Ana Maris; Marciano-Cabral, Francine

    2017-10-01

    Naegleria fowleri is a free-living amoeba found in freshwater lakes and ponds and is the causative agent of primary amoebic meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system (CNS). PAM occurs when amoebae attach to the nasal epithelium and invade the CNS, a process that involves binding to, and degradation of, extracellular matrix (ECM) components. This degradation is mediated by matrix metalloproteinases (MMPs), enzymes that have been described in other pathogenic protozoa, and that have been linked to their increased motility and invasive capability. These enzymes also are upregulated in tumorigenic cells and have been implicated in metastasis of certain tumours. In the present study, in vitro experiments linked MMPs functionally to the degradation of the ECM. Gelatin zymography demonstrated enzyme activity in N. fowleri whole cell lysates, conditioned media and media collected from invasion assays. Western immunoblotting indicated the presence of the metalloproteinases MMP-2 (gelatinase A), MMP-9 (gelatinase B) and MMP-14 [membrane type-1 matrix metalloproteinase (MT1-MMP)]. Highly virulent mouse-passaged amoebae expressed higher levels of MMPs than weakly virulent axenically grown amoebae. The functional relevance of MMPs in media was indicated through the use of the MMP inhibitor, 1,10-phenanthroline. The collective in vitro results suggest that MMPs play a critical role in vivo in invasion of the CNS and that these enzymes may be amenable targets for limiting PAM.

  12. Effect of kudiezi injection on stent thrombosis and matrix metalloproteinase in patients with PCI

    International Nuclear Information System (INIS)

    Wang Zhihui; Zhang Jing; Xing Yue

    2010-01-01

    Objective: To investigate the effects of Kudiezi injection on stent thrombosis and the levels of matrix metalloproteinase (MMPs) and thromboxane B 2 (TXB2) in elderly patients with percutaneous coronary intervention (PCI), and investigate the mechanism of Kudiezi on the decrease of stent thrombosis. Methods: Forty elderly patients were divided into two groups (Kudiezi group and control group) after PCI. Kudiezi were administered into patients in Kudiezi group and the patients in control group were treated with regular medication. The angioraphic and clinic follow-up outcomes of 40 elderly patients with PCI there retrospectively analyze. Stent thrombosis (ST) was confirmed by angiography. The levels MMPs and TXB2 in Kudiezi group (n=20) and control group (n=20) were determined before stent implantation and after 6 months. Major cardiac events (restenosis, cardiac death, myocardiac infarction, revasculation) were observed during follow-up. Results: The levels of MMPs and TXB2 in Kudiezi and control group decreased significantly after PCI. The levels of MMPs and TXB2 in Kudiezi group were less than that in control group after PCI. The levels of MMPs and TXB 2 in all patients group were significantly different between pre-procedure and post-procedure (P 2 . (authors)

  13. Matrix Metalloproteinases: Inflammatory Regulators of Cell Behaviors in Vascular Formation and Remodeling

    Directory of Open Access Journals (Sweden)

    Qishan Chen

    2013-01-01

    Full Text Available Abnormal angiogenesis and vascular remodeling contribute to pathogenesis of a number of disorders such as tumor, arthritis, atherosclerosis, restenosis, hypertension, and neurodegeneration. During angiogenesis and vascular remodeling, behaviors of stem/progenitor cells, endothelial cells (ECs, and vascular smooth muscle cells (VSMCs and its interaction with extracellular matrix (ECM play a critical role in the processes. Matrix metalloproteinases (MMPs, well-known inflammatory mediators are a family of zinc-dependent proteolytic enzymes that degrade various components of ECM and non-ECM molecules mediating tissue remodeling in both physiological and pathological processes. MMPs including MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, and MT1-MMP, are stimulated and activated by various stimuli in vascular tissues. Once activated, MMPs degrade ECM proteins or other related signal molecules to promote recruitment of stem/progenitor cells and facilitate migration and invasion of ECs and VSMCs. Moreover, vascular cell proliferation and apoptosis can also be regulated by MMPs via proteolytically cleaving and modulating bioactive molecules and relevant signaling pathways. Regarding the importance of vascular cells in abnormal angiogenesis and vascular remodeling, regulation of vascular cell behaviors through modulating expression and activation of MMPs shows therapeutic potential.

  14. Zymography as a Research Tool in the Study of Matrix Metalloproteinase Inhibitors.

    Science.gov (United States)

    Ren, Zongli; Chen, Juanjuan; Khalil, Raouf A

    2017-01-01

    Matrix metalloproteinases (MMPs) are proteolytic enzymes that degrade various components of the extracellular matrix (ECM) and play a role in tissue remodeling. Changes in MMPs have been observed in cancer, connective tissue disorders, and vascular disease, and both endogenous tissue inhibitors of MMPs (TIMPs) and synthetic MMP inhibitors (MMPIs) have been evaluated as modulators of MMP activity in various biological systems. Zymography is a simple technique that is commonly used to assess MMP activity and the efficacy of MMPIs. Also, reverse zymography is a modified technique to study the activity of endogenous TIMPs. However, problems are often encountered during the zymography procedure, which could interfere with accurate assessment of MMP activity in control specimens, and thus make it difficult to determine the pathological changes in MMPs and their responsiveness to MMPIs. Simplified protocols for preparation of experimental solutions, tissue preparation, regular and reverse zymography procedures, and zymogram analysis are presented. Additional helpful tips to troubleshoot problems in the zymography technique and to enhance the quality of the zymograms should make it more feasible to determine the changes in MMPs and assess the efficacy of MMPIs in modulating MMP activity in various biological systems and pathological conditions.

  15. Strategies to Target Matrix Metalloproteinases as Therapeutic Approach in Cancer.

    Science.gov (United States)

    Piperigkou, Zoi; Manou, Dimitra; Karamanou, Konstantina; Theocharis, Achilleas D

    2018-01-01

    Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that are capable of degrading numerous extracellular matrix (ECM) components thus participating in physiological and pathological processes. Apart from the remodeling of ECM, they affect cell-cell and cell-matrix interactions and are implicated in the development and progression of various diseases such as cancer. Numerous studies have demonstrated that MMPs evoke epithelial to mesenchymal transition (EMT) of cancer cells and affect their signaling, adhesion, migration and invasion to promote cancer cell aggressiveness. Various studies have suggested MMPs as suitable targets for treatment of malignancies, and several MMP inhibitors (MMPIs) have been developed. Although initial trials have failed to establish MMPIs as anticancer agents due to lack of specificity and side effects, new MMPIs have been developed with improved action that are currently being investigated. Furthermore, novel strategies that target MMPs for improving drug delivery and regulating their activity in tumors are presented. This review summarizes the implication of MMPs in cancer progression and discusses the advancements in their targeting.

  16. Neuronal targeting, internalization, and biological activity of a recombinant atoxic derivative of botulinum neurotoxin A

    Science.gov (United States)

    Botulinum neurotoxins (BoNT) have the unique capacity to cross epithelial barriers, target neuromuscular junctions, and translocate active metalloprotease component to the cytosol of motor neurons. We have taken advantage of the molecular carriers responsible for this trafficking to create a family ...

  17. Expression and effect of inhibition of aminopeptidase-A during nephrogenesis.

    NARCIS (Netherlands)

    Dijkman, H.B.P.M.; Assmann, K.J.M.; Steenbergen, E.; Wetzels, J.F.M.

    2006-01-01

    Aminopeptidase-A (APA) is a metalloprotease that cleaves N-terminal aspartyl and glutamyl residues from peptides. Its best-known substrate is angiotensin II (Ang II), the most active compound of the renin-angiotensin system (RAS). The RAS is involved in renal development. Most components of the RAS

  18. Adamalysins in biology and disease

    NARCIS (Netherlands)

    van Goor, Harry; Melenhorst, Wynand B. W. H.; Turner, Anthony J.; Holgate, Stephen T.

    2009-01-01

    ADAMs (a disintegrin and metalloprotease) are membrane-bound enzymes, capable of shedding a multitude of proteins from the surface of the cell. They are therefore considered crucial modulators of physiological and pathophysiological processes. The structure and function of ADAMs is related to those

  19. The cysteine-rich domain of human ADAM 12 supports cell adhesion through syndecans and triggers signaling events that lead to beta1 integrin-dependent cell spreading

    DEFF Research Database (Denmark)

    Iba, K; Albrechtsen, R; Gilpin, B

    2000-01-01

    The ADAMs (a disintegrin and metalloprotease) family of proteins is involved in a variety of cellular interactions, including cell adhesion and ecto- domain shedding. Here we show that ADAM 12 binds to cell surface syndecans. Three forms of recombinant ADAM 12 were used in these experiments: the ...

  20. Measurement of MMP-9 and -12 degraded elastin (ELM) provides unique information on lung tissue degradation

    DEFF Research Database (Denmark)

    Skjøt-Arkil, Helene; Clausen, Rikke E; Nguyen, Quoc Hai Trieu

    2012-01-01

    Elastin is an essential component of selected connective tissues that provides a unique physiological elasticity. Elastin may be considered a signature protein of lungs where matrix metalloprotease (MMP) -9-and -12, may be considered the signature proteases of the macrophages, which in part...

  1. A proteomic approach for identification of secreted proteins during the differentiation of 3T3-L1 preadipocytes to adipocytes

    DEFF Research Database (Denmark)

    Kratchmarova, Irina; Kalume, Dario E; Blagoev, Blagoy

    2002-01-01

    additional secreted proteins including resistin, secreted acidic cysteine-rich glycoprotein/osteonectin, stromal cell-derived factor-1, cystatin C, gelsolin, and matrix metalloprotease-2 were identified by this method. To our knowledge, this is the first study to identify several novel secreted proteins...

  2. Multiplicity of carbohydrate-binding sites in β-prism fold lectins ...

    Indian Academy of Sciences (India)

    PRAKASH KUMAR

    roles of plant lectins are yet to be fully elucidated. It is believed that they are involved in root–nodule symbiosis ..... NP_012158.1. Saccharomyces cerevisiae. 1. 696. 32. 1. Putative metalloprotease. 93. XP_445234.1. Candida glabrata. 1. 683. 34. 1. Unnamed protein product. 94. CAB63793.1. Schizosaccharomyces pombe.

  3. Journal of Biosciences | Indian Academy of Sciences

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Biosciences. Malay Mandal. Articles written in Journal of Biosciences. Volume 28 Issue 2 March 2003 pp 205-213 Articles. Role of matrix metalloprotease-2 in oxidant activation of Ca ATPase by hydrogen peroxide in pulmonary vascular smooth muscle plasma membrane · Malay Mandal Sudip ...

  4. Characterization of the Serralysin-like gene of 'Ca. Liberibacter solanacearum' associated with Potato Zebra Chip disease

    Science.gov (United States)

    The non-culturable bacterium ‘Candidatus Liberibacter solanacearum’ (Lso) is the causative agent of zebra chip disease in potato. Computational analysis of the Lso genome revealed a serralysin-like gene based on conserved domains characteristic of genes encoding metalloprotease enzymes similar to se...

  5. Congenital thrombotic thrombocytopenic purpura caused by new compound heterozygous mutations of the ADAMTS13 gene

    DEFF Research Database (Denmark)

    Rank, Cecilie Utke; Kremer Hovinga, Johanna; Taleghani, Magnus Mansouri

    2014-01-01

    Upshaw-Schulman syndrome (USS) is due to severe congenital deficiency of von Willebrand factor (VWF)-cleaving protease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 domains, nr 13) activity resulting in the presence of unusually large forms of VWF in the circulation, caus...

  6. ADAM12 alleviates the skeletal muscle pathology in mdx dystrophic mice

    DEFF Research Database (Denmark)

    Kronqvist, Pauliina; Kawaguchi, Nobuko; Albrechtsen, Reidar

    2002-01-01

    we examined the role of the transmembrane ADAM12, a disintegrin and metalloprotease, which is normally associated with development and regeneration of skeletal muscle. We demonstrate that ADAM12 overexpression in the dystrophin-deficient mdx mice alleviated the muscle pathology in these animals...

  7. Breakdown of the innate immune system by bacterial proteases

    NARCIS (Netherlands)

    Laarman, A.J.

    2011-01-01

    Bacteria have developed many strategies to circumvent our immune system to survive and colonize human tissues. One of these strategies is by secreting proteases that specifically target the innate immune system. Aureolysin is a metalloprotease from Staphylococcus aureus which target the main

  8. sarA negatively regulates Staphylococcus epidermidis biofilm formation by modulating expression of 1 MDa extracellular matrix binding protein and autolysis‐dependent release of eDNA

    DEFF Research Database (Denmark)

    Christner, Martin; Heinze, Constanze; Busch, Michael

    2012-01-01

    to biofilm formation in mutant 1585ΔsarA. Increased eDNA amounts indirectly resulted from upregulation of metalloprotease SepA, leading to boosted processing of autolysin AtlE, in turn inducing augmented autolysis and release of eDNA. Hence, this study identifies sarA as a negative regulator of Embp‐ and e...

  9. SarA is a negative regulator of Staphylococcus epidermidis biofilm formation

    DEFF Research Database (Denmark)

    Martin, Christer; Heinze, C.; Busch, M.

    2012-01-01

    to biofilm formation in mutant 1585ΔsarA. Increased eDNA amounts indirectly resulted from up-regulation of metalloprotease SepA, leading to boosted processing of major autolysin AtlE, in turn inducing augmented autolysis and release of chromosomal DNA. Hence, this study identifies sarA as a negative...

  10. Promotion of mitochondrial membrane complex assembly by a proteolytically inactive yeast Lon

    NARCIS (Netherlands)

    Rep, M; van Dijl, J M; Suda, K; Schatz, G; Grivell, L A; Suzuki, C K

    1996-01-01

    Afg3p and Rca1p are adenosine triphosphate (ATP)-dependent metalloproteases in yeast mitochondria. Cells lacking both proteins exhibit defects in respiration-dependent growth, degradation of mitochondrially synthesized proteins, and assembly of inner-membrane complexes. Defects in growth and protein

  11. A Potent Peptidomimetic Inhibitor of Botulinum Neurotoxin Serotype A has a Very Different Conformation than SNAP-25 Substrate

    National Research Council Canada - National Science Library

    Zuniga, Jorge E; Schmidt, James J; Fenn, Timothy; Burnett, James C; Arac, Demet; Gussio, Rick; Stafford, Robert G; Badie, Shirin S; Bavari, Sina; Brunger, Axel T

    2008-01-01

    Botulinum neurotoxin serotype A is the most lethal of all known toxins. Here, we report the crystal structure, along with SAR data, of the zinc metalloprotease domain of BoNT/A bound to a potent peptidomimetic inhibitor (K(i)=41 nM...

  12. Targeting prolyl endopeptidase with valproic acid as a potential modulator of neutrophilic inflammation

    NARCIS (Netherlands)

    Abdul Roda, Mojtaba; Sadik, Mariam; Gaggar, Amit; Hardison, Matthew T; Jablonsky, Michael J; Braber, Saskia; Blalock, James Edwin; Redegeld, Frank A; Folkerts, Gert; Jackson, Patricia L

    2014-01-01

    A novel neutrophil chemoattractant derived from collagen, proline-glycine-proline (PGP), has been recently characterized in chronic obstructive pulmonary disease (COPD). This peptide is derived via the proteolytic activity of matrix metalloproteases (MMP's)-8/9 and PE, enzymes produced by

  13. from Cerastes cerastes venom gland

    African Journals Online (AJOL)

    Sequence analysis and alignment using bioinformatic programs indicated that samples 1, 2 and 3 bear significant homology to the metalloprotease family of snake venom sequences deposited in the Genbank. Translation to the amino acid sequence and alignment using protein database showed strong homology with ...

  14. Journal of Biosciences | Indian Academy of Sciences

    Indian Academy of Sciences (India)

    Exposure of bovine pulmonary artery smooth muscle plasma membrane suspension with the oxidant H2O2 (1 mM) stimulated Ca2+ATPase activity. We sought to determine the role of matrix metalloprotease-2 (MMP-2) in stimulating Ca2+ATPase activity by H2O2 in the smooth muscle plasma membrane. The smooth ...

  15. Detection of Pseudomonas fluorescens from broth, water and ...

    African Journals Online (AJOL)

    Loop mediated isothermal amplification is rapid, highly sensitive and specifically developed method for detection of bacterial infections. AprX gene for alkaline metalloprotease of Pseudomonas fluorescens was used to design four primers and loop mediated isothermal amplification (LAMP) conditions were standardized for ...

  16. Compensation for dystrophin-deficiency

    DEFF Research Database (Denmark)

    Moghadaszadeh, Behzad; Albrechtsen, Reidar; Guo, Ling T

    2003-01-01

    Mouse models for genetic diseases are among the most powerful tools available for developing and testing new treatment strategies. ADAM12 is a disintegrin and metalloprotease, previously demonstrated to significantly alleviate the pathology of mdx mice, a model for Duchenne muscular dystrophy...

  17. Regulation of human ADAM 12 protease by the prodomain. Evidence for a functional cysteine switch

    DEFF Research Database (Denmark)

    Loechel, F; Overgaard, M T; Oxvig, C

    1999-01-01

    The ADAMs (a disintegrin and metalloprotease) are a family of multidomain proteins that are believed to play key roles in cell-cell and cell-matrix interactions. We have shown recently that human ADAM 12-S (meltrin alpha) is an active metalloprotease. It is synthesized as a zymogen, with the prod......The ADAMs (a disintegrin and metalloprotease) are a family of multidomain proteins that are believed to play key roles in cell-cell and cell-matrix interactions. We have shown recently that human ADAM 12-S (meltrin alpha) is an active metalloprotease. It is synthesized as a zymogen......, with the prodomain maintaining the protease in a latent form. We now provide evidence that the latency mechanism of ADAM 12 can be explained by the cysteine switch model, in which coordination of Zn2+ in the active site of the catalytic domain by a cysteine residue in the prodomain is critical for inhibition...... of the protease. Replacing Cys179 with other amino acids results in an ADAM 12 proform that is proteolytically active, but latency can be restored by placing cysteine at other positions in the propeptide. None of the amino acids adjacent to the crucial cysteine residue is essential for blocking activity...

  18. Tetanus toxin light chain expression in Sertoli cells of transgenic mice causes alterations of the actin cytoskeleton and disrupts spermatogenesis

    NARCIS (Netherlands)

    Eisel, Ulrich; Reynolds, Kay; Riddick, Michelle; Zimmer, Anne; Niemann, Heiner; Zimmer, Andreas; Gruss, P.

    Tetanus toxin is a powerful neurotoxin known to inhibit neurotransmitter release. The tetanus toxin light chain is a metalloprotease that cleaves some members of the synaptobrevin gene family with high specificity. Here, we report the expression of a synthetic gene encoding the tetanus toxin light

  19. Impact of viral enhancin genes on potency of Lymantria dispar multiple nucleopolyhedrovirus in L. dispar following disruption of the peritrophic matrix

    Science.gov (United States)

    Kelli Hoover; Merideth A. Humphries; Alyssa R. Genfron; James M. Slavicek

    2010-01-01

    Enhancins are metalloproteases found in many betabaculoviruses and several alphabaculoviruses, which enhance alphabaculovirus potency by degrading a protein component of the peritrophic matrix (PM), facilitating passage of virions through this structure. Earlier studies on betabaculovirus enhancins within heterologous systems suggested that enhancins facilitate virion...

  20. Polymorphism, genetic exchange and intragenic recombination of the aureolysin gene among Staphylococcus aureus strains

    NARCIS (Netherlands)

    Sabat, Artur J.; Wladyka, Benedykt; Kosowska-Shick, Klaudia; Grundmann, Hajo; van Dijl, Jan Maarten; Kowal, Julia; Appelbaum, Peter C.; Dubin, Adam; Hryniewicz, Waleria

    2008-01-01

    Background: Staphylococcus aureus expresses several proteases, which are thought to contribute to the virulence of this bacterium. Here we focus on aureolysin, the major thermolysin-like metalloprotease. Despite the importance of aureolysin in the physiology and pathogenesis of S. aureus, relatively

  1. Cross-domain inhibition of TACE ectodomain

    DEFF Research Database (Denmark)

    Tape, Christopher J; Willems, Sofie H; Dombernowsky, Sarah L

    2011-01-01

    Proteolytic release from the cell surface is an essential activation event for many growth factors and cytokines. TNF-a converting enzyme (TACE) is a membrane-bound metalloprotease responsible for solubilizing many pathologically significant membrane substrates and is an attractive therapeutic...

  2. Association of ADAM33 gene polymorphisms with adult-onset ...

    Indian Academy of Sciences (India)

    2011-08-19

    Aug 19, 2011 ... Abstract. ADAM33, a member of the ADAM (a disintegrin and metalloprotease) gene family, is an asthma susceptibility gene origi- nally identified by positional cloning. In the present study, we investigated the possible association of five single-nucleotide polymorphisms (SNPs) in the ADAM33 (rs511898, ...

  3. New Insights into the Role of Matrix Metalloproteinases in Preeclampsia.

    Science.gov (United States)

    Espino Y Sosa, Salvador; Flores-Pliego, Arturo; Espejel-Nuñez, Aurora; Medina-Bastidas, Diana; Vadillo-Ortega, Felipe; Zaga-Clavellina, Veronica; Estrada-Gutierrez, Guadalupe

    2017-07-20

    Preeclampsia is a severe pregnancy complication globally, characterized by poor placentation triggering vascular dysfunction. Matrix metalloproteinases (MMPs) exhibit proteolytic activity implicated in the efficiency of trophoblast invasion to the uterine wall, and a dysregulation of these enzymes has been linked to preeclampsia. A decrease in MMP-2 and MMP-9 interferes with the normal remodeling of spiral arteries at early pregnancy stages, leading to the initial pathophysiological changes observed in preeclampsia. Later in pregnancy, an elevation in MMP-2 and MMP-9 induces abnormal release of vasoactive factors conditioning hypertension. Although these two enzymes lead the scene, other MMPs like MMP-1 and MMP-14 seem to have a role in this pathology. This review gathers published recent evidence about the implications of different MMPs in preeclampsia, and the potential use of these enzymes as emergent biomarkers and biological therapeutic targets, focusing on studies involving human subjects.

  4. Matched molecular pair-based data sets for computer-aided medicinal chemistry

    Science.gov (United States)

    Bajorath, Jürgen

    2014-01-01

    Matched molecular pairs (MMPs) are widely used in medicinal chemistry to study changes in compound properties including biological activity, which are associated with well-defined structural modifications. Herein we describe up-to-date versions of three MMP-based data sets that have originated from in-house research projects. These data sets include activity cliffs, structure-activity relationship (SAR) transfer series, and second generation MMPs based upon retrosynthetic rules. The data sets have in common that they have been derived from compounds included in the ChEMBL database (release 17) for which high-confidence activity data are available. Thus, the activity data associated with MMP-based activity cliffs, SAR transfer series, and retrosynthetic MMPs cover the entire spectrum of current pharmaceutical targets. Our data sets are made freely available to the scientific community. PMID:24627802

  5. Internal strain drives spontaneous periodic buckling in collagen and regulates remodeling

    Science.gov (United States)

    Dittmore, Andrew; Silver, Jonathan; Sarkar, Susanta K.; Marmer, Barry; Goldberg, Gregory I.; Neuman, Keir C.

    2016-01-01

    Fibrillar collagen, an essential structural component of the extracellular matrix, is remarkably resistant to proteolysis, requiring specialized matrix metalloproteinases (MMPs) to initiate its remodeling. In the context of native fibrils, remodeling is poorly understood; MMPs have limited access to cleavage sites and are inhibited by tension on the fibril. Here, single-molecule recordings of fluorescently labeled MMPs reveal cleavage-vulnerable binding regions arrayed periodically at ∼1-µm intervals along collagen fibrils. Binding regions remain periodic even as they migrate on the fibril, indicating a collective process of thermally activated and self-healing defect formation. An internal strain relief model involving reversible structural rearrangements quantitatively reproduces the observed spatial patterning and fluctuations of defects and provides a mechanism for tension-dependent stabilization of fibrillar collagen. This work identifies internal–strain-driven defects that may have general and widespread regulatory functions in self-assembled biological filaments. PMID:27402741

  6. Matched molecular pair-based data sets for computer-aided medicinal chemistry [v2; ref status: indexed, http://f1000r.es/309

    Directory of Open Access Journals (Sweden)

    Ye Hu

    2014-02-01

    Full Text Available Matched molecular pairs (MMPs are widely used in medicinal chemistry to study changes in compound properties including biological activity, which are associated with well-defined structural modifications. Herein we describe up-to-date versions of three MMP-based data sets that have originated from in-house research projects. These data sets include activity cliffs, structure-activity relationship (SAR transfer series, and second generation MMPs based upon retrosynthetic rules. The data sets have in common that they have been derived from compounds included in the ChEMBL database (release 17 for which high-confidence activity data are available. Thus, the activity data associated with MMP-based activity cliffs, SAR transfer series, and retrosynthetic MMPs cover the entire spectrum of current pharmaceutical targets. Our data sets are made freely available to the scientific community.

  7. Exercise-induced regulation of matrix metalloproteinases in the skeletal muscle of subjects with type 2 diabetes

    DEFF Research Database (Denmark)

    Scheede-Bergdahl, Celena; Bergdahl, Andreas; Schjerling, Peter

    2014-01-01

    -training. At baseline, there were no effects of diabetes on MMP or TIMP mRNA or protein. mRNA and protein response to training was similar in both groups, except active MMP-2 protein was elevated post training in T2DM only. Our results indicate that exercise-induced stimulation of MMPs is preserved in skeletal muscle......Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMP) play a critical role during vascular remodelling, in both health and disease. Impaired MMP regulation is associated with many diabetes-related complications. This study examined whether exercise-induced regulation of MMPs...... is maintained in the skeletal muscle of patients with uncomplicated type 2 diabetes (T2DM). Subjects [12 T2DM, 9 healthy control subjects (CON)] underwent 8 weeks of physical training. Messenger RNA (mRNA) was measured at baseline, during and after 8 weeks of training. Protein was measured pre- and post...

  8. Matched molecular pair-based data sets for computer-aided medicinal chemistry [v1; ref status: indexed, http://f1000r.es/2w9

    Directory of Open Access Journals (Sweden)

    Ye Hu

    2014-02-01

    Full Text Available Matched molecular pairs (MMPs are widely used in medicinal chemistry to study changes in compound properties including biological activity, which are associated with well-defined structural modifications. Herein we describe up-to-date versions of three MMP-based data sets that have originated from in-house research projects. These data sets include activity cliffs, structure-activity relationship (SAR transfer series, and second generation MMPs based upon retrosynthetic rules. The data sets have in common that they have been derived from compounds included in the latest release of the ChEMBL database for which high-confidence activity data are available. Thus, the activity data associated with MMP-based activity cliffs, SAR transfer series, and retrosynthetic MMPs cover the entire spectrum of current pharmaceutical targets. Our data sets are made freely available to the scientific community.

  9. A study for radiation-related tumor microenvironment

    International Nuclear Information System (INIS)

    Son, Young Sook; Hong, Seok Il; Kim, Young Soon; Jin Yong Jae; Lee, Tae Hee; Chung, Eun Kyung; Yi, Jae Yeun; Park, Myung Jin; Kim, Yun Young; Kang, Sin Keun

    1999-04-01

    In this study, we attempted to elucidate the mechanism involved in radiation-induced modification and changes of biological factors and physicochemical factors of tumor microenvironment and develop techniques and agents for the modification of tumor microenvironment which is favorable for efficient radio-cancer therapy based on our basic study. We established in vitro tumor invasion and angiogenesis model, elucidated the importance of MMPs activation and the MMPs/TIMPs complex in the invasive transition of tumor. Furthermore we showed the signaling pathway for MMPs induction through EGF receptor and TGF beta 1 stimulated E-M transition. We also established primary culture of human endothelial cells and tubule forming condition which is utilized for the detection of novel angiogenic factors. We also identified hypoxia induced signaling pathway and showed that GBE improved blood perfusion which may increase the effectiveness of radio-cancer therapy

  10. A study for radiation-related tumor microenvironment

    Energy Technology Data Exchange (ETDEWEB)

    Son, Young Sook; Hong, Seok Il; Kim, Young Soon; Jin Yong Jae; Lee, Tae Hee; Chung, Eun Kyung; Yi, Jae Yeun; Park, Myung Jin; Kim, Yun Young; Kang, Sin Keun

    1999-04-01

    In this study, we attempted to elucidate the mechanism involved in radiation-induced modification and changes of biological factors and physicochemical factors of tumor microenvironment and develop techniques and agents for the modification of tumor microenvironment which is favorable for efficient radio-cancer therapy based on our basic study. We established in vitro tumor invasion and angiogenesis model, elucidated the importance of MMPs activation and the MMPs/TIMPs complex in the invasive transition of tumor. Furthermore we showed the signaling pathway for MMPs induction through EGF receptor and TGF beta 1 stimulated E-M transition. We also established primary culture of human endothelial cells and tubule forming condition which is utilized for the detection of novel angiogenic factors. We also identified hypoxia induced signaling pathway and showed that GBE improved blood perfusion which may increase the effectiveness of radio-cancer therapy.

  11. 2-Methoxy-2,4-diphenyl-3(2H)-furanone-labeled gelatin zymography and reverse zymography: a rapid real-time method for quantification of matrix metalloproteinases-2 and -9 and tissue inhibitors of metalloproteinases.

    Science.gov (United States)

    Min, Danqing; Lyons, James Guy; Jia, Junhong; Lo, Lisa; McLennan, Susan V

    2006-02-01

    Measurement of matrix metalloproteinases (MMPs) and their specific tissue inhibitors of metalloproteinases (TIMPs) by the techniques of zymography and reverse zymography provide useful information regarding the status of matrix accumulation or breakdown. This report describes the use of 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF), a fluorescent compound which can be used to label gelatin as a substrate for detection of the gelatin degrading MMP-2 and -9 by zymography. In addition, a modification of the zymographic technique by addition of excess MMPs enables the use of the MDPF-labeled gelatin substrate for the identification and quantification of TIMPs by reverse zymography. Both systems are real-time sensitive reliable quantification techniques, easily used for measurement of these MMPs and TIMPs in clinical, biological, and tissue culture samples.

  12. Matrix Metalloproteinases and Neurotrauma: Evolving Roles in Injury and Reparative Processes

    Science.gov (United States)

    Zhang, Haoqian; Adwanikar, Hita; Werb, Zena; Noble-Haeusslein, Linda J.

    2010-01-01

    Matrix metalloproteinases (MMPs) are involved in a wide range of proteolytic events in fetal development and normal tissue remodeling as well as wound healing and inflammation. In the CNS, they have been implicated in a variety of neurodegenerative diseases ranging from multiple sclerosis to Alzheimer disease and are integral to stroke-related cell damage. Although studies implicate increased activity of MMPs in pathogenesis in the CNS, there is also a growing literature to support their participation in events that support recovery processes. Here the authors provide a brief overview of MMPs and their regulation, address their complex roles following traumatic injuries to the adult and developing CNS, and consider their time- and context-dependent signatures that influence both injury and reparative processes. PMID:20400713

  13. Matrix metalloproteinases and neurotrauma: evolving roles in injury and reparative processes.

    Science.gov (United States)

    Zhang, Haoqian; Adwanikar, Hita; Werb, Zena; Noble-Haeusslein, Linda J

    2010-04-01

    Matrix metalloproteinases (MMPs) are involved in a wide range of proteolytic events in fetal development and normal tissue remodeling as well as wound healing and inflammation. In the CNS, they have been implicated in a variety of neurodegenerative diseases ranging from multiple sclerosis to Alzheimer disease and are integral to stroke-related cell damage. Although studies implicate increased activity of MMPs in pathogenesis in the CNS, there is also a growing literature to support their participation in events that support recovery processes. Here the authors provide a brief overview of MMPs and their regulation, address their complex roles following traumatic injuries to the adult and developing CNS, and consider their time- and context-dependent signatures that influence both injury and reparative processes.

  14. Matrix Metalloproteinase-9 and Haemozoin: Wedding Rings for Human Host and Plasmodium falciparum Parasite in Complicated Malaria.

    Science.gov (United States)

    Prato, Mauro; Giribaldi, Giuliana

    2011-01-01

    It is generally accepted that the combination of both Plasmodium falciparum parasite and human host factors is involved in the pathogenesis of complicated severe malaria, including cerebral malaria (CM). Among parasite products, the malarial pigment haemozoin (HZ) has been shown to impair the functions of mononuclear and endothelial cells. Different CM models were associated with enhanced levels of matrix metalloproteinases (MMPs), a family of proteolytic enzymes able to disrupt subendothelial basement membrane and tight junctions and shed, activate, or inactivate cytokines, chemokines, and other MMPs through cleavage from their precursors. Among MMPs, a good candidate for targeted therapy might be MMP-9, whose mRNA and protein expression enhancement as well as direct proenzyme activation by HZ have been recently investigated in a series of studies by our group and others. In the present paper the role of HZ and MMP-9 in complicated malaria, as well as their interactions, will be discussed.

  15. Matrix Metalloproteinase-9 and Haemozoin: Wedding Rings for Human Host and Plasmodium falciparum Parasite in Complicated Malaria

    Directory of Open Access Journals (Sweden)

    Mauro Prato

    2011-01-01

    Full Text Available It is generally accepted that the combination of both Plasmodium falciparum parasite and human host factors is involved in the pathogenesis of complicated severe malaria, including cerebral malaria (CM. Among parasite products, the malarial pigment haemozoin (HZ has been shown to impair the functions of mononuclear and endothelial cells. Different CM models were associated with enhanced levels of matrix metalloproteinases (MMPs, a family of proteolytic enzymes able to disrupt subendothelial basement membrane and tight junctions and shed, activate, or inactivate cytokines, chemokines, and other MMPs through cleavage from their precursors. Among MMPs, a good candidate for targeted therapy might be MMP-9, whose mRNA and protein expression enhancement as well as direct proenzyme activation by HZ have been recently investigated in a series of studies by our group and others. In the present paper the role of HZ and MMP-9 in complicated malaria, as well as their interactions, will be discussed.

  16. Genetic polymorphisms of matrix metalloproteinases and their inhibitors in potentially malignant and malignant lesions of the head and neck

    Directory of Open Access Journals (Sweden)

    Asotra Kamlesh

    2010-02-01

    Full Text Available Abstract Matrix metalloproteinases (MMPs are a family of zinc-dependent proteinases that are capable of cleaving all extra cellular matrix (ECM substrates. Degradation of matrix is a key event in progression, invasion and metastasis of potentially malignant and malignant lesions of the head and neck. It might have an important polymorphic association at the promoter regions of several MMPs such as MMP-1 (-1607 1G/2G, MMP-2 (-1306 C/T, MMP-3 (-1171 5A/6A, MMP-9 (-1562 C/T and TIMP-2 (-418 G/C or C/C. Tissue inhibitors of metalloproteinases (TIMPs are naturally occurring inhibitors of MMPs, which inhibit the activity of MMPs and control the breakdown of ECM. Currently, many MMP inhibitors (MMPIs are under development for treating different malignancies. Useful markers associated with molecular aggressiveness might have a role in prognostication of malignancies and to better recognize patient groups that need more antagonistic treatment options. Furthermore, the introduction of novel prognostic markers may also promote exclusively new treatment possibilities, and there is an obvious need to identify markers that could be used as selection criteria for novel therapies. The objective of this review is to discuss the molecular functions and polymorphic association of MMPs and TIMPs and the possible therapeutic aspects of these proteinases in potentially malignant and malignant head and neck lesions. So far, no promising drug target therapy has been developed for MMPs in the lesions of this region. In conclusion, further research is required for the development of their potential diagnostic and therapeutic possibilities.

  17. Expression of matrix metalloproteinases and components of the endocannabinoid system in the knee joint are associated with biphasic pain progression in a rat model of osteoarthritis

    Directory of Open Access Journals (Sweden)

    Pajak A

    2017-08-01

    Full Text Available Agnieszka Pajak, Magdalena Kostrzewa, Natalia Malek, Michal Korostynski, Katarzyna Starowicz Institute of Pharmacology, Polish Academy of Sciences, Krakow, Poland Abstract: Matrix metalloproteinases (MMPs are considered important in articular cartilage breakdown during osteoarthritis (OA. Similarly, the endocannabinoid system (ECS is implicated in joint function and modulation of nociceptive processing. Functional interplay between ECS and MMPs has been recently indicated. Here, we tested if changes in the expression of selected MMPs and major ECS elements temporally correlate with the intensity of OA-related pain. Knee OA was induced in male Wistar rats by intra-articular sodium monoiodoacetate injection. OA-like pain behavior was tested using the dynamic weight bearing. Joint tissue samples at different time points after OA induction were subjected to gene (quantitative polymerase chain reaction and protein (Western blot expression analyses. Monoiodoacetate-induced nocifensive responses in rats showed a biphasic progression pattern. The alterations in expression of selected MMPs elegantly corresponded to the two-stage development of OA pain. The most substantial changes in the expression of the ECS system were revealed at a later stage of OA progression. Alterations within ECS are involved in the process of adaptation to persistent painful stimuli. The accumulation of MMPs in osteoarthritic cartilage may have a role in the biphasic progression of OA-related pain. Temporal association of changes in ECS and MMPs expression shows a potential therapeutic approach that utilizes the concept of combining indirect ECS-mediated MMP inhibition and ECS modulation of pain transduction. Keywords: osteoarthritis, monoiodoacetate, pain, matrix metalloproteinases, endocannabinoid system

  18. Tomato Sl3-MMP, a member of the Matrix metalloproteinase family, is required for disease resistance against Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000.

    Science.gov (United States)

    Li, Dayong; Zhang, Huijuan; Song, Qiuming; Wang, Lu; Liu, Shixia; Hong, Yongbo; Huang, Lei; Song, Fengming

    2015-06-14

    Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases. MMPs have been characterized in detail in mammals and shown to play key roles in many physiological and pathological processes. Although MMPs in some plant species have been identified, the function of MMPs in biotic stress responses remains elusive. A total of five MMP genes were identified in tomato genome. qRT-PCR analysis revealed that expression of Sl-MMP genes was induced with distinct patterns by infection of Botrytis cinerea and Pseudomonas syringae pv. tomato (Pst) DC3000 and by treatment with defense-related hormones such as salicylic acid, jasmonic acid and ethylene precursor 1-amino cyclopropane-1-carboxylic acid. Virus-induced gene silencing (VIGS)-based knockdown of individual Sl-MMPs and disease assays indicated that silencing of Sl3-MMP resulted in reduced resistance to B. cinerea and Pst DC3000, whereas silencing of other four Sl-MMPs did not affect the disease resistance against these two pathogens. The Sl3-MMP-silenced tomato plants responded with increased accumulation of reactive oxygen species and alerted expression of defense genes after infection of B. cinerea. Transient expression of Sl3-MMP in leaves of Nicotiana benthamiana led to an enhanced resistance to B. cinerea and upregulated expression of defense-related genes. Biochemical assays revealed that the recombinant mature Sl3-MMP protein had proteolytic activities in vitro with distinct preferences for specificity of cleavage sites. The Sl3-MMP protein was targeted onto the plasma membrane of plant cells when transiently expressed in onion epidermal cells. VIGS-based knockdown of Sl3-MMP expression in tomato and gain-of-function transient expression of Sl3-MMP in N. benthamiana demonstrate that Sl3-MMP functions as a positive regulator of defense response against B. cinerea and Pst DC3000.

  19. Effects of Hormones on the Expression of Matrix Metalloproteinases and Their Inhibitors in Bovine Spermatozoa

    Directory of Open Access Journals (Sweden)

    Sang-Hwan Kim

    2013-03-01

    Full Text Available Proteases and protease inhibitors play key roles in most physiological processes, including cell migration, cell signaling, and cell surface and tissue remodeling. Among these, the matrix metalloproteinase (MMPs pathway is one of the most efficient biosynthetic pathways for controlling the activation of enzymes responsible for protein degradation. This also indicates the association of MMPs with the maturation of spermatozoa. In an attempt to investigate the effect of MMP activation and inhibitors in cultures with various hormones during sperm capacitation, we examined and monitored the localization and expression of MMPs (MMP-2 and MMP-9, tissue inhibitors of metalloproteinases (TIMP-2 and TIMP-3, as well as their expression profiles. Matured spermatozoa were collected from cultures with follicle-stimulating hormone (FSH, luteinizing hormone (LH, and Lutalyse at 1 h, 6 h, 18 h, and 24 h. ELISA detected the expression of MMP-2, MMP-9, TIMP-2, and TIMP-3 in all culture media, regardless of medium type (FSH-supplemented fertilization Brackett-Oliphant medium (FFBO, LH-supplemented FBO (LFBO, or Lutalyse-supplemented FBO (LuFBO. TIMP-2 and TIMP-3 expression patterns decreased in LFBO and LuFBO. MMP-2 and MMP-9 activity in FBO and FFBO progressively increased from 1 h to 24 h but was not detected in LFBO and LuFBO. The localization and expression of TIMP-2 and TIMP-3 in sperm heads was also measured by immunofluorescence analysis. However, MMPs were not detected in the sperm heads. MMP and TIMP expression patterns differed according to the effect of various hormones. These findings suggest that MMPs have a role in sperm viability during capacitation. In conjunction with hormones, MMPs play a role in maintaining capacitation and fertilization by controlling extracellular matrix inhibitors of sperm.

  20. Complexity in differentiating the expression of truncated or matured forms of MMP-2 and MMP-9 through zymography in rat brain tissues after acute ischaemic stroke.

    Science.gov (United States)

    Alam, Mustafa; Shuaib, Ashfaq

    2013-05-30

    Matrix metalloproteinases (MMPs) play an important role in the pathogenesis of ischaemic stroke. In particular, the mature forms of MMPs 2 and 9 have similar sizes and share gelatine as a common substrate. Both MMPs are upregulated in ischaemic stroke and play detrimental roles during stroke pathogenesis. Throughout this study, we demonstrated that pro-MMP-2 and pro-MMP-9 from ischaemic rat brain tissue homogenate is detected either through immunoblotting or zymography because of the remarkable size difference between these enzymes (72 versus 95 kDa, respectively). However, the mature MMP-2 and MMP-9 cannot be discriminated through zymography because of the almost identical sizes of these forms (66 and 67 kDa, respectively). The use of gelatine zymography on ischaemic rat brain tissue homogenate revealed a 65-kDa MMP band, corresponding to the heterogeneous band of mature MMP-2 and/or MMP-9. Furthermore, we also detected mature MMPs of 65 kDa generated from both recombinant human MMP-2 and MMP-9. Using a pull down assay in rat brain tissue homogenate with gelatine-agarose beads, we showed increased activities for both the pro and mature forms of MMP-2 and MMP-9. However, we could not determine the origin of the respective mature MMPs from the heterogeneous band. Thus, in this study, we demonstrated that the identification and quantification of mature MMP-2 and MMP-9 could not be achieved using zymography alone. Therefore, the development of a reliable technique to identify and measure the respective MMPs is needed to test new stroke therapies targeting MMP-2 and MMP-9. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Matrix Metalloproteinases in Cerebral Vasospasm following Aneurysmal Subarachnoid Hemorrhage

    Directory of Open Access Journals (Sweden)

    Vivek Mehta

    2013-01-01

    Full Text Available Delayed cerebral vasospasm is a significant cause of morbidity and mortality following aneurysmal subarachnoid hemorrhage (SAH. While the cellular mechanisms underlying vasospasm remain unclear, it is believed that inflammation may play a critical role in vasospasm. Matrix metalloproteinasees (MMPs are a family of extracellular and membrane-bound proteases capable of degrading the blood-rain barrier (BBB. As such, MMP upregulation following SAH may result in a proinflammatory extravascular environment capable of inciting delayed cerebral vasospasm. This paper presents an overview of MMPs and describes existing data pertinent to delayed cerebral vasospasm.

  2. Kinetic analysis of the inhibition of matrix metalloproteinases: lessons from the study of tissue inhibitors of metalloproteinases.

    Science.gov (United States)

    Willenbrock, Frances; Thomas, Daniel A; Amour, Augustin

    2010-01-01

    Tissue inhibitors of metalloproteinases (TIMPs) are a group of highly potent inhibitors of matrix metalloproteinases (MMPs) and disintegrin metalloproteinases (ADAMs). The high affinity and "tight-binding" nature of the inhibition of MMPs or ADAMs by TIMPs presents challenges for the determination of both equilibrium and dissociation rate constants of these inhibitory events. Methodologies that enable some of these challenges to be overcome are described in this chapter and represent valuable lessons for the in vitro assessment of MMP or ADAM inhibitors within a drug discovery context.

  3. Myelin Formation during Development of the CNS Is Delayed in Matrix Metalloproteinase-9 and -12 Null Mice

    DEFF Research Database (Denmark)

    Larsen, Peter Hjørringgaard; DaSilva, Angelika G.; Conant, Kathrine

    2006-01-01

    The matrix metalloproteinases (MMPs) are implicated in several activities within the nervous system. Although many functions of abnormally elevated MMPs are undesirable, the discrete expression of particular MMP members can have beneficial roles. We previously found that MMP-9 expressed locally...... was correlated with fewer mature oligodendrocytes, but similar precursor cell numbers, in MMP null animals compared with wild type. Because an important growth factor for oligodendrocyte maturation is insulin-like growth factor-1 (IGF-1), we addressed whether this was involved in the deficient myelination in MMP...

  4. Positive Correlation between Matrix Metalloproteinases and Epithelial-to-Mesenchymal Transition and its Association with Clinical Outcome in Bladder Cancer Patients.

    Science.gov (United States)

    Singh, R; Mandhani, A; Agrawal, V; Garg, Minal

    2018-01-18

    Involvement of matrix metalloproteinases (MMPs) in the pathogenesis of urothelial carcinoma elects them to be sensitive marker for clinical and prognostic implications. MMPs regulate tumor growth and invasion by inducing epithelial-to-mesenchymal transition (EMT) which is characterized by the complex reprogramming of epithelial cells and ultimately bring about major changes in the structural organization of bladder urothelium. The present study has been undertaken to evaluate the clinical relevance of MMPs in two distinct types of bladder cancer disease. Expression analysis of MMPs namely MMP-2, MMP-7, MMP-9 and EMT markers including epithelial marker, E-cadherin; mesenchymal markers, N-cadherin and Vimentin; and EMT-activating transcriptional factors (EMT-ATFs), Snail, Slug, Twist and Zeb was done in 64 cases of bladder tumor tissues [{Non-muscle invasive bladder cancer (NMIBC): 35 cases} and {Muscle invasive bladder cancer (MIBC): 29 cases}] by real-time quantitative polymerase chain reaction (RT-qPCR). Immunohistochemistry (IHC) staining was done in matched bladder tumor tissues to evaluate the protein expression and localization of E-cadherin, N-cadherin, Vimentin, Snail, and Slug. Our data showed overexpression of MMP-2, MMP-7 and MMP-9 at transcriptome level in 32.8%, 25% and 37.5% bladder tumor cases respectively. These tumor tissues were examined for higher expression of mesenchymal markers (N-cadherin and Vimentin) at mRNA and protein level and exhibited statistical association with tumor stage and tumor grade (p = 0.02, p = 0.04, Mann-Whitney test). Significant statistical correlation in tumor tissues with overexpressed MMPs has also been observed between gain of transcriptional factors and weak expression of E-cadherin with tumor stage, grade, gender, presence of hematuria and smoking history of the patients. Gene expression patterns of EMT markers in bladder tumors with overexpressed MMPs and their significant association with clinical profile

  5. Studies of microparticles in patients with the antiphospholipid syndrome (APS).

    Science.gov (United States)

    Vikerfors, A; Mobarrez, F; Bremme, K; Holmström, M; Ågren, A; Eelde, A; Bruzelius, M; Antovic, A; Wallén, H; Svenungsson, E

    2012-06-01

    To study circulating platelet, monocyte and endothelial microparticles (PMPs, MMPs and EMPs) in patients with antiphospholipid syndrome (APS) in comparison with healthy controls. Fifty-two patients with APS and 52 healthy controls were investigated. MPs were measured on a flow cytometer (Beckman Gallios) and defined as particles sized APS patients versus controls (p APS patients. We observed a high number of EMPs expressing TF in APS patients. The numbers of MMPs and total EMPs were also higher as compared with healthy controls but in contrast to previous reports, the number of PMPs did not differ between groups.

  6. Influência do pH na atividade funcional de enzimas endógenas (metaloproteinases -2 e -9) que degradam a matriz de colágeno da dentina coronária e radicular humana

    OpenAIRE

    Stella da Silva Ferreira

    2015-01-01

    As metaloproteinases da matriz (MMPs) são uma família de endopeptidades cálcio e zinco dependentes que participam da degradação de praticamente todos os componentes da matriz extracelular. Com o pressuposto de que estas enzimas podem estar relacionadas à progressão da erosão dental e que os agentes ácidos causadores da erosão podem influenciar na ativação das MMPs, o objetivo desse estudo in vitro foi avaliar a influência do pH sobre a atividade funcional das MMP-2 e -9 presentes na dentina c...

  7. (-1607 2G/1G) and MMP-3 (-1612 5A/6A)

    Indian Academy of Sciences (India)

    Hari Om Singh

    2017-07-04

    Jul 4, 2017 ... The pathogenesis of HIV-associated neurocognitive disorder (HAND) is modulated by host genetic susceptibility factors such as Matrix metalloproteinases (MMPs). Promoter polymorphism of MMP-1 and MMP-3 may modify the expression of the gene. Hence, we evaluated the association of ...

  8. GYY4137, a Hydrogen Sulfide Donor Modulates miR194-Dependent Collagen Realignment in Diabetic Kidney.

    Science.gov (United States)

    John, A M Sashi Papu; Kundu, Sourav; Pushpakumar, Sathnur; Fordham, Maura; Weber, Gregory; Mukhopadhyay, Manas; Sen, Utpal

    2017-09-07

    The relationship between hydrogen sulfide (H 2 S), microRNAs (miRs), matrix metalloproteinases (MMPs) and poly-ADP-ribose-polymerase-1 (PARP-1) in diabetic kidney remodeling remains mostly obscured. We aimed at investigating whether alteration of miR-194-dependent MMPs and PARP-1 causes renal fibrosis in diabetes kidney, and whether H 2 S ameliorates fibrosis. Wild type, diabetic Akita mice as well as mouse glomerular endothelial cells (MGECs) were used as experimental models, and GYY4137 as H 2 S donor. In diabetic mice, plasma H 2 S levels were decreased while ROS and expression of its modulator (ROMO1) were increased. In addition, alteration of MMPs-9, -13 and -14 expression, PARP-1, HIF1α, and increased collagen biosynthesis as well as collagen cross-linking protein, P4HA1 and PLOD2 were observed along with diminished vascular density in diabetic kidney. These changes were ameliorated by GYY4137. Further, downregulated miRNA-194 was normalized by GYY4137 in diabetic kidney. Similar results were obtained in in vitro condition. Interestingly, miR-194 mimic also diminished ROS production, and normalized ROMO1, MMPs-9, -13 and -14, and PARP-1 along with collagen biosynthesis and cross-linking protein in HG condition. We conclude that decrease H 2 S diminishes miR-194, induces collagen deposition and realignment leading to fibrosis and renovascular constriction in diabetes. GYY4137 mitigates renal fibrosis in diabetes through miR-194-dependent pathway.

  9. Degradation of the organic phase of bone by osteoclasts

    DEFF Research Database (Denmark)

    Henriksen, Kim; Sørensen, Mette G; Nielsen, Rasmus H

    2006-01-01

    Osteoclasts degrade bone matrix by secretion of hydrochloric acid and proteases. We studied the processes involved in the degradation of the organic matrix of bone in detail and found that lysosomal acidification is involved in this process and that MMPs are capable of degrading the organic matrix...

  10. First-time report of metalloproteinases in fish bile and their potential as bioindicators regarding environmental contamination.

    Science.gov (United States)

    Hauser-Davis, R A; Lima, A A; Ziolli, R L; Campos, R C

    2012-04-01

    Gallbladder bile from 2 fish species, mullet (Mugil liza) and tilapias (Tilapia rendalli), contain substantial matrix metalloproteinases (MMPs). Extensive purification studies were conducted in order to obtain workable samples for SDS-PAGE and zymography analysis. Proteinase activities were assayed by gelatin substrate zymography. Several protein bands were observed, corresponding to molecular weights of 200, 136, 43, 36, 34, 29, 23 and 14 kDa in mullet bile and 179, 97, 79, 61, 54, 45, 36, 33 and 21 kDa in tilapia bile. Specific inhibitor studies were conducted, in which MMPS were inhibited by EDTA and 1,10 phenanthroline, but not by serine and cysteine protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF) and transepoxysuccinyl-l-leucylamido-l-guanidino butane (E-64), confirming the proteinase identities as MMPs. Differences in proteinase expression were observed in fish from a contaminated and reference site. Some studies regarding MMPs in different fish tissues exist, however this is the first study conducted in fish bile, and their involvement in detoxification processes and organism protection against the effects of aquatic contaminants may be a possibility. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. Amniotic Fluid MMP-9 and Neurotrophins in Autism Spectrum Disorders

    DEFF Research Database (Denmark)

    Abdallah, Morsi; Pearce, Brad D; Larsen, Nanna

    2012-01-01

    Evidence suggests that some developmental disorders, such as autism spectrum disorders (ASDs), are caused by errors in brain plasticity. Given the important role of matrix metalloproteinases (MMPs) and neurotrophins (NTs) in neuroplasticity, amniotic fluid samples for 331 ASD cases and 698...

  12. Defective Connective Tissue Remodeling in Smad3 Mice Leads to Accelerated Aneurysmal Growth Through Disturbed Downstream TGF-β Signaling

    Directory of Open Access Journals (Sweden)

    I. van der Pluijm, PhD

    2016-10-01

    Smad3 deficiency leads to imbalanced activation of downstream genes, no activation of MMPs in VSMCs, and immune responses resulting in rapid aortic wall dilatation and rupture. Our findings uncover new possibilities for treatment of SMAD3 patients; instead of targeting TGF-β signaling, immune suppression may be more beneficial.

  13. Journal of Biosciences | Indian Academy of Sciences

    Indian Academy of Sciences (India)

    The pathogenesis of HIV-associated neurocognitive disorder (HAND) is modulated by host genetic susceptibility factorssuch as Matrix metalloproteinases (MMPs). ... Department of Molecular Biology, National AIDS Research Institute, Pune 411 026, India; Department of Clinical Sciences, National AIDS Research Institute, ...

  14. Visualization of Protease Activity In Vivo Using an Activatable Photo-Acoustic Imaging Probe Based on CuS Nanoparticles

    Science.gov (United States)

    Yang, Kai; Zhu, Lei; Nie, Liming; Sun, Xiaolian; Cheng, Liang; Wu, Chenxi; Niu, Gang; Chen, Xiaoyuan; Liu, Zhuang

    2014-01-01

    Herein, we for the first time report a novel activatable photoacoustic (PA) imaging nano-probe for in vivo detection of cancer-related matrix metalloproteinases (MMPs). A black hole quencher 3 (BHQ3) which absorbs red light is conjugated to near-infrared (NIR)-absorbing copper sulfide (CuS) nanoparticles via a MMP-cleavable peptide linker. The obtained CuS-peptide-BHQ3 (CPQ) nano-probe exhibits two distinctive absorption peaks at 630 nm and 930 nm. Inside the tumor microenviorment where MMPs present, the MMP-sensitive peptide would be cleaved, releasing BHQ3 from the CuS nanoparticles, the former of which as a small molecule is then rapidly cleared out from the tumor, whereas the latter of which as large nanoparticles would retain inside the tumor for a much longer period of time. As the result, the PA signal at 680 nm which is contributed by BHQ3 would be quickly diminished while that at 930 nm would be largely retained. The PA signal ratio of 680 nm / 930 nm could thus serve as an in vivo indicator of MMPs activity inside the tumor. Our work presents a novel strategy of in vivo sensing of MMPs based on PA imaging, which should offer remarkably improved detection depth compared with traditional optical imaging techniques. PMID:24465271

  15. Matrix metalloproteinases in gastric inflammation and cancer : clinical relevance and prognostic impact

    NARCIS (Netherlands)

    Kubben, Francois Jozef Gerard Marie

    2007-01-01

    The studies in this thesis describe the clinical impact of several matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in H. pylori-induced gastritis and gastric cancer. In patients with H. pylori-induced gastritis, significantly increased mucosal MMP-9 levels were

  16. Natural antioxidant polyphenols on inflammation management: Anti-glycation activity vs metalloproteinases inhibition.

    Science.gov (United States)

    Crascì, Lucia; Lauro, Maria Rosaria; Puglisi, Giovanni; Panico, Annamaria

    2016-09-20

    The diet polyphenols are a secondary metabolites of plants able to act on inflammation process. Their anti-inflammatory activity is articulated through several mechanisms that are related to their antioxidative and radical scavengers properties. Our work is focused on a novel approach to inflammatory disease management, based on anti-glycative and matrix metalloproteinases (MMPs) inhibition effects, as a connected phenomena. To better understand these correlation, polyphenols Structure-Activity Relationship (SAR) studies were also reported. The antioxidant polyphenols inhibit the AGEs at different levels of the glycation process in the following ways: (1) prevention of Amadori adduct oxidation; (2) trapping reactive dycarbonyl compounds; (3) attenuation of receptor for AGEs (RAGE) expression. Moreover, several flavonoids with radical scavenging property showed also MMPs inhibition interact directly with MMPs or indirectly via radical scavengers and AGEs reduction. The essential polyphenols features involved in these mechanisms are C2-C3 double bond and number and position of hydroxyl, glycosyl and O-methyl groups. These factors induce a change in molecular planarity interfering with the hydrogen bond formation, electron delocalization and metal ion chelation. In particular, C2-C3 double bond improve the antioxidant and MMPs inhibition, while the hydroxylation, glycosylation and methylation induce a positive and negative correlation, respectively.

  17. Aneurysmal disease – current management concepts

    African Journals Online (AJOL)

    increased proteolytic activity of the metallo-matrix proteinases. (MMPs) resulting in degeneration of elastin (smoking related). • atherosclerotic damage to elastin and collagen. • genetic predisposition – increased prevalence of AAAs in the inherited collagen disorders Marfan's syndrome and Ehlers-. Danlos type IV disease, ...

  18. Venous aneurysm complicating arteriovenous fistula access and matrix metalloproteinases

    Directory of Open Access Journals (Sweden)

    Serra Raffaele

    2015-01-01

    Full Text Available Introduction: An arteriovenous fistula (AVF for placed for hemodialysis may be burdened by one particular complication-the formation of a venous aneurysm. It has been shown that matrix metalloproteinases (MMPs and neutrophil gelatinase-associated lipocalin (NGAL could represent markers of disease in both venous and arterial vessels.

  19. TIMP2 gene polymorphisms are associated with hypertension in ...

    Indian Academy of Sciences (India)

    2014-08-06

    Aug 6, 2014 ... Under normal physiological conditions, the activities of MMPs are regu- lated at the level of transcription, activation of the precursor zymogens, TIMPs, and interaction with specific extracellu- lar matrix (ECM) components and are likely to be reflected in the biomechanical properties of this connective tissue.

  20. Muscle-derived matrix metalloproteinase regulates stem cell proliferation in planarians.

    Science.gov (United States)

    Dingwall, Caitlin B; King, Ryan S

    2016-09-01

    Matrix metalloproteinases (MMPs) are a large family of regulatory enzymes that function in extracellular matrix degradation and facilitate a diverse range of cellular processes. Despite the significant focus on the activities of MMPs in human disease, there is a lack of substantial knowledge regarding their normal physiological roles and their role in regulating aspects of stem cell biology. The freshwater planarian Schmidtea mediterranea (S. mediterranea) is an excellent system in which to study robust and nearly unlimited regeneration, guided by a population of mitotically active stem cells, termed neoblasts. We characterized MMPs in the context of planarian stem cells, specifically exploring the role of S. mediterranea MT-MMPB. Using in situ hybridization and available functional genomic tools, we observed that mt-mmpB is expressed in the dorsoventral muscle cells, and its loss results in a reduction in animal size accompanied by a decrease in mitotic cells, suggesting that it plays a unique role in regulating stem cell proliferation. The novel findings of this study bring to light the unique and critical roles that muscles play in regulating neoblast function, and more broadly, highlight the importance of MMPs in stem cell biology. Developmental Dynamics 245:963-970, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  1. MMP-1 polymorphism and its relationship to pathological processes

    Indian Academy of Sciences (India)

    Madhsudhan

    Matrix metalloproteinases (MMPs) are a family of zinc (Zn)-dependent endopeptidases that are collectively capable of cleaving virtually all ..... stromal effectors of human carcinoma progression: therapeutic implications; Matrix Biol. .... Lièvre A, Milet J, Carayol J, Le Corre D, Milan C, Pariente A, Nalet. B, Lafon J, Faivre J, ...

  2. Synthetic inhibitors of matrix metalloproteinases prevent sulfur mustard-induced epidermal-dermal separation in human skin pieces

    NARCIS (Netherlands)

    Mol, M.A.E.; Alblas, S.W.; Hammer, A.; Benschop, H.P.

    2000-01-01

    Degradation of proteins of the basement membrane zone (BMZ) in the skin depends on the activity of proteolytic enzymes, particularly those belonging to the group of matrix metalloproteinases (MMPs). In the present study we have investigated the contribution of these enzymes to the epidermal-dermal

  3. CCR5 delta32, matrix metalloproteinase-9 and disease activity in multiple sclerosis

    DEFF Research Database (Denmark)

    Sellebjerg, Finn; Madsen, Hans O; Jensen, Claus V

    2000-01-01

    Chemokines and matrix metalloproteinases (MMPs) appear to be crucial in leukocyte recruitment to the central nervous system in multiple sclerosis (MS). CCR5 delta32, a truncated allele of the CC chemokine receptor CCR5 gene encoding a non-functional receptor, did not confer protection from MS. CCR5...... targeting CCR5 or treatment with MMP inhibitors may attenuate disease activity in MS...

  4. Relaxin stimulates MMP-2 and alpha-smooth muscle actin expression by human periodontal ligament cells.

    NARCIS (Netherlands)

    Henneman, S.; Bildt, M.M.; Degroot, J.; Kuijpers-Jagtman, A.M.; Hoff, J.W. Von den

    2008-01-01

    The main cells in the periodontal ligament (PDL) are the fibroblasts, which play an important role in periodontal remodelling. Matrix metalloproteinases (MMPs) are largely responsible for the degradation of extracellular matrix proteins in the PDL. Previous studies have indicated that MMP production

  5. Relaxin stimulates MMP-2 and α-smooth muscle actin expression by human periodontal ligament cells

    NARCIS (Netherlands)

    Henneman, S.; Bildt, M.M.; Groot, J. de; Kuijpers-Jagtman, A.M.; Von den Hoff, J.W.

    2008-01-01

    The main cells in the periodontal ligament (PDL) are the fibroblasts, which play an important role in periodontal remodelling. Matrix metalloproteinases (MMPs) are largely responsible for the degradation of extracellular matrix proteins in the PDL. Previous studies have indicated that MMP production

  6. Correlation of matrix metalloproteinase (MMP)-1, -2, -3, and -9 expressions with demographic and radiological features in primary lumbar intervertebral disc disease.

    Science.gov (United States)

    Basaran, Recep; Senol, Mehmet; Ozkanli, Seyma; Efendioglu, Mustafa; Kaner, Tuncay

    2017-07-01

    Degeneration of IVD is a progressive and irreversible process and can be evaluated with immunohistochemical examination or radiological grading. MMPs are a family of proteolytic enzymes and involved in the degradation of the matrix components of the IVD. We aimed to compare MMP-1, -2, -3, and -9 expressions with demographic features, visual analogue scale (VAS), Oswestry Disability Index (ODI) and radiological (MRI) grades. The study involved 60 participants. We recorded data about age, complaint, radiological imaging, expression levels of MMP-1, -2, -3, and -9, ODI and VAS for back pain retrospectively. Intervertebral disc degeneration was graded on a 0-5 scale according to the Pfirrmann classification. As a result of the study, the median age was 52.09±12.74years. There were statistical significances between age and MMP-1, and MMP-2. There was a close correlation between grade and MMP-9. We found correlation between the VAS and the MMP-9 expression. In addition, there was relationship between expression of MMP-2 and MMP-1, MMP-3, MMP-9. In conclusion, the expressions of MMP-1 and -2 are increased with aging. There was no relationship between radiological evaluation of IVDD and aging. Increased expression of MMPs affected IVDD positively. The relationship with MMPs is not explained. This study adds to our understanding of the interaction between MMPs and IVDD. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Key metalloproteinases are expressed by specific cell types in experimental autoimmune encephalomyelitis

    DEFF Research Database (Denmark)

    Toft-Hansen, Henrik; Nuttall, Robert K; Edwards, Dylan R

    2004-01-01

    animal model, experimental autoimmune encephalomyelitis (EAE). We used real-time RT-PCR to profile the expression of all 22 known mouse MMPs, seven ADAMs, and all four known TIMPs in spinal cord from SJL/J mice and mice with adoptively transferred myelin basic protein (MBP)-specific EAE. A significant...

  8. Journal of Biosciences | Indian Academy of Sciences

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Biosciences; Volume 34; Issue 2 ... Review Volume 34 Issue 2 June 2009 pp 313-320 ... Matrix metalloproteinases (MMPs) are a family of zinc (Zn)-dependent endopeptidases that are collectively capable of cleaving virtually all extracellular matrix (ECM) substrates and play an important role in ...

  9. New intracellular activities of matrix metalloproteinases shine in the moonlight.

    Science.gov (United States)

    Jobin, Parker G; Butler, Georgina S; Overall, Christopher M

    2017-11-01

    Adaption of a single protein to perform multiple independent functions facilitates functional plasticity of the proteome allowing a limited number of protein-coding genes to perform a multitude of cellular processes. Multifunctionality is achievable by post-translational modifications and by modulating subcellular localization. Matrix metalloproteinases (MMPs), classically viewed as degraders of the extracellular matrix (ECM) responsible for matrix protein turnover, are more recently recognized as regulators of a range of extracellular bioactive molecules including chemokines, cytokines, and their binders. However, growing evidence has convincingly identified select MMPs in intracellular compartments with unexpected physiological and pathological roles. Intracellular MMPs have both proteolytic and non-proteolytic functions, including signal transduction and transcription factor activity thereby challenging their traditional designation as extracellular proteases. This review highlights current knowledge of subcellular location and activity of these "moonlighting" MMPs. Intracellular roles herald a new era of MMP research, rejuvenating interest in targeting these proteases in therapeutic strategies. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Matrix metalloproteinase-2 is a consistent prognostic factor in gastric cancer.

    NARCIS (Netherlands)

    Kubben, F.J.G.M.; Sier, C.F.M.; Duijn, W. van; Griffioen, G.; Hanemaaijer, R.; Velde, C.J. van de; Krieken, J.H.J.M. van; Lamers, C.B.H.W.; Verspaget, H.W.

    2006-01-01

    In a pioneer study, we showed 10 years ago that enhanced tissue levels of the matrix metalloproteinases (MMPs) MMP-2 and MMP-9 in gastric cancers, as determined by zymography, were related with worse overall survival of the patients. To corroborate these observations, we now assessed MMP-2 and MMP-9

  11. Effect of the anti-tumor necrosis factor-α antibody infliximab on the ex vivo mucosal matrix metalloproteinase-proteolytic phenotype in inflammatory bowel disease

    NARCIS (Netherlands)

    Meijer, M.J.; Mieremet-Ooms, M.A.C.; Duijn, W. van; Zon, A.M. van der; Hanemaaijer, R.; Verheijen, J.H.; Hogezand, R.A. van; Lamers, C.B.H.W.; Verspaget, H.W.

    2007-01-01

    Background: Previous studies have shown an upregulation of matrix metalloproteinases (MMPs) in intestinal tissue of patients with inflammatory bowel disease (IBD) and significant clinical improvement after administration of the anti-TNF-α antibody infliximab. The aims of our study were to determine

  12. Matrix metalloproteinase-2 is a consistent prognostic factor in gastric cancer

    NARCIS (Netherlands)

    Kubben, F.J.G.M.; Sier, C.F.M.; Duijn, W. van; Griffioen, G.; Hanemaaijer, R.; Velde, C.J.H. van de; Krieken, J.H.J.M. van; Lamers, C.B.H.W.; Verspaget, H.W.

    2006-01-01

    In a pioneer study, we showed 10 years ago that enhanced tissue levels of the matrix metalloproteinases (MMPs) MMP-2 and MMP-9 in gastric cancers, as determined by zymography, were related with worse overall survival of the patients. To corroborate these observations, we now assessed MMP-2 and MMP-9

  13. Nobiletin Inhibits Expression of Inflammatory Mediators and ...

    African Journals Online (AJOL)

    chondrocytes thereby enhancing the expression and release of NO and PGE2 via up-regulation of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) [7]. Studies have shown that NO up-regulates the production of MMPs and inflammatory cytokines in OA [8]. Moreover, studies have suggested activated.

  14. A study on the expression levels of matrix metalloproteinases and ...

    African Journals Online (AJOL)

    Conclusion: MMP-2, MMP-7, and MMP-9 are potential targets for therapeutic control of UC. Keywords: Glandular epithelium, Inflammatory cells, Inhibitors, Matrix metalloproteinases (MMPs),. Tissue inhibitors of metalloproteinases, Ulcerative colitis. Tropical Journal of Pharmaceutical Research is indexed by Science ...

  15. Chaiwongsa et al., Afr J Tradit Complement Altern Med. (2013) 10(1 ...

    African Journals Online (AJOL)

    AJTCAM

    MMPs activities in the culture media were analyzed by zymographic techniques. Dexamethasone was used as the positive control. It was found that compound D at the concentration of 10 - 100 μM significantly decreased the mRNA expressions of MMP-1, -2, -3, and -13 which was induced by IL-1Я (P<0.05) concomitantly ...

  16. Active Compound of Zingiber Cassumunar Roxb. Down-Regulates ...

    African Journals Online (AJOL)

    MMPs activities in the culture media were analyzed by zymographic techniques. Dexamethasone was used as the positive control. It was found that compound D at the concentration of 10 - 100 µM significantly decreased the mRNA expressions of MMP-1, -2, -3, and -13 which was induced by IL-1ß (P<0.05) concomitantly ...

  17. Induced sputum MMP-1, -3 & -8 concentrations during treatment of tuberculosis.

    Directory of Open Access Journals (Sweden)

    Cesar A Ugarte-Gil

    Full Text Available Tuberculosis (TB destroys lung tissues and this immunopathology is mediated in part by Matrix Metalloproteinases (MMPs. There are no data on the relationship between local tissue MMPs concentrations, anti-tuberculosis therapy and sputum conversion.Induced sputum was collected from 68 TB patients and 69 controls in a cross-sectional study. MMPs concentrations were measured by Luminex array, TIMP concentrations by ELISA and were correlated with a disease severity score (TBscore. 46 TB patients were then studied longitudinally at the 2nd, 8th week and end of treatment.Sputum MMP-1,-2,-3,-8,-9 and TIMP-1 and -2 concentrations are increased in TB. Elevated MMP-1 and -3 concentrations are independently associated with higher TB severity scores (p<0.05. MMP-1, -3 and -8 concentrations decreased rapidly during treatment (p<0.05 whilst there was a transient increase in TIMP-1/2 concentrations at week 2. MMP-2, -8 and -9 and TIMP-2 concentrations were higher at TB diagnosis in patients who remain sputum culture positive at 2 weeks and MMP-3, -8 and TIMP-1 concentrations were higher in these patients at 2nd week of TB treatment.MMPs are elevated in TB patients and associate with disease severity. This matrix-degrading phenotype resolves rapidly with treatment. The MMP profile at presentation correlates with a delayed treatment response.

  18. Involvement of matrix metalloproteinases and their inhibitors in peripheral synovitis and down-regulation by tumor necrosis factor alpha blockade in spondylarthropathy

    NARCIS (Netherlands)

    Vandooren, Bernard; Kruithof, Elli; Yu, David T. Y.; Rihl, Markus; Gu, Jieruo; de Rycke, Leen; van den Bosch, Filip; Veys, Eric M.; de Keyser, Filip; Baeten, Dominique

    2004-01-01

    OBJECTIVE: To investigate the role of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in spondylarthropathy (SpA) synovitis. METHODS: Paired samples of synovial biopsy tissue as well as serum and synovial fluid (SF) from 41 patients with SpA and 20

  19. Chemopreventive effect of Cousinia shulabadensis Attar ...

    African Journals Online (AJOL)

    Here, in this study, we investigated the cytotoxicity effect of Cousinia shulabadensis Attar & Ghahraman extract as well as its impact on MMPs activity using a model of cell line (Fibrosarcoma-Wehi164). To assess anti-invasiveness potentials, a modified zymoanalysis method was used to measure MMP-2 and MMP-9 ...

  20. Matrix Metalloproteinase Stromelysin-1 Triggers a Cascade of Molecular Alterations that leads to stable epithelial-to-Mesenchymal Conversion and a Premalignant Phenotype in Mammary Epithelial Cells

    Energy Technology Data Exchange (ETDEWEB)

    Lochter, A.; Galosy, S.; Muschler, J.; Freedman, N.; Werb, Z.; Bissell, M.J.

    1997-08-11

    Matrix metalloproteinases (MMPs) regulate ductal morphogenesis, apoptosis, and neoplastic progression in mammary epithelial cells. To elucidate the direct effects of MMPs on mammary epithelium, we generated functionally normal cells expressing an inducible autoactivating stromelysin-1 (SL-1) transgene. Induction of SL-1 expression resulted in cleavage of E-cadherin, and triggered progressive phenotypic conversion characterized by disappearance of E-cadherin and catenins from cell-cell contacts, downregulation of cytokeratins, upregulation of vimentin, induction of keratinocyte growth factor expression and activation, and upregulation of endogenous MMPs. Cells expressing SL-1 were unable to undergo lactogenic differentiation and became invasive. Once initiated, this phenotypic conversion was essentially stable, and progressed even in the absence of continued SL-1 expression. These observations demonstrate that inappropriate expression of SL-1 initiates a cascade of events that may represent a coordinated program leading to loss of the differentiated epithelial phenotype and gain of some characteristics of tumor cells. Our data provide novel insights into how MMPs function in development and neoplastic conversion.

  1. Increased MMP-2 activity during intervertebral disc degeneration is correlated to MMP-14 levels

    NARCIS (Netherlands)

    Rutges, J. P. H. J.; Kummer, J. A.; Oner, F. C.; Verbout, A. J.; Roestenburg, H. J. A.; Dhert, W. J. A.; Creemers, L. B.

    Intervertebral disc (IVD) degeneration is associated with the increased expression of several matrix metalloproteinases (MMPs), in particular MMP-2. However, little is known about the actual activity of MMP-2 in healthy and degenerated discs, or what mechanisms are involved in its activation. A

  2. Matrix metalloproteinases and tissue inhibitors of metalloproteinases in gingival crevicular fluid during orthodontic tooth movement

    NARCIS (Netherlands)

    Bildt, Miriam; Bloemen, M; Kuijpers-Jagtman, A.M.; Von Den Hoff, Johannes W

    2009-01-01

    Orthodontic tooth movement requires extensive re-modelling of the periodontium. Matrix metalloproteinases (MMPs) degrade the extracellular matrix during re-modelling, while their activity is regulated by the tissue inhibitors of metalloproteinases (TIMPs). The aim of this study was to investigate

  3. Matrix metalloproteinases and tissue inhibitors of metalloproteinases in gingival crevicular fluid during orthodontic tooth movement.

    NARCIS (Netherlands)

    Bildt, M.M.; Bloemen, M.; Kuijpers-Jagtman, A.M.; Hoff, J.W. Von den

    2009-01-01

    Orthodontic tooth movement requires extensive re-modelling of the periodontium. Matrix metalloproteinases (MMPs) degrade the extracellular matrix during re-modelling, while their activity is regulated by the tissue inhibitors of metalloproteinases (TIMPs). The aim of this study was to investigate

  4. Increased matrix metalloproteinase-8 and -9 activity in patients with infarct rupture after myocardial infarction

    NARCIS (Netherlands)

    Borne, S.W.M. van den; Cleutjens, J.P.M.; Hanemaaijer, R.; Creemers, E.E.; Smits, J.F.M.; Daemen, M.J.A.P.; Blankesteijn, W.M.

    2009-01-01

    Background: Infarct rupture is a usually fatal complication of myocardial infarction (MI), for which no molecular mechanism has been described in humans. Experimental evidence in mouse models suggests that the degradation of the extracellular matrix by matrix metalloproteinases (MMPs) plays an

  5. Tissue levels of matrix metalloproteinases MMP-2 and MMP-9 are related to the overall survival of patients with gastric carcinoma

    NARCIS (Netherlands)

    Sier, C.F.M.; Kubben, F.J.G.M.; Ganesh, S.; Heerding, M.M.; Griffioen, G.; Hanemaaijer, R.; Krieken, J.H.J.M. van; Lamers, C.B.H.W.; Verspaget, H.W.

    1996-01-01

    Proteinases are involved in tumour invasion and metastasis. Several matrix metalloproteinases (MMPs) have been shown to be increased in various human carcinomas. We assessed the levels of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) in 50 gastric carcinomas and corresponding mucosa using

  6. In situ localization of gelatinolytic activity in the extracellular matrix of metastases of colon cancer in rat liver using quenched fluorogenic DQ-gelatin

    NARCIS (Netherlands)

    Mook, Olaf R. F.; van Overbeek, Claudia; Ackema, Eleonora G.; van Maldegem, Febe; Frederiks, Wilma M.

    2003-01-01

    Matrix metalloproteinases (MMPs) such as gelatinases are believed to play an important role in invasion and metastasis of cancer. In this study we investigated the possible role of MMP-2 and MMP-9 in an experimental model of colon cancer metastasis in rat liver. We demonstrated with gelatin

  7. Involvement of matrix metalloproteinases in chronic Q fever

    NARCIS (Netherlands)

    Jansen, A.F.M.; Schoffelen, T.; Textoris, J.; Mege, J.L.; Bleeker-Rovers, C.P.; Roest, H.I.J.; Wever, P.C.; Joosten, L.A.B.; Netea, M.G.; Vosse, van de E.; Deuren, van M.

    2017-01-01

    Objectives: Chronic Q fever is a persistent infection with the intracellular Gram-negative bacterium Coxiella burnetii, which can lead to complications of infected aneurysms. Matrix metalloproteinases (MMPs) cleave extracellular matrix and are involved in infections as well as aneurysms. We aimed to

  8. Matrix metalloproteinase-12 (MMP-12) in osteoclasts

    DEFF Research Database (Denmark)

    Hou, Peng; Troen, Tine; Ovejero, Maria C

    2004-01-01

    Osteoclasts require matrix metalloproteinase (MMP) activity and cathepsin K to resorb bone, but the critical MMP has not been identified. Osteoclasts express MMP-9 and MMP-14, which do not appear limiting for resorption, and the expression of additional MMPs is not clear. MMP-12, also called...

  9. Doxycycline inhibits collagen synthesis by bovine chondrocytes cultured in alginate

    NARCIS (Netherlands)

    Beekman, B.; Verzijl, N.; Roos, J.A.D.M.de; Koopman, J.L.; Tekoppele, J.M.

    1997-01-01

    Doxycycline is known for its ability to inhibit matrix metalloproteinases (MMPs), a family of enzymes that play a role in cartilage breakdown in arthritides. Its prophylactic effect in reducing joint degradation in osteoarthritis is mainly attributed to this property. In this study, we show that

  10. Transforming Growth Factor-Beta and Matrix Metalloproteinases: Functional Interactions in Tumor Stroma-Infiltrating Myeloid Cells

    Directory of Open Access Journals (Sweden)

    Jelena Krstic

    2014-01-01

    Full Text Available Transforming growth factor-beta (TGF-β is a pleiotropic factor with several different roles in health and disease. In tumorigenesis, it may act as a protumorigenic factor and have a profound impact on the regulation of the immune system response. Matrix metalloproteinases (MMPs are a family that comprises more than 25 members, which have recently been proposed as important regulators acting in tumor stroma by regulating the response of noncellular and cellular microenvironment. Tumor stroma consists of several types of resident cells and infiltrating cells derived from bone marrow, which together play crucial roles in the promotion of tumor growth and metastasis. In cancer cells, TGF-β regulates MMPs expression, while MMPs, produced by either cancer cells or residents’ stroma cells, activate latent TGF-β in the extracellular matrix, together facilitating the enhancement of tumor progression. In this review we will focus on the compartment of myeloid stroma cells, such as tumor-associated macrophages, neutrophils, and dendritic and mast cells, which are potently regulated by TGF-β and produce large amounts of MMPs. Their interplay and mutual implications in the generation of pro-tumorigenic cancer microenvironment will be analyzed.

  11. Application of vacuum metallurgy to separate pure metal from mixed metallic particles of crushed waste printed circuit board scraps.

    Science.gov (United States)

    Zhan, Lu; Xu, Zhenming

    2008-10-15

    The principle of separating pure metal from mixed metallic particles (MMPs) byvacuum metallurgy is that the vapor pressures of various metals at the same temperature are different As a result, the metal with high vapor pressure and low boiling point can be separated from the mixed metals through distillation or sublimation, and then it can be recycled through condensation under a certain condition. The vacuum metallurgy separation (VMS) of MMPs of crushed waste printed circuit boards (WPCBs) has been studied in this paper. Theoretical analyses show that the MMPs (copper, zinc, bismuth, lead, and indium, for example) can be separated by vacuum metallurgy. The copper particles (0.15-0.20 mm) and zinc particles (<0.30 mm) were chosen to simulate the MMPs of crushed WPCBs. Experimental results show that the separated efficiency of zinc in the copper-rich particles achieves 96.19 wt % when the vacuum pressure is 0.01-0.10 Pa, the heating temperature is 1123 K, and the heating time is 105 min. Under this operation condition, the separated efficiency of zinc in the copper-rich particles from crushed WPCBs achieves 97.00 wt % and the copper purity increases from 90.68 to 99.84 wt %.

  12. Insights into the degradation of human elastin by matrilysin-1

    DEFF Research Database (Denmark)

    Heinz, Andrea; Taddese, Samuel; Sippl, Wolfgang

    2011-01-01

    Human matrilysin-1 (MMP-7) is one of the most potent elastases besides macrophage elastase in the family of matrix metalloproteinases (MMPs). It has been reported to provide macrophages with the highest elastinolytic capacity and plays key roles in diseases such as emphysema and cancer. Describin...

  13. The effect of chronic periodontitis on serum levels of matrix ...

    African Journals Online (AJOL)

    A complex network of chemokines and pro- and anti-inflammatory mediators is involved in the initiation and progression of chronic periodontitis. Matrix metalloproteinases (MMPs), the main enzymes responsible for matrix degradation, are important for periodontal tissue destruction, but their activity can be inhibited by tissue ...

  14. Increments in cytokines and matrix metalloproteinases in skeletal muscle after injection of tissue-damaging toxins from the venom of the snake Bothrops asper

    Directory of Open Access Journals (Sweden)

    Alexandra Rucavado

    2002-01-01

    Full Text Available Envenomations by the snake Bothrops asper are characterized by prominent local tissue damage (i.e. myonecrosis, blistering, hemorrhage and edema. Various phospholipases A2 and metalloproteinases that induce local pathological alterations have been purified from this venom. Since these toxins induce a conspicuous inflammatory response, it has been hypothesized that inflammatory mediators may contribute to the local pathological alterations described. This study evaluated the local production of cytokines and matrix metalloproteinases (MMPs as a consequence of intramuscular injections of an Asp-49 myotoxic phospholipase A2 (myotoxin III (MT-III and a P-I type hemorrhagic metalloproteinase (BaP1 isolated from B. asper venom. Both enzymes induced prominent tissue alterations and conspicuous increments in interleukin (IL-1β, IL-6 and a number of MMPs, especially gelatinase MMP-9, rapidly after injection. In contrast, no increments in tumor necrosis factor-α (TNF-α and interferon-γ were detected. In agreement, MT-III and BaP1 did not induce the synthesis of TNF-α by resident peritoneal macrophages in vitro. Despite the conspicuous expression of latent forms of MMPs in muscle, evidenced by zymography, there were no increments in activated MMP-2 and only a small increase in activated MMP-9, as detected by a functional enzymatic assay. This suggests that MMP activity was regulated by a highly controlled activation of latent forms and, probably, by a concomitant synthesis of MMP inhibitors. Since no hemorrhage nor dermonecrosis were observed after injection of MT-III, despite a prominent increase in MMP expression, and since inflammatory exudate did not enhance hemorrhage induced by BaP1, it is suggested that endogenous MMPs released in the tissue are not responsible for the dermonecrosis and hemorrhage characteristic of B. asper envenomation. Moreover, pretreatment of mice with the peptidomimetic MMP inhibitor batimastat did not reduce myotoxic nor

  15. Matrix metalloproteinases in a sea urchin ligament with adaptable mechanical properties.

    Directory of Open Access Journals (Sweden)

    Ana R Ribeiro

    Full Text Available Mutable collagenous tissues (MCTs of echinoderms show reversible changes in tensile properties (mutability that are initiated and modulated by the nervous system via the activities of cells known as juxtaligamental cells. The molecular mechanism underpinning this mechanical adaptability has still to be elucidated. Adaptable connective tissues are also present in mammals, most notably in the uterine cervix, in which changes in stiffness result partly from changes in the balance between matrix metalloproteinases (MMPs and tissue inhibitors of metalloproteinases (TIMPs. There have been no attempts to assess the potential involvement of MMPs in the echinoderm mutability phenomenon, apart from studies dealing with a process whose relationship to the latter is uncertain. In this investigation we used the compass depressor ligaments (CDLs of the sea-urchin Paracentrotus lividus. The effect of a synthetic MMP inhibitor - galardin - on the biomechanical properties of CDLs in different mechanical states ("standard", "compliant" and "stiff" was evaluated by dynamic mechanical analysis, and the presence of MMPs in normal and galardin-treated CDLs was determined semi-quantitatively by gelatin zymography. Galardin reversibly increased the stiffness and storage modulus of CDLs in all three states, although its effect was significantly lower in stiff than in standard or compliant CDLs. Gelatin zymography revealed a progressive increase in total gelatinolytic activity between the compliant, standard and stiff states, which was possibly due primarily to higher molecular weight components resulting from the inhibition and degradation of MMPs. Galardin caused no change in the gelatinolytic activity of stiff CDLs, a pronounced and statistically significant reduction in that of standard CDLs, and a pronounced, but not statistically significant, reduction in that of compliant CDLs. Our results provide evidence that MMPs may contribute to the variable tensility of the

  16. Planarians as a model to assess in vivo the role of matrix metalloproteinase genes during homeostasis and regeneration.

    Directory of Open Access Journals (Sweden)

    Maria Emilia Isolani

    Full Text Available Matrix metalloproteinases (MMPs are major executors of extracellular matrix remodeling and, consequently, play key roles in the response of cells to their microenvironment. The experimentally accessible stem cell population and the robust regenerative capabilities of planarians offer an ideal model to study how modulation of the proteolytic system in the extracellular environment affects cell behavior in vivo. Genome-wide identification of Schmidtea mediterranea MMPs reveals that planarians possess four mmp-like genes. Two of them (mmp1 and mmp2 are strongly expressed in a subset of secretory cells and encode putative matrilysins. The other genes (mt-mmpA and mt-mmpB are widely expressed in postmitotic cells and appear structurally related to membrane-type MMPs. These genes are conserved in the planarian Dugesia japonica. Here we explore the role of the planarian mmp genes by RNA interference (RNAi during tissue homeostasis and regeneration. Our analyses identify essential functions for two of them. Following inhibition of mmp1 planarians display dramatic disruption of tissues architecture and significant decrease in cell death. These results suggest that mmp1 controls tissue turnover, modulating survival of postmitotic cells. Unexpectedly, the ability to regenerate is unaffected by mmp1(RNAi. Silencing of mt-mmpA alters tissue integrity and delays blastema growth, without affecting proliferation of stem cells. Our data support the possibility that the activity of this protease modulates cell migration and regulates anoikis, with a consequent pivotal role in tissue homeostasis and regeneration. Our data provide evidence of the involvement of specific MMPs in tissue homeostasis and regeneration and demonstrate that the behavior of planarian stem cells is critically dependent on the microenvironment surrounding these cells. Studying MMPs function in the planarian model provides evidence on how individual proteases work in vivo in adult tissues

  17. Planarians as a model to assess in vivo the role of matrix metalloproteinase genes during homeostasis and regeneration.

    Science.gov (United States)

    Isolani, Maria Emilia; Abril, Josep F; Saló, Emili; Deri, Paolo; Bianucci, Anna Maria; Batistoni, Renata

    2013-01-01

    Matrix metalloproteinases (MMPs) are major executors of extracellular matrix remodeling and, consequently, play key roles in the response of cells to their microenvironment. The experimentally accessible stem cell population and the robust regenerative capabilities of planarians offer an ideal model to study how modulation of the proteolytic system in the extracellular environment affects cell behavior in vivo. Genome-wide identification of Schmidtea mediterranea MMPs reveals that planarians possess four mmp-like genes. Two of them (mmp1 and mmp2) are strongly expressed in a subset of secretory cells and encode putative matrilysins. The other genes (mt-mmpA and mt-mmpB) are widely expressed in postmitotic cells and appear structurally related to membrane-type MMPs. These genes are conserved in the planarian Dugesia japonica. Here we explore the role of the planarian mmp genes by RNA interference (RNAi) during tissue homeostasis and regeneration. Our analyses identify essential functions for two of them. Following inhibition of mmp1 planarians display dramatic disruption of tissues architecture and significant decrease in cell death. These results suggest that mmp1 controls tissue turnover, modulating survival of postmitotic cells. Unexpectedly, the ability to regenerate is unaffected by mmp1(RNAi). Silencing of mt-mmpA alters tissue integrity and delays blastema growth, without affecting proliferation of stem cells. Our data support the possibility that the activity of this protease modulates cell migration and regulates anoikis, with a consequent pivotal role in tissue homeostasis and regeneration. Our data provide evidence of the involvement of specific MMPs in tissue homeostasis and regeneration and demonstrate that the behavior of planarian stem cells is critically dependent on the microenvironment surrounding these cells. Studying MMPs function in the planarian model provides evidence on how individual proteases work in vivo in adult tissues. These results

  18. Understanding the binding of inhibitors of matrix metalloproteinases by molecular docking, quantum mechanical calculations, molecular dynamics simulations, and a MMGBSA/MMBappl study.

    Science.gov (United States)

    Singh, Tanya; Adekoya, Olayiwola Adedotun; Jayaram, B

    2015-04-01

    Matrix metalloproteinases (MMPs) consist of a class of proteins required for normal tissue function. Their over expression is associated with many disease states and hence the interest in MMPs as drug targets. Almost all MMP inhibitors have been reported to fail in clinical trials due to lack of specificity. Zinc in the binding site of metalloproteinases performs essential biological functions and contributes to the binding affinity of inhibitors. The multiple possibilities for coordination geometry and the consequent charge on the zinc atom indicate that parameters developed are not directly transferable across different families of zinc metalloproteinases with different zinc coordination geometries, active sites and ligand architectures which makes it difficult to evaluate metal-ligand interactions. In order to assist in drug design endeavors for MMP targets, a computationally tractable pathway is presented, comprising docking of small molecule inhibitors against the target MMPs, derivation of quantum mechanical charges on the zinc ion in the active site and the amino acids coordinating with zinc including the inhibitor molecule, molecular dynamics simulations on the docked ligand-MMP complexes and evaluation of binding affinities of the ligand-MMP complexes via an accurate scoring function for zinc containing metalloprotein-ligand complexes. The above pathway was applied to study the interaction of inhibitor Batimastat with MMPs, which resulted in a high correlation between the predicted binding free energies and experiment, suggesting the potential applicability of the pathway. We then proceeded to formulate a few design principles which identify the key protein residues for generating molecules with high affinity and specificity against each of the MMPs.

  19. Gene expression levels of matrix metalloproteinases in human atherosclerotic plaques and evaluation of radiolabeled inhibitors as imaging agents for plaque vulnerability

    International Nuclear Information System (INIS)

    Müller, Adrienne; Krämer, Stefanie D.; Meletta, Romana; Beck, Katharina; Selivanova, Svetlana V.; Rancic, Zoran; Kaufmann, Philipp A.; Vos, Bernhard; Meding, Jörg; Stellfeld, Timo; Heinrich, Tobias K.; Bauser, Marcus; Hütter, Joachim; Dinkelborg, Ludger M.; Schibli, Roger; Ametamey, Simon M.

    2014-01-01

    Introduction: Atherosclerotic plaque rupture is the primary cause for myocardial infarction and stroke. During plaque progression macrophages and mast cells secrete matrix-degrading proteolytic enzymes, such as matrix metalloproteinases (MMPs). We studied levels of MMPs and tissue inhibitor of metalloproteinases-3 (TIMP-3) in relation to the characteristics of carotid plaques. We evaluated in vitro two radiolabeled probes targeting active MMPs towards non-invasive imaging of rupture-prone plaques. Methods: Human carotid plaques obtained from endarterectomy were classified into stable and vulnerable by visual and histological analysis. MMP-1, MMP-2, MMP-8, MMP-9, MMP-10, MMP-12, MMP-14, TIMP-3, and CD68 levels were investigated by quantitative polymerase chain reaction. Immunohistochemistry was used to localize MMP-2 and MMP-9 with respect to CD68-expressing macrophages. Western blotting was applied to detect their active forms. A fluorine-18-labeled MMP-2/MMP-9 inhibitor and a tritiated selective MMP-9 inhibitor were evaluated by in vitro autoradiography as potential lead structures for non-invasive imaging. Results: Gene expression levels of all MMPs and CD68 were elevated in plaques. MMP-1, MMP-9, MMP-12 and MMP-14 were significantly higher in vulnerable than stable plaques. TIMP-3 expression was highest in stable and low in vulnerable plaques. Immunohistochemistry revealed intensive staining of MMP-9 in vulnerable plaques. Western blotting confirmed presence of the active form in plaque lysates. In vitro autoradiography showed binding of both inhibitors to stable and vulnerable plaques. Conclusions: MMPs differed in their expression patterns among plaque phenotypes, providing possible imaging targets. The two tested MMP-2/MMP-9 and MMP-9 inhibitors may be useful to detect atherosclerotic plaques, but not the vulnerable lesions selectively

  20. Association of cytokine and matrix metalloproteinase profiles with disease activity and function in ankylosing spondylitis

    Science.gov (United States)

    2012-01-01

    Introduction The pathology of ankylosing spondylitis (AS) suggests that certain cytokines and matrix metalloproteinases (MMPs) might provide useful markers of disease activity. Serum levels of some cytokines and MMPs have been found to be elevated in active disease, but there is a general lack of information about biomarker profiles in AS and how these are related to disease activity and function. The purpose of this study was to investigate whether clinical measures of disease activity and function in AS are associated with particular profiles of circulating cytokines and MMPs. Methods Measurement of 30 cytokines, five MMPs and four tissue inhibitors of metalloproteinases was carried out using Luminex® technology on a well-characterised population of AS patients (n = 157). The relationship between biomarker levels and measures of disease activity (Bath ankylosing spondylitis disease activity index (BASDAI)), function (Bath ankylosing spondylitis functional index) and global health (Bath ankylosing spondylitis global health) was investigated. Principal component analysis was used to reduce the large number of biomarkers to a smaller set of independent components, which were investigated for their association with clinical measures. Further analyses were carried out using hierarchical clustering, multiple regression or multivariate logistic regression. Results Principal component analysis identified eight clusters consisting of various combinations of cytokines and MMPs. The strongest association with the BASDAI was found with a component consisting of MMP-8, MMP-9, hepatocyte growth factor and CXCL8, and was independent of C-reactive protein levels. This component was also associated with current smoking. Hierarchical clustering revealed two distinct patient clusters that could be separated on the basis of MMP levels. The high MMP cluster was associated with increased C-reactive protein, the BASDAI and the Bath ankylosing spondylitis functional index. Conclusions

  1. Extracellular matrix metalloproteinase inducer expression in the baboon endometrium: menstrual cycle and endometriosis.

    Science.gov (United States)

    Braundmeier, A G; Fazleabas, A T; Nowak, R A

    2010-12-01

    Extracellular matrix metalloproteinase inducer (EMMPRIN; BSG) regulates tissue remodeling through matrix metalloproteinases (MMPs). In human and non-human primates, endometrial remodeling is important for menstruation and the pathogenesis of endometriosis. We hypothesized that as in humans, BSG and MMPs are expressed in the endometrium of cycling baboons, and their expression is hormonally regulated by ovarian hormones, but endometriosis disrupts this regulation. BSG expression was evaluated in the baboon endometrium by q-PCR and immunohistochemistry. In the endometrium of control cycling animals, BSG mRNA levels were highest in late secretory stage tissue. BSG protein localized to glandular epithelial cells during the proliferative phase; whereas, secretory stage tissues expressed BSG in glandular and luminal epithelia with weak stromal staining. Several MMPs were differentially expressed throughout the menstrual cycle with the highest levels found during menstruation. In ovariectomized animals, BSG endometrial mRNA levels were highest with treatment of both estrogen and progesterone than that with only estrogen. Estrogen alone resulted in BSG protein localization primarily in the endometrial glandular epithelia, while estrogen and progesterone treatment displayed BSG protein localization in both the glandular and stromal cells. Exogenous hormone treatment resulted in differential expression patterns of all MMPs compared with the control cycling animals. In the eutopic endometrium of endometriotic animals, BSG mRNA levels and protein were elevated early but decreased later in disease progression. Endometriosis elevated the expression of all MMPs except MMP7 compared with the control animals. In baboons, BSG and MMP endometrial expression is regulated by both ovarian hormones, and their expression patterns are dysregulated in endometriotic animals.

  2. Serratia marcescens suppresses host cellular immunity via the production of an adhesion-inhibitory factor against immunosurveillance cells.

    Science.gov (United States)

    Ishii, Kenichi; Adachi, Tatsuo; Hamamoto, Hiroshi; Sekimizu, Kazuhisa

    2014-02-28

    Injection of a culture supernatant of Serratia marcescens into the bloodstream of the silkworm Bombyx mori increased the number of freely circulating immunosurveillance cells (hemocytes). Using a bioassay with live silkworms, serralysin metalloprotease was purified from the culture supernatant and identified as the factor responsible for this activity. Serralysin inhibited the in vitro attachment of both silkworm hemocytes and murine peritoneal macrophages. Incubation of silkworm hemocytes or murine macrophages with serralysin resulted in degradation of the cellular immune factor BmSPH-1 or calreticulin, respectively. Furthermore, serralysin suppressed in vitro phagocytosis of bacteria by hemocytes and in vivo bacterial clearance in silkworms. Disruption of the ser gene in S. marcescens attenuated its host killing ability in silkworms and mice. These findings suggest that serralysin metalloprotease secreted by S. marcescens suppresses cellular immunity by decreasing the adhesive properties of immunosurveillance cells, thereby contributing to bacterial pathogenesis.

  3. Binding of ADAM12, a marker of skeletal muscle regeneration, to the muscle-specific actin-binding protein, alpha -actinin-2, is required for myoblast fusion

    DEFF Research Database (Denmark)

    Galliano, M F; Huet, C; Frygelius, J

    2000-01-01

    of differentiation. Using the yeast two-hybrid screen, we found that the muscle-specific alpha-actinin-2 strongly binds to the cytoplasmic tail of ADAM12. In vitro binding assays with GST fusion proteins confirmed the specific interaction. The major binding site for alpha-actinin-2 was mapped to a short sequence......ADAM12 belongs to the transmembrane metalloprotease ADAM ("a disintegrin and metalloprotease") family. ADAM12 has been implicated in muscle cell differentiation and fusion, but its precise function remains unknown. Here, we show that ADAM12 is dramatically up-regulated in regenerated, newly formed...... in a dominant negative fashion by inhibiting fusion of C2C12 cells, whereas expression of a cytosolic ADAM12 lacking the major alpha-actinin-2 binding site had no effect on cell fusion. Our results suggest that interaction of ADAM12 with alpha-actinin-2 is important for ADAM12 function....

  4. Effect of protease inhibitors on exflagellation in Plasmodium falciparum.

    Science.gov (United States)

    Rupp, Ingrid; Bosse, Rebecca; Schirmeister, Tanja; Pradel, Gabriele

    2008-04-01

    Enzymes involved in sexual differentiation and fertilization of the human malaria parasite Plasmodium falciparum represent potential targets for transmission blocking strategies. Parasite proteases are putatively involved in several steps during fertilization, but the types of proteases, their targets and modes of action remain hitherto unknown. We investigated the involvement of proteases in gametogenesis via exflagellation and immunofluorescence assays, using a variety of commercially available as well as newly designed protease inhibitors. The assays revealed a blockade of microgamete formation by the cysteine/serine protease inhibitors TLCK and TPCK. The serine protease inhibitor PMSF, the falcipain-targeting inhibitor RV112D, and the aspartic protease inhibitor EPNP also significantly decreased formation of microgametes. The metalloprotease inhibitor 1,10-phenanthroline, on the other hand, inhibited exflagellation by interfering with microgamete motility. Furthermore, EPNP reduced the activation of male and female gametocytes. Our data point to a major involvement of serine proteases and a non-thermolysin-like zinc metalloprotease in microgametocyte exflagellation.

  5. Serratia marcescens Suppresses Host Cellular Immunity via the Production of an Adhesion-inhibitory Factor against Immunosurveillance Cells*

    Science.gov (United States)

    Ishii, Kenichi; Adachi, Tatsuo; Hamamoto, Hiroshi; Sekimizu, Kazuhisa

    2014-01-01

    Injection of a culture supernatant of Serratia marcescens into the bloodstream of the silkworm Bombyx mori increased the number of freely circulating immunosurveillance cells (hemocytes). Using a bioassay with live silkworms, serralysin metalloprotease was purified from the culture supernatant and identified as the factor responsible for this activity. Serralysin inhibited the in vitro attachment of both silkworm hemocytes and murine peritoneal macrophages. Incubation of silkworm hemocytes or murine macrophages with serralysin resulted in degradation of the cellular immune factor BmSPH-1 or calreticulin, respectively. Furthermore, serralysin suppressed in vitro phagocytosis of bacteria by hemocytes and in vivo bacterial clearance in silkworms. Disruption of the ser gene in S. marcescens attenuated its host killing ability in silkworms and mice. These findings suggest that serralysin metalloprotease secreted by S. marcescens suppresses cellular immunity by decreasing the adhesive properties of immunosurveillance cells, thereby contributing to bacterial pathogenesis. PMID:24398686

  6. Localization of Tissue Inhibitor of Metalloproteinases 1 (TIMP-1) in Human Colorectal Adenoma and Adenocarcinoma

    DEFF Research Database (Denmark)

    Holten-Andersen, Mads N.; Hansen, Ulla; Brünner, Nils

    2005-01-01

    Tissue inhibitor of matrix metalloproteases 1 (TIMP-1) inhibits the proteolytic activity of matrix metalloproteases and hereby prevents cancer invasion. However, TIMP-1 also possesses other functions such as inhibition of apoptosis, induction of malignant transformation and stimulation of cell......-growth. We have previously demonstrated that TIMP-1 is elevated in blood from colorectal cancer patients and that high TIMP-1 levels predict poor prognosis. To clarify the role of TIMP-1 in colorectal tumorigenesis, the expression pattern of TIMP-1 in benign and malignant colorectal tumors was studied....... In all of 24 cases of colorectal adenocarcinoma TIMP-1 mRNA was detected by in situ hybridization. In all cases TIMP-1 expression was found in fibroblast-like cells located at the invasive front but was seen only sporadically in normal mucosa. No TIMP-1 mRNA was seen in any of the cases in benign...

  7. ADAM10 mediates vascular injury induced by Staphylococcus aureus α-hemolysin.

    Science.gov (United States)

    Powers, Michael E; Kim, Hwan Keun; Wang, Yang; Bubeck Wardenburg, Juliane

    2012-08-01

    Staphylococcus aureus is a leading cause of bacteremia and sepsis. The interaction of S. aureus with the endothelium is central to bloodstream infection pathophysiology yet remains ill-understood. We show herein that staphylococcal α-hemolysin, a pore-forming cytotoxin, is required for full virulence in a murine sepsis model. The α-hemolysin binding to its receptor A-disintegrin and metalloprotease 10 (ADAM10) upregulates the receptor's metalloprotease activity on endothelial cells, causing vascular endothelial-cadherin cleavage and concomitant loss of endothelial barrier function. These cellular injuries and sepsis severity can be mitigated by ADAM10 inhibition. This study therefore provides mechanistic insight into toxin-mediated endothelial injury and suggests new therapeutic approaches for staphylococcal sepsis.

  8. Activation of ADAM 12 protease by copper

    DEFF Research Database (Denmark)

    Loechel, F; Wewer, Ulla M.

    2001-01-01

    Conversion of latent proteases to the active form occurs by various mechanisms characteristic for different protease families. Here we report that the disintegrin metalloprotease ADAM 12-S is activated by Cu(II). Copper activation is distinct from the cysteine switch component of latency: elimina......Conversion of latent proteases to the active form occurs by various mechanisms characteristic for different protease families. Here we report that the disintegrin metalloprotease ADAM 12-S is activated by Cu(II). Copper activation is distinct from the cysteine switch component of latency......: elimination of the ADAM 12 cysteine switch by a point mutation in the propeptide had no effect on copper activation, whereas mutation of an unpaired cysteine residue in the catalytic domain resulted in a mutant form of ADAM 12-S that was insensitive to copper. This suggests a multi-step activation mechanism...... for ADAM 12 involving both furin cleavage and copper binding....

  9. Regulation of ADAM12 cell-surface expression by protein kinase C epsilon

    DEFF Research Database (Denmark)

    Sundberg, Christina; Thodeti, Charles Kumar; Kveiborg, Marie

    2004-01-01

    The ADAM (a disintegrin and metalloprotease) family consists of multidomain cell-surface proteins that have a major impact on cell behavior. These transmembrane-anchored proteins are synthesized as proforms that have (from the N terminus): a prodomain; a metalloprotease-, disintegrin......-like-, cysteine-rich, epidermal growth factor-like, and transmembrane domain; and a cytoplasmic tail. The 90-kDa mature form of human ADAM12 is generated in the trans-Golgi through cleavage of the prodomain by a furin-peptidase and is stored intracellularly until translocation to the cell surface...... as a constitutively active protein. However, little is known about the regulation of ADAM12 cell-surface translocation. Here, we used human RD rhabdomyosarcoma cells, which express ADAM12 at the cell surface, in a temporal pattern. We report that protein kinase C (PKC) epsilon induces ADAM12 translocation to the cell...

  10. ADAM 12 protease induces adipogenesis in transgenic mice

    DEFF Research Database (Denmark)

    Kawaguchi, Nobuko; Xu, Xiufeng; Tajima, Rie

    2002-01-01

    in the perivascular space in muscle tissue of 1- to 2-week-old transgenic mice whereas mature lipid-laden adipocytes were seen at 3 to 4 weeks. Moreover, female transgenics expressing ADAM 12-S exhibited increases in body weight, total body fat mass, abdominal fat mass, and herniation, but were normoglycemic and did......ADAM 12 (meltrin-alpha) is a member of the ADAM (a disintegrin and metalloprotease) family. ADAM 12 functions as an active metalloprotease, supports cell adhesion, and has been implicated in myoblast differentiation and fusion. Human ADAM 12 exists in two forms: the prototype membrane......-anchored protein, ADAM 12-L, and a shorter secreted form, ADAM 12-S. Here we report the occurrence of adipocytes in the skeletal muscle of transgenic mice in which overexpression of either form is driven by the muscle creatine kinase promoter. Cells expressing a marker of early adipogenesis were apparent...

  11. Thioredoxin and Its Reductase Are Present on Synaptic Vesicles, and Their Inhibition Prevents the Paralysis Induced by Botulinum Neurotoxins

    Directory of Open Access Journals (Sweden)

    Marco Pirazzini

    2014-09-01

    Full Text Available Botulinum neurotoxins consist of a metalloprotease linked via a conserved interchain disulfide bond to a heavy chain responsible for neurospecific binding and translocation of the enzymatic domain in the nerve terminal cytosol. The metalloprotease activity is enabled upon disulfide reduction and causes neuroparalysis by cleaving the SNARE proteins. Here, we show that the thioredoxin reductase-thioredoxin protein disulfide-reducing system is present on synaptic vesicles and that it is functional and responsible for the reduction of the interchain disulfide of botulinum neurotoxin serotypes A, C, and E. Specific inhibitors of thioredoxin reductase or thioredoxin prevent intoxication of cultured neurons in a dose-dependent manner and are also very effective inhibitors of the paralysis of the neuromuscular junction. We found that this group of inhibitors of botulinum neurotoxins is very effective in vivo. Most of them are nontoxic and are good candidates as preventive and therapeutic drugs for human botulism.

  12. Appearance and distribution of regioisomers in metallo- and serine-protease-catalysed acylation of sucrose in N,N-dimethylformamide

    DEFF Research Database (Denmark)

    Lie, Aleksander; Meyer, Anne S.; Pedersen, Lars Haastrup

    2014-01-01

    The appearance and distribution of monoester regioisomers were investigated in the virtually irreversible acylation of sucrose with the enol ester, vinyl laurate, as acyl donor catalysed by serine proteases and a metalloprotease in the hydrophilic, aprotic solvent N,N-dimethylformamide. Sucrose l...... without protein in the reaction mixture appeared to be catalysed in the presence of aluminosilicate molecular sieves. Non-catalytic protein in the reaction medium seemed to lower the catalytic activity of the molecular sieves....

  13. A Novel Approach to Managing Invasive Termite Species Using Genetically Engineered Bacteria

    Science.gov (United States)

    2008-08-01

    respectively). Controls for the inhibitors were the enzymes 1 unit/µl thermolysin (Sigma #P1512), 10 units/ml carboxypeptidase-A (Sigma #C9268), 500...protease inhibitors against the samples. The inhibitors used were EDTA (ethylenediaminetetraacetic acid, 8 mM), a metalloproteases inhibitor , and PMSF...phenylmethylsulfonyl fluoride, 10 mM), a serine protease inhibitor , both of which were purchased from Sigma Aldrich (#EDS-100 g and #P7626-1 G

  14. The Medicinal Chemistry of Botulinum, Ricin and Anthrax Toxins

    Science.gov (United States)

    2005-01-01

    metalloprotease) inhibitors , activity [40]. The process of enzymatic hydrolysis of captopril and phosphoramidon, do not inhibit the catalytic thermolysin ...followed by a discussion on the mechanisms of action of each at the molecular level, and a review of current potential inhibitors under investigation...methodologies used to develop inhibitors of other enzymes of which there are seven different serotypes, are some of the may be employed to develop

  15. Structural basis for the inhibition of M1 family aminopeptidases by the natural product actinonin: Crystal structure in complex with E. coli aminopeptidase N

    OpenAIRE

    Ganji, Roopa Jones; Reddi, Ravikumar; Gumpena, Rajesh; Marapaka, Anil Kumar; Arya, Tarun; Sankoju, Priyanka; Bhukya, Supriya; Addlagatta, Anthony

    2015-01-01

    Actinonin is a pseudotripeptide that displays a high affinity towards metalloproteases including peptide deformylases (PDFs) and M1 family aminopeptidases. PDF and M1 family aminopeptidases belong to thermolysin-metzincin superfamily. One of the major differences in terms of substrate binding pockets between these families is presence (in M1 aminopeptidases) or absence (in PDFs) of an S1 substrate pocket. The binding mode of actinonin to PDFs has been established previously; however, it is no...

  16. Subunit Organization of a Synechocystis Hetero-Oligomeric Thylakoid FtsH Complex Involved in Photosystem II Repair

    Czech Academy of Sciences Publication Activity Database

    Boehm, M.; Yu, J.; Krynická, Vendula; Barker, M.; Tichý, Martin; Komenda, Josef; Nixon, P. J.; Nield, J.

    2012-01-01

    Roč. 24, č. 9 (2012), s. 3669-3683 ISSN 1040-4651 R&D Projects: GA MŠk(CZ) ED2.1.00/03.0110; GA ČR GBP501/12/G055 Institutional support: RVO:61388971 Keywords : photosystem II * FtsH metalloproteases * subunit Subject RIV: EE - Microbiology, Virology Impact factor: 9.251, year: 2012

  17. Sulfur mustard-stimulated proteases and their inhibitors in a cultured normal human epidermal keratinocytes model: A potential approach for anti-vesicant drug development

    Directory of Open Access Journals (Sweden)

    Xiannu Jin

    Full Text Available Protease stimulation in cultured normal human epidermal keratinocytes (NHEK due to sulfur mustard (SM exposure is well documented. However, the specific protease(s stimulated by SM and the protease substrates remain to be determined. In this study, we observed that SM stimulates several proteases and the epidermal-dermal attachment protein laminin-5 is one of the substrates. We propose that following SM exposure of the skin, laminin-5 degradation causes the detachment of the epidermis from the dermis and, therefore, vesication. We utilized gelatin zymography, Western blotting, immuno-fluorescence staining, and real-time polymerase chain reaction (RT-PCR analyses to study the SM-stimulated proteases and laminin-5 degradation in NHEK. Two major protease bands (64 kDa and 72 kDa were observed by zymography in SM-exposed cells. Addition of serine protease inhibitor (aprotinin, 100 μM, or the metalloprotease inhibitor (amastatin, 100 μM to NHEK cultures prior to SM exposure decreased the SM-stimulated protease bands seen by zymography. These inhibitors completely or partially prevented SM-induced laminin-5 γ2 degradation as seen by Western blotting as well as immuno-fluorescence staining. Our results from Western blotting and RT-PCR studies also indicated that the membrane-type matrix metalloproteinase-1 (MT-MM-1 may be involved in SM-induced skin blistering.To summarize, our results in the NHEK model indicate the following: (a SM stimulates multiple proteases including serine protease(s, and metalloproteases; (b SM decreases the level of laminin-5 γ2, which is prevented by either a serine protease inhibitor or a metalloprotease inhibitor and (c MT-MMP-1 maybe one of the proteases that is involved in skin blistering due to SM exposure. Keywords: Sulfur mustard, Serine protease, Metalloprotease, Protease inhibiter, Zymography, Laminin-5 γ2

  18. Mitochondrial Debris as a Discriminator Between Inflammatory and Infectious Complications of Blast Injuries: The Enemy Within

    Science.gov (United States)

    2012-01-01

    Weathington N, et al. Matrix metalloprotease-9 dysregulation in lower airway secretions of cystic fibrosis patients. Am J Physiol Lung Cell Mol Physiol...in austere environments such as forward deployments. In this regard we have ‘fine-tuned’ our PCR-based approach to diagnosis by constantly improving...Multiplex PCR Allows Rapid and Accurate Diagnosis of Bloodstream Infections in Newborns and Children with Suspected Sepsis. Journal of Clinical

  19. Heparan sulfate regulates ADAM12 through a molecular switch mechanism

    DEFF Research Database (Denmark)

    Sørensen, Hans P; Vives, Romain R; Manetopoulos, Christina

    2008-01-01

    The disintegrin and metalloproteases (ADAMs) are emerging as therapeutic targets in human disease, but specific drug design is hampered by potential redundancy. Unlike other metzincins, ADAM pro domains remain bound to the mature enzyme to regulate activity. Here ADAM12, a protease that promotes....... These data present a novel concept that might allow targeting of ADAM12 and suggest that other ADAMs may have specific regulatory activity embedded in their pro and catalytic domain structures....

  20. Aeromonas hydrophila Extracelullar Products Extraction and its Effects on Tilapia Roja (Oreochromis spp. and Cachama Blanca (Piaractus brachypomus

    Directory of Open Access Journals (Sweden)

    M. Rodríguez

    2005-07-01

    of 128 UH/μL and a proteolitic activity of 38.4 UP/μL and produced high fish mortality and severe multiorganic lesions. Electrophoresis revealed bands from 50 to 52 kDa y 63 to 68 kDa, that could correspond to the b hemolisina or a hemolisina y other from 30 to 36 kDa that could correspond to the thermostable metalloprotease.

  1. Generation of antibodies against disintegrin and cysteine-rich domains by DNA immunization: An approach to neutralize snake venom-induced haemorrhage

    Directory of Open Access Journals (Sweden)

    Sidgi Syed Anwer Abdo Hasson

    2017-03-01

    Conclusions: Antibodies generated against the E. ocellatus venom prothrombin activator-like metalloprotease and disintegrin-cysteine-rich domains modulated and inhibited the catalytic activity both in vitro and in vivo of venom metalloproteinase disintegrin cysteine rich molecules. Thus, generating of venom specific-toxin antibodies by DNA immunization offer a more rational treatment of snake envenoming than conventional antivenom.

  2. Correlating In Vitro Target-Oriented Screening and Docking: Inhibition of Matrix Metalloproteinases Activities by Flavonoids.

    Science.gov (United States)

    Crascì, Lucia; Basile, Livia; Panico, Annamaria; Puglia, Carmelo; Bonina, Francesco P; Basile, Pierluigi Maria; Rizza, Luisa; Guccione, Salvatore

    2017-07-01

    Metalloproteases are a family of zinc-containing endopeptidases involved in a variety of pathological disorders. The use of flavonoid derivatives as potential metalloprotease inhibitors has recently increased.Particular plants growing in Sicily are an excellent yielder of the flavonoids luteolin, apigenin, and their respective glycoside derivatives (7-O-rutinoside, 7-O-glucoside, and 7-O-glucuronide).The inhibitory activity of luteolin, apigenin, and their respective glycoside derivatives on the metalloproteases MMP-1, MMP-3, MMP-13, MMP-8, and MMP-9 was assessed and rationalized correlating in vitro target-oriented screening and in silico docking.The flavones apigenin, luteolin, and their respective glucosides have good ability to interact with metalloproteases and can also be lead compounds for further development. Glycones are more active on MMP-1, -3, -8, and -13 than MMP-9. Collagenases MMP-1, MMP-8, and MMP-13 are inhibited by compounds having rutinoside glycones. Apigenin and luteolin are inactive on MMP-1, -3, and -8, which can be interpreted as a better selectivity for both -9 and -13 peptidases. The more active compounds are apigenin-7-O-rutinoside on MMP-1 and luteolin-7-O-rutinoside on MMP-3. The lowest IC 50 values were also found for apigenin-7-O-glucuronide, apigenin-7-O-rutinoside, and luteolin-7-O-glucuronide. The glycoside moiety might allow for a better anchoring to the active site of MMP-1, -3, -8, -9, and -13. Overall, the in silico data are substantially in agreement with the in vitro ones (fluorimetric assay). Georg Thieme Verlag KG Stuttgart · New York.

  3. Apoptosis and Tumor Invasion in Breast Cancer

    Science.gov (United States)

    2000-08-01

    while down- regulating the synthesis of at least one inhibitor S~ < of the matrix metalloproteases (TIMP-1) and f / / ,,"* , cystatin C . We are...UNCLASSIFIED AD Award Number: DAMD17-97-1-7268 TITLE: Apoptosis and Tumor Invasion in Breast Cancer PRINCIPAL INVESTIGATOR: Martin Tenniswood, Ph.D...preparing the same or similar computer software, or ( c ) used by a party other than the Government, except that the Government may release or disclose

  4. Hyaluronan-CD44 Interactions Decrease the Metastatic Potential of Breast Cancer Cells

    Science.gov (United States)

    2008-06-01

    cancer cells in vivo. This study focuses on understanding the mechanisms by which CD44 inhibits breast cancer cell invasion. We have found that the differential interaction between CD44 and soluble or embedded Hyaluronan leads to differential phosphorylation of ERK and Pak. These differentially activated signaling pathways molecularly govern a phenotypic switch between invasiveness or proliferation. Additionally, this interactions also leads to decreased transcription of the metalloprotease MMP9 when on collagen IV gels but not collagen I gels. Together, these mechanisms

  5. Targeting prolyl endopeptidase with valproic acid as a potential modulator of neutrophilic inflammation

    OpenAIRE

    Abdul Roda, Mojtaba; Sadik, Mariam; Gaggar, Amit; Hardison, Matthew T; Jablonsky, Michael J; Braber, Saskia; Blalock, James Edwin; Redegeld, Frank A; Folkerts, Gert; Jackson, Patricia L

    2014-01-01

    A novel neutrophil chemoattractant derived from collagen, proline-glycine-proline (PGP), has been recently characterized in chronic obstructive pulmonary disease (COPD). This peptide is derived via the proteolytic activity of matrix metalloproteases (MMP's)-8/9 and PE, enzymes produced by neutrophils and present in COPD serum and sputum. Valproic acid (VPA) is an inhibitor of PE and could possibly have an effect on the severity of chronic inflammation. Here the interaction site of VPA to PE a...

  6. Use of MALDI-TOF Mass Spectrometry for Specificity Studies of Biomedically Important Proteases

    OpenAIRE

    Siigur, Jüri; Trummal, Katrin; TÕnismägi, Külli; Samel, Mari; Siigur, Ene; Vija, Heikki; Tammiste, Indrek; Subbi, Juhan

    2002-01-01

    Proteases play crucial role starting from fertilization until to cell death. Our studies of the two Viperidae venoms (Levantine viper Vipera lebetina, Common viper Vipera berus) have demonstrated the existence of biomedically important proteases, both coagulants and anticoagulants that may be useful as diagnostic tools or potential therapeutics. We showed that venoms of both snakes contain: (i) metalloproteases and serine proteases that degrade fibrinogen, but not fibrin; (ii) factor X activa...

  7. Purification and Antithrombotic Potential of a Fibrinolytic Enzyme from Shiitake Culinary- Medicinal Mushroom, Lentinus edodes GNA01 (Agaricomycetes).

    Science.gov (United States)

    Choi, Jun-Hui; Kim, Kyung-Je; Kim, Seung

    2018-01-01

    We purified Lentinus edodes GNA01 fibrinolytic enzyme (LEFE) and identified it as a novel metalloprotease. LEFE was purified to homogeneity through a 2-step procedure, with an 8.28-fold increase in specific activity and 5.3% recovery. The molecular mass of LEFE was approximately 38 kDa, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its optimal pH, optimal temperature, pH stability, and thermal stability were 5, 30°C, 6-7, and 40°C, respectively. LEFE was inhibited by zinc and magnesium ions, and by EDTA and EGTA, indicating that LEFE is a metalloprotease. The protease exhibited fibrinolytic activity and a degradative effect on clot formation and blood clots. The protease prolonged activated partial thromboplastin time, prothrombin time, and coagulation time as induced by platelet aggregators (collagen and epinephrine). Taken together, our results indicate that L. edodes GNA01 produces a metalloprotease/fibrinolytic enzyme and that this enzyme might be applied as a new thrombolytic and antithrombotic agent for thrombosis-related cardiovascular disorders.

  8. PROTEOLYTIC PROCESSING OF VON WILLEBRAND FACTOR BY ADAMTS13 AND LEUKOCYTE PROTEASES

    Directory of Open Access Journals (Sweden)

    Stefano Lancellotti

    2013-09-01

    Full Text Available ADAMTS13 is a 190 kDa zinc protease encoded by a gene located on chromosome 9q34.   This protease specifically hydrolyzes von Willebrand factor (VWF multimers, thus causing VWF size reduction. ADAMTS13 belongs to the A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats (ADAMTS family, involved in proteolytic processing of many matrix proteins. ADAMTS13 consists of numerous domains including a metalloprotease domain, a disintegrin domain, several thrombospondin type 1 (TSP1 repeats, a cysteine-rich domain, a spacer domain and 2 CUB (Complement c1r/c1s, sea Urchin epidermal growth factor, and Bone morphogenetic protein domains. ADAMTS13 cleaves a single peptide bond (Tyr1605-Met1606 in the central A2 domain of the VWF molecule. This proteolytic cleavage is essential to reduce the size of ultra-large VWF polymers, which, when exposed to high shear stress in the microcirculation, are prone to form with platelets clumps, which cause severe syndromes called thrombotic microangiopathies (TMAs. In this review, we a discuss the current knowledge of structure-function aspects of ADAMTS13 and its involvement in the pathogenesis of TMAs, b address the recent findings concerning proteolytic processing of VWF multimers by different proteases, such as the leukocyte-derived serine and metallo-proteases and c indicate the direction of future investigations

  9. ADAMTS9-Regulated Pericellular Matrix Dynamics Governs Focal Adhesion-Dependent Smooth Muscle Differentiation

    Directory of Open Access Journals (Sweden)

    Timothy J. Mead

    2018-04-01

    Full Text Available Summary: Focal adhesions anchor cells to extracellular matrix (ECM and direct assembly of a pre-stressed actin cytoskeleton. They act as a cellular sensor and regulator, linking ECM to the nucleus. Here, we identify proteolytic turnover of the anti-adhesive proteoglycan versican as a requirement for maintenance of smooth muscle cell (SMC focal adhesions. Using conditional deletion in mice, we show that ADAMTS9, a secreted metalloprotease, is required for myometrial activation during late gestation and for parturition. Through knockdown of ADAMTS9 in uterine SMC, and manipulation of pericellular versican via knockdown or proteolysis, we demonstrate that regulated pericellular matrix dynamics is essential for focal adhesion maintenance. By influencing focal adhesion formation, pericellular versican acts upstream of cytoskeletal assembly and SMC differentiation. Thus, pericellular versican proteolysis by ADAMTS9 balances pro- and anti-adhesive forces to maintain an SMC phenotype, providing a concrete example of the dynamic reciprocity of cells and their ECM. : Mead et al. identify a proteolytic mechanism that actively maintains a pericellular microenvironment conducive to uterine smooth muscle activation prior to parturition. They show that pericellular matrix proteolysis by the secreted metalloprotease ADAMTS9 is crucial for maintenance of focal adhesions in uterine smooth muscle cells, and its absence impairs parturition. Keywords: metalloprotease, extracellular matrix, smooth muscle, proteoglycan, myometrium, parturition, uterus, focal adhesion, proteolysis, interference reflection microscopy

  10. Posttranscriptional stimulation of endothelial cell matrix metalloproteinases 2 and 1 by endothelioma cells.

    Science.gov (United States)

    Taraboletti, G; Sonzogni, L; Vergani, V; Hosseini, G; Ceruti, R; Ghilardi, C; Bastone, A; Toschi, E; Borsotti, P; Scanziani, E; Giavazzi, R; Pepper, M S; Stetler-Stevenson, W G; Bani, M R

    2000-08-01

    Matrix metalloproteinases (MMPs) play a critical role in the development of hemangioma-like vascular tumors in mice injected with murine eEnd.1 endothelioma cells. The current study was designed to (a) characterize the presence of MMPs in the vascular tumor, (b) define whether these MMPs originate from the transformed cells or from the recruited stromal cells and (c) study the stimulatory effect of eEnd.1 cells on the production of MMPs by endothelial cells. Several gelatinases were present in the eEnd.1 tumor extract, including latent and activated MMP-2 (72-kDa gelatinase A, EC 3.4.24. 24) and MMP-9 (92-kDa gelatinase B, EC 3.4.24.35). Immunohistochemical analysis of the tumor revealed focal reactivity for MMP-2. No gelatinase was produced by cultured eEnd.1 cells, or by six of nine related endothelioma cell lines, suggesting that stroma cells, particularly endothelial cells recruited by the tumor cells, rather than eEnd.1 cells themselves, are the source of the gelatinases observed in the tumors in vivo. The conditioned medium of eEnd.1 cells stimulated the release of MMP-2 and MMP-1 (interstitial collagenase, EC 3.4.24.7) by endothelial cells, but not of the inhibitor TIMP-2. The increased production of MMP-2 and MMP-1, observed at the protein level (zymogram and Western blot analysis), occurred through a posttranscriptional mechanism, since no increase in mRNA was observed and the stimulation was not prevented by inhibitors of protein synthesis. The inhibitory effects of monensin and brefeldin A, inhibitors of protein secretion, and the decrease in cell-associated MMP-2 in stimulated endothelial cells indicated that regulation occurred mostly at the level of protease secretion. MMPs are known to be regulated at different levels; this study indicates that, in endothelial cells, the stimulation of MMPs can also occur at the level of secretion, a mechanism that provides a rapid mobilization of these crucial enzymes in the early phases of angiogenesis. Copyright

  11. Detection of functional matrix metalloproteinases by zymography.

    Science.gov (United States)

    Hu, Xueyou; Beeton, Christine

    2010-11-08

    Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups based on their structure and substrate specificity (collagenases, gelatinases, membrane type [MT-MMP], stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease. Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle. This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel. Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary

  12. Ramiprilate inhibits functional matrix metalloproteinase activity in Crohn's disease fistulas

    DEFF Research Database (Denmark)

    Efsen, Eva; Saermark, Torben; Hansen, Alastair

    2011-01-01

    Increased expression of matrix metalloproteinase (MMP)-2, -3 and -9 has been demonstrated in Crohn's disease fistulas, but it is unknown whether these enzymes are biologically active and represent a therapeutic target. Therefore, we investigated the proteolytic activity of MMPs in fistula tissue...... and examined the effect of inhibitors, including clinically available drugs that beside their main action also suppress MMPs. Fistula specimens were obtained by surgical excision from 22 patients with Crohn's disease and from 10 patients with fistulas resulting from other causes. Colonic endoscopic biopsies......-diamine-tetraacetic acid (EDTA), the synthetic broad-spectrum inhibitor, GM6001, the angiotensin-converting enzyme (ACE) inhibitor, ramiprilate, and the tetracycline, doxycycline. In Crohn's disease fistulas, about 50% of the total protease activity was attributable to MMP activity. The average total MMP activity...

  13. Glomerular Protein Levels of Matrix Metalloproteinase-1 and Tissue Inhibitor of Metalloproteinase-1 Are Lower in Diabetic Subjects

    Science.gov (United States)

    Cornish, Toby C.; Bagnasco, Serena M.; Macgregor, Anne M.; Lu, Jie; Selvin, Elizabeth; Halushka, Marc K.

    2009-01-01

    Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) regulate extracellular matrix turnover throughout the body, including in renal glomeruli. We investigated protein levels of multiple MMPs (MMP-1, MMP-2, MMP-3, and MMP-9) and TIMP-1 in glomeruli and investigated whether disease phenotypes were associated with levels of these proteins. Renal cortex was collected from 100 adult autopsy subjects arrayed across 17 tissue microarrays. Immunohistochemical staining intensity for each MMP and TIMP-1 was determined using quantitative color deconvolution techniques. We observed significantly decreased glomerular MMP-1 and TIMP-1 staining in subjects with diabetes, hypertension, and an estimated glomerular filtration rate contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 57:995–1001, 2009) PMID:19506087

  14. The radioenzymatic assay of matrix metalloproteinase-1 activity

    International Nuclear Information System (INIS)

    Wu Shiliang; Xu Lan; Zhou Yinghui; Wang Weiping; Shi Ning

    1998-12-01

    The radioenzymatic assay method for tissue collagenase, a metalloproteinase, activity in matrix was established. The matrix collagenase is the most vital catabolic enzyme of collagen in tissue. It mainly acts on type- I, II, III matrix collagen and is also called matrix metalloproteinase-1 (MMPs-1). The assay method for the matrix collagenase was as follows: After type-I collagen was prepared from calf skin and identified with HPLC, it was marked with 3 H-acetic anhydride as the substrate. Then a series of assays were performed in animal experiments and human cases, which showed that the matrix collagenase (MMPs-1) activity assay is feasible and gives reliable results in clinical biochemistry study

  15. Lack of relationship between TIMP-1 tumour cell immunoreactivity, treatment efficacy and prognosis in patients with advanced epithelial ovarian cancer

    DEFF Research Database (Denmark)

    Steffensen, Karina Dahl; Waldstrøm, Marianne; Christensen, Rikke Kølby

    2010-01-01

    BACKGROUND: Tissue inhibitor of metalloproteinase 1 (TIMP-1) is a natural inhibitor of the matrix metalloproteinases (MMPs) which are proteolytic enzymes involved in degradation of extracellular matrix thereby favoring tumour cell invasion and metastasis. TIMP-1 activity in tumour tissue may ther...... immunoreactivity in tumour tissue from patients with primary epithelial ovarian cancer did not correlate with patient survival or response to combination platinum/cyclophosphamide therapy.......BACKGROUND: Tissue inhibitor of metalloproteinase 1 (TIMP-1) is a natural inhibitor of the matrix metalloproteinases (MMPs) which are proteolytic enzymes involved in degradation of extracellular matrix thereby favoring tumour cell invasion and metastasis. TIMP-1 activity in tumour tissue may...... therefore play an essential role in the progression of a malignant tumour.The primary aim of the present study was to evaluate TIMP-1 protein immunoreactivity in tissue from primary ovarian cancer patients and associate these findings with the course of the disease including response to treatment...

  16. Selective small-molecule inhibitors as chemical tools to define the roles of matrix metalloproteinases in disease.

    Science.gov (United States)

    Meisel, Jayda E; Chang, Mayland

    2017-11-01

    The focus of this article is to highlight novel inhibitors and current examples where the use of selective small-molecule inhibitors has been critical in defining the roles of matrix metalloproteinases (MMPs) in disease. Selective small-molecule inhibitors are surgical chemical tools that can inhibit the targeted enzyme; they are the method of choice to ascertain the roles of MMPs and complement studies with knockout animals. This strategy can identify targets for therapeutic development as exemplified by the use of selective small-molecule MMP inhibitors in diabetic wound healing, spinal cord injury, stroke, traumatic brain injury, cancer metastasis, and viral infection. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. In Vivo Anti-Cancer Mechanism of Low-Molecular-Weight Fucosylated Chondroitin Sulfate (LFCS from Sea Cucumber Cucumaria frondosa

    Directory of Open Access Journals (Sweden)

    Xiaoxiao Liu

    2016-05-01

    Full Text Available The low-molecular-weight fucosylated chondroitin sulfate (LFCS was prepared from native fucosylated chondroitin sulfate (FCS, which was extracted and isolated from sea cucumber Cucumaria frondosa, and the anti-cancer mechanism of LFCS on mouse Lewis lung carcinoma (LLC was investigated. The results showed that LFCS remarkably inhibited LLC growth and metastasis in a dose-dependent manner. LFCS induced cell cycle arrest by increasing p53/p21 expression and apoptosis through activation of caspase-3 activity in LLC cells. Meanwhile, LFCS suppressed the expression of vascular endothelial growth factor (VEGF, increased the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1 and downregulated the matrix metalloproteinases (MMPs level. Furthermore, LFCS significantly suppressed the activation of ERK1/2/p38 MAPK/NF-κB pathway, which played a prime role in expression of MMPs. All of these data indicate LFCS may be used as anti-cancer drug candidates and deserve further study.

  18. TIMP-1 expression in human colorectal cancer is associated with TGF-B1, LOXL2, INHBA1, TNF-AIP6 and TIMP-2 transcript profiles

    DEFF Research Database (Denmark)

    Offenberg, Hanne Kjær; Brunner, Nils; Mansilla, Francisco

    2008-01-01

    -regulated in CRC samples compared with normal tissue, while TIMP-2 was down-regulated. Eight MMPs were up-regulated in CRC compared with normal tissue. Correlating up-regulated genes with the TIMP-1 transcript, we identified 13 that were also up-regulated in cancerous tissue. Among these were genes associated...... it is regulated. To further elucidate potential mechanisms of regulation of this protein, we did a number of experiments to look at associations between the transcript profile of TIMP-1 with known matrix metalloproteinases (MMPs) as well as with expression profiles of other genes differentially regulated in human...... colorectal cancer (CRC) and the other TIMPs 2-4, which have also been associated with the progression of colorectal cancer. Genome-wide expression profiling of 172 CRC and normal mucosa samples was used to identify transcript changes for the genes under investigation. We found that TIMP-1 was up...

  19. MMP-12 catalytic domain recognizes and cleaves at multiple sites in human skin collagen type I and type III

    DEFF Research Database (Denmark)

    Taddese, Samuel; Jung, Michael C; Ihling, Christian

    2010-01-01

    . The catalytic domain of MMP-12 binds to the triple helix and cleaves the typical sites -Gly(775)-Leu(776)- in alpha-2 type I collagen and -Gly(775)-Ile(776)- in alpha-1 type I and type III collagens and at multiple other sites in both collagen types. Moreover, it was observed that the region around...... metalloproteinases (MMPs) are required to initiate further degradation by other enzymes. Several MMPs are known to degrade collagens, but the action of MMP-12 has not yet been studied in detail. In this work, the potential of MMP-12 in recognizing sites in human skin collagen types I and III has been investigated...... on the collagenolysis process. Taken together, the results of the present work confirm that the catalytic domain of MMP-12 alone binds and degrades collagens I and III....

  20. Structural differences of matrix metalloproteinases with potential implications for inhibitor selectivity examined by the GRID/CPCA approach

    DEFF Research Database (Denmark)

    Terp, Gitte Elgaard; Cruciani, Gabriele; Christensen, Inge Thøger

    2002-01-01

    The matrix metalloproteinases (MMPs) are a family of proteolytic enzymes, which have been the focus of a lot of research in recent years because of their involvement in various disease conditions. In this study, structures of 10 enzymes (MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP12, MMP13, MMP14......, and MMP20) were examined with the intention of highlighting regions that could be potential sites for obtaining selectivity. For this purpose, the GRID/CPCA approach as implemented in GOLPE was used. Counterions were included to take into account the different electrostatic properties of the proteins......, and the GRID calculations were performed, allowing the protein side chains to move in response to interaction with the probes. In the search for selectivity, the MMPs are known to be a very difficult case because the enzymes of this family are very similar. The well-known differences in the S1' pocket were...

  1. Circulating levels of matrix metalloproteinases and tissue inhibitors of metalloproteinases in patients with incisional hernia

    DEFF Research Database (Denmark)

    Henriksen, Nadia A; Sørensen, Lars T; Jorgensen, Lars N

    2013-01-01

    for MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-12, MMP-13, TIMP-1, TIMP-2, and TIMP-4 using a multiplex sandwich fluorescent immunoassay supplemented with gelatin zymography. The screening indicated differences in serum MMP-9 and TIMP-1 levels. Consequently, MMP-9 and TIMP-1 levels were......Incisional hernia formation is a common complication to laparotomy and possibly associated with alterations in connective tissue metabolism. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are closely involved in the metabolism of the extracellular matrix. Our...... aim was to study serum levels of multiple MMPs and TIMPs in patients with and without incisional hernia. Out of 305 patients who underwent laparotomy, 79 (25.9%) developed incisional hernia over a median follow-up period of 3.7 years. Pooled sera from a subset (n = 72) of these patients were screened...

  2. Simultaneous demonstration of gelatinolytic activity, morphology, and immunohistochemical reaction using zymography film.

    Science.gov (United States)

    Kanomata, Naoki; Hasebe, Takahiro; Moriya, Takuya; Ochiai, Atsushi

    2013-12-01

    In situ zymography has been used to assess gelatinolytic activity, which is mainly due to matrix metalloproteinases (MMPs) in cancer tissues. MMPs play an important role in cancer invasion and metastasis. Film in situ zymography (FIZ) enables the in situ evaluation of gelatinolytic activity with high reproducibility. In this article, we report a study of FIZ, in a case of breast cancer with an invasive carcinoma component showing clear gelatinolytic activity, and in a non-invasive carcinoma component showing little gelatinolytic activity. Immunohistochemistry on FIZ was also performed. The simultaneous detection of gelatinolytic activity and immunohistochemical reaction was established in a single film. Immunohistochemistry on FIZ may have good potential for the investigation of cancer microenvironment.

  3. Patients with rosacea have increased risk of dementia

    DEFF Research Database (Denmark)

    Egeberg, Alexander; Hansen, Peter R; Gislason, G. H.

    2016-01-01

    Objective Rosacea is a common chronic inflammatory skin disorder where upregulation of matrix metalloproteinases (MMPs) and antimicrobial peptides (AMPs) is observed. Notably, inflammation, MMPs, and AMPs are also involved in the etiopathogenesis of neurodegenerative disorders including certain...... forms of dementia such as Alzheimer disease (AD). Based on several clinical observations, we investigated the association between rosacea and dementia, including AD in Danish registers. Methods All Danish citizens aged ≥18 years between January 1, 1997 and December 31, 2012 were linked at the individual...... level through administrative registers. Cox regression was used to calculate unadjusted and adjusted hazard ratios (HRs). Results The study comprised a total of 5,591,718 individuals, including 82,439 patients with rosacea. A total of 99,040 individuals developed dementia (any form) in the study period...

  4. Collagenolytic Matrix Metalloproteinases in Chronic Obstructive Lung Disease and Cancer

    Directory of Open Access Journals (Sweden)

    Denzel Woode

    2015-02-01

    Full Text Available Chronic obstructive pulmonary disease (COPD and lung cancer result in significant morbidity and mortality worldwide. In addition to the role of environmental smoke exposure in the development of both diseases, recent epidemiological studies suggests a connection between the development of COPD and lung cancer. Furthermore, individuals with concomitant COPD and cancer have a poor prognosis when compared with individuals with lung cancer alone. The modulation of molecular pathways activated during emphysema likely lead to an increased susceptibility to lung tumor growth and metastasis. This review summarizes what is known in the literature examining the molecular pathways affecting matrix metalloproteinases (MMPs in this process as well as external factors such as smoke exposure that have an impact on tumor growth and metastasis. Increased expression of MMPs provides a unifying link between lung cancer and COPD.

  5. Influence of Polyphenol Extract from Evening Primrose (Oenothera Paradoxa Seeds on Proliferation of Caco-2 Cells and on Expression, Synthesis and Activity of Matrix Metalloproteinases and Their Inhibitors

    Directory of Open Access Journals (Sweden)

    Szewczyk Karolina

    2014-09-01

    Full Text Available Evening primrose (Oenothera paradoxa Hudziok seeds are a rich source of not only a valuable oil containing an essential fatty acid - ᵧ-linolenic acid (GLA - but also polyphenols which can be obtained from the biomass remaining after oil pressing. The aim of our studies was to evaluate the influence of a polyphenol extract from defatted seeds of evening primrose on human colorectal adenocarcinoma Caco-2 cell proliferation and matrix metalloproteinases (MMPs synthesis and activity. To assess the effect of evening primrose extract on Caco-2 cell proliferation, crystal violet staining and sulforhodamine B (SRB assays were used whereas mRNA expression and activity of MMPs were evaluated by RT-PCR and gelatin zymography.

  6. Tumor necrosis factor-α-accelerated degradation of type I collagen in human skin is associated with elevated matrix metalloproteinase (MMP)-1 and MMP-3 ex vivo

    DEFF Research Database (Denmark)

    Ågren, Magnus S; Schnabel, Reinhild; Christensen, Lise H

    2015-01-01

    Tumor necrosis factor (TNF)-α induces matrix metalloproteinases (MMPs) that may disrupt skin integrity. We have investigated the effects and mechanisms of exogenous TNF-α on collagen degradation by incubating human skin explants in defined serum-free media with or without TNF-α (10ng/ml) in the a......Tumor necrosis factor (TNF)-α induces matrix metalloproteinases (MMPs) that may disrupt skin integrity. We have investigated the effects and mechanisms of exogenous TNF-α on collagen degradation by incubating human skin explants in defined serum-free media with or without TNF-α (10ng...... tissue-derived collagenolytic activity with TNF-α exposure was blocked by neutralizing MMP-1 monoclonal antibody and was not due to down-regulation of tissue inhibitor of metalloproteinase-1. TNF-α increased production (pendogenous MMP-1...

  7. Collagenolytic Matrix Metalloproteinases in Chronic Obstructive Lung Disease and Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Woode, Denzel; Shiomi, Takayuki; D’Armiento, Jeanine, E-mail: jmd12@cumc.columbia.edu [Department of Anesthesiology, Columbia University, College of Physicians and Surgeons, New York, NY 10033 (United States)

    2015-02-05

    Chronic obstructive pulmonary disease (COPD) and lung cancer result in significant morbidity and mortality worldwide. In addition to the role of environmental smoke exposure in the development of both diseases, recent epidemiological studies suggests a connection between the development of COPD and lung cancer. Furthermore, individuals with concomitant COPD and cancer have a poor prognosis when compared with individuals with lung cancer alone. The modulation of molecular pathways activated during emphysema likely lead to an increased susceptibility to lung tumor growth and metastasis. This review summarizes what is known in the literature examining the molecular pathways affecting matrix metalloproteinases (MMPs) in this process as well as external factors such as smoke exposure that have an impact on tumor growth and metastasis. Increased expression of MMPs provides a unifying link between lung cancer and COPD.

  8. Reducing Haemorrhagic Transformation after Thrombolysis for Stroke: A Strategy Utilising Minocycline

    Directory of Open Access Journals (Sweden)

    David J. Blacker

    2013-01-01

    Full Text Available Haemorrhagic transformation (HT of recently ischaemic brain is a feared complication of thrombolytic therapy that may be caused or compounded by ischaemia-induced activation of matrix metalloproteinases (MMPs. The tetracycline antibiotic minocycline inhibits matrix MMPs and reduces macroscopic HT in rodents with stroke treated with tissue plasminogen activator (tPA. The West Australian Intravenous Minocycline and TPA Stroke Study (WAIMATSS aims to determine the safety and efficacy of adding minocycline to tPA in acute ischaemic stroke. The WAIMATSS is a multicentre, prospective, and randomised pilot study of intravenous minocycline, 200 mg 12 hourly for 5 doses, compared with standard care, in patients with ischaemic stroke treated with intravenous tPA. The primary endpoint is HT diagnosed by brain CT and MRI. Secondary endpoints include clinical outcome measures. Some illustrative cases from the early recruitment phase of this study will be presented, and future perspectives will be discussed.

  9. Exploitation of Conformational Dynamics in Imparting Selective Inhibition for Related Matrix Metalloproteinases.

    Science.gov (United States)

    Mahasenan, Kiran V; Bastian, Maria; Gao, Ming; Frost, Emma; Ding, Derong; Zorina-Lichtenwalter, Katerina; Jacobs, John; Suckow, Mark A; Schroeder, Valerie A; Wolter, William R; Chang, Mayland; Mobashery, Shahriar

    2017-06-08

    Matrix metalloproteinases (MMPs) have numerous physiological functions and share a highly similar catalytic domain. Differential dynamical information on the closely related human MMP-8, -13, and -14 was integrated onto the benzoxazinone molecular template. An in silico library of 28,099 benzoxazinones was generated and evaluated in the context of the molecular-dynamics information. This led to experimental evaluation of 19 synthesized compounds and identification of selective inhibitors, which have potential utility in delineating the physiological functions of MMPs. Moreover, the approach serves as an example of how dynamics of closely related active sites may be exploited to achieve selective inhibition by small molecules and should find applications in other enzyme families with similar active sites.

  10. Increasing extracellular matrix collagen level and MMP activity induces cyst development in polycystic kidney disease

    Directory of Open Access Journals (Sweden)

    Liu Bin

    2012-09-01

    Full Text Available Abstract Background Polycystic Kidney Disease (PKD kidneys exhibit increased extracellular matrix (ECM collagen expression and metalloproteinases (MMPs activity. We investigated the role of these increases on cystic disease progression in PKD kidneys. Methods We examined the role of type I collagen (collagen I and membrane bound type 1 MMP (MT1-MMP on cyst development using both in vitro 3 dimensional (3D collagen gel culture and in vivo PCK rat model of PKD. Results We found that collagen concentration is critical in controlling the morphogenesis of MDCK cells cultured in 3D gels. MDCK cells did not form 3D structures at collagen I concentrations lower than 1 mg/ml but began forming tubules when the concentration reaches 1 mg/ml. Significantly, these cells began to form cyst when collagen I concentration reached to 1.2 mg/ml, and the ratios of cyst to tubule structures increased as the collagen I concentration increased. These cells exclusively formed cyst structures at a collagen I concentration of 1.8 mg/ml or higher. Overexpression of MT1-MMP in MDCK cells significantly induced cyst growth in 3D collagen gel culture. Conversely, inhibition of MMPs activity with doxycycline, a FDA approved pan-MMPs inhibitor, dramatically slowed cyst growth. More importantly, the treatment of PCK rats with doxycycline significantly decreased renal tubule cell proliferation and markedly inhibited the cystic disease progression. Conclusions Our data suggest that increased collagen expression and MMP activity in PKD kidneys may induce cyst formation and expansion. Our findings also suggest that MMPs may serve as a therapeutic target for the treatment of human PKD.

  11. A role for matrix metalloproteinase-9 in pathogenesis of urogenital Chlamydia muridarum infection in mice

    OpenAIRE

    Imtiaz, Muhammad T.; Distelhorst, John T.; Schripsema, Justin H.; Sigar, Ira M.; Kasimos, John N.; Lacy, Shanon R.; Ramsey, Kyle H.

    2007-01-01

    Matrix metalloproteinases (MMPs) are a family of host-derived enzymes involved in the turnover of extracellular matrix (ECM) molecules and the processing of cytokines, chemokines and growth factors. We have previously reported that global inhibition of MMP in Chlamydia muridarum urogenital tract infection of susceptible strains of female mice impeded ascension of C. muridarum into the upper genital tract, blunted acute inflammatory responses and reduced the rate of formation of chronic diseas...

  12. Aspirin Inhibits IKK-β-mediated Prostate Cancer Cell Invasion by Targeting Matrix Metalloproteinase-9 and Urokinase-Type Plasminogen Activator

    OpenAIRE

    Chongjun Shi; Nini Zhang; Yang Feng; Jiewei Cao; Xuyi Chen; Bin Liu

    2017-01-01

    Background/Aims: Aspirin has been demonstrated to possess potent chemopreventive and anticancer effects on prostate cancer. However, the more detailed molecular mechanisms of aspirin to suppress prostate cancer cell invasion have not been clearly elucidated. Methods: Transwell assays were performed to evaluate the effects of aspirin on cell invasion. Matrix metalloproteinases (MMPs) and serine proteinases activities in cell media were examined by gelatin zymography and ELISA. In addition, inh...

  13. Copper modulates zinc metalloproteinase-dependent ectodomain shedding of key signaling and adhesion proteins and promotes the invasion of prostate cancer epithelial cells

    OpenAIRE

    Parr-Sturgess, Catherine A.; Tinker, Claire L.; Hart, Claire A.; Brown, Michael D.; Clarke, Noel W.; Parkin, Edward T.

    2012-01-01

    A disintegrin and metalloproteinases (ADAMs) and matrix metalloproteinases (MMPs) are zinc metalloproteinases (ZMPs) that catalyze the "ectodomain shedding" of a range of cell surface proteins including signaling and adhesion molecules. These "sheddases" are associated with the invasion and metastasis of a range of cancers. Increased serum and tumor tissue levels of copper are also observed in several cancers, although little is known about how the metal might promote disease progression at t...

  14. Rescue of proinflammatory cytokine-inhibited chondrogenesis by the antiarthritic effect of melatonin in synovium mesenchymal stem cells via suppression of reactive oxygen species and matrix metalloproteinases.

    Science.gov (United States)

    Liu, Xiaozhen; Xu, Yong; Chen, Sijin; Tan, Zifang; Xiong, Ke; Li, Yan; Ye, Yun; Luo, Zong-Ping; He, Fan; Gong, Yihong

    2014-03-01

    Cartilage repair by mesenchymal stem cells (MSCs) often occurs in diseased joints in which the inflamed microenvironment impairs chondrogenic maturation and causes neocartilage degradation. In this environment, melatonin exerts an antioxidant effect by scavenging free radicals. This study aimed to investigate the anti-inflammatory and chondroprotective effects of melatonin on human MSCs in a proinflammatory cytokine-induced arthritic environment. MSCs were induced toward chondrogenesis in the presence of interleukin-1β (IL-1β) or tumor necrosis factor α (TNF-α) with or without melatonin. Levels of intracellular reactive oxygen species (ROS), hydrogen peroxide, antioxidant enzymes, and cell viability were then assessed. Deposition of glycosaminoglycans and collagens was also determined by histological analysis. Gene expression of chondrogenic markers and matrix metalloproteinases (MMPs) was assessed by real-time polymerase chain reaction. In addition, the involvement of the melatonin receptor and superoxide dismutase (SOD) in chondrogenesis was investigated using pharmacologic inhibitors. The results showed that melatonin significantly reduced ROS accumulation and increased SOD expression. Both IL-1β and TNF-α had an inhibitory effect on the chondrogenesis of MSCs, but melatonin successfully restored the low expression of cartilage matrix and chondrogenic genes. Melatonin prevented cartilage degradation by downregulating MMPs. The addition of luzindole and SOD inhibitors abrogated the protective effect of melatonin associated with increased levels of ROS and MMPs. These results demonstrated that proinflammatory cytokines impair the chondrogenesis of MSCs, which was rescued by melatonin treatment. This chondroprotective effect was potentially correlated to decreased ROS, preserved SOD, and suppressed levels of MMPs. Thus, melatonin provides a new strategy for promoting cell-based cartilage regeneration in diseased or injured joints. Copyright © 2013 Elsevier

  15. Development and Comparison of TACAMO Icon Design Formats

    Science.gov (United States)

    1992-03-01

    Wilhelm Von Leibniz (1646-1716) planted the seeds of symbolic logic and computer design (Kreiling, 1968, p. 100). Leibniz ’ dream was that someday a...outlined in Appendix B.2. C. PICTORIAL SYMBOLS AND INITIAL MMPS ICON DESIGNS Over 300 years ago, the German philosopher and mathematician Baron Gottfried ...Annual Meeting 1988, 357-361, Santa Monica, CA: Human Factors Society. Kreiling, Frederick C. " Leibniz ," Scientific American, 1968, 220, 95-100. Laurel

  16. A serum biomarker reflecting collagen type I degradation (C1M) is an independent risk factor for acute myocardial infarction in postmenopausal women: results from the PERF study

    DEFF Research Database (Denmark)

    Bertelsen, D.M.; Nielsen, Signe Holm; Neergaard, J.S.

    2017-01-01

    Cardiovascular disease (CVD) is the leading cause of death in postmenopausal women, and symptoms of ischemic heart disease (IHD) and acute myocardial infarction (AMI) are often overlooked. With the loss of estrogen production collagen stability is affected with potential of an increased risk of u...... of unstable plaques in coronary vessels. Collagen type I, a major component of the cardiac extracellular matrix (ECM), is cleaved by matrix metalloproteinases (MMPs) and known to be active remodeled in CVD....

  17. Effect of statin therapy on serum activity of proteinases and cytokines in patients with abdominal aortic aneurysm

    OpenAIRE

    Muehling, Bernd

    2008-01-01

    Bernd Muehling1, Alexander Oberhuber1, Hubert Schelzig1, Gisela Bischoff1, Nikolaus Marx2, Ludger Sunder-Plassmann1, Karl H Orend11Department of Thoracic and Vascular surgery; 2Department of Internal Medicine, University of Ulm, Ulm, GermanyBackground and aims: Metalloproteinases (MMPs) are considered to be key enzymes in the pathogenesis of abdominal aortic aneurysms (AAA), with elevated levels in diseased aorta and in patient sera. Statins seem to exert an inhibitory effect on MMP activity ...

  18. Differences in extracellular matrix remodeling in the placenta of mares that retain fetal membranes and mares that deliver fetal membranes physiologically.

    Science.gov (United States)

    Rapacz-Leonard, A; Kankofer, M; Leonard, M; Wawrzykowski, J; Dąbrowska, M; Raś, A; Paździor-Czapula, K; Janowski, T

    2015-10-01

    In mammals, placenta separation at term may involve degradation of the extracellular matrix by matrix metalloproteinases (MMPs). The activity of MMPs is modulated by TIMPs. We hypothesized that the placentas of mares that deliver fetal membranes physiologically and those that retain fetal membranes (FMR) differ in terms of histology; mRNA expression of MMP-2 and MMP-9; protein expression of MMP-2, MMP-9, and TIMP-2; and the potential activity of both MMPs. Placenta biopsies were taken from mares (n = 9; 4 FMR, 5 controls) immediately after foal expulsion. Retention was defined as failure to expel all fetal membranes within 3 h of expulsion. All mares were monitored for time of expulsion. The degree of allantochorial/endometrial adhesion was determined in FMR mares, and biopsies from all mares were histologically examined. mRNA expression, protein immunolocalization, protein amount and potential enzyme activity were determined with RT-PCR, immunohistochemistry, Western Blotting and zymography, respectively. FMR mares had strong to extremely strong allantochorial/endometrial adhesion, and significantly more connective tissue in the allantochorial villi than controls. The range of MMP-2 mRNA expression levels was more than 13 times greater in FMR mares than in controls. Protein content of both MMPs and TIMP-2 differed significantly between groups. The range of potential MMP-2 and MMP-9 activity was larger in FMR mares, and MMP-2 potential activity was 1.4 times higher in controls (P = 0.02). These results indicate differences in extracellular matrix remodeling in FMR mares and controls, and suggest dysregulation of MMP expression and activation in FMR mares. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Cerebrospinal fluid matrix metalloproteinases are elevated in cerebral adrenoleukodystrophy and correlate with MRI severity and neurologic dysfunction.

    Science.gov (United States)

    Thibert, Kathryn A; Raymond, Gerald V; Nascene, David R; Miller, Weston P; Tolar, Jakub; Orchard, Paul J; Lund, Troy C

    2012-01-01

    X-linked adrenoleukodystrophy results from mutations in the ABCD1 gene disrupting the metabolism of very-long-chain fatty acids. The most serious form of ALD, cerebral adrenoleukodystrophy (cALD), causes neuroinflammation and demyelination. Neuroimaging in cALD shows inflammatory changes and indicates blood-brain-barrier (BBB) disruption. We hypothesize that disruption may occur through the degradation of the extracellular matrix defining the BBB by matrix metalloproteinases (MMPs). MMPs have not been evaluated in the setting of cALD. We used a multiplex assay to correlate the concentration of MMPs in cerebrospinal fluid and plasma to the severity of brain inflammation as determined by the ALD MRI (Loes) score and the neurologic function score. There were significant elevations of MMP2, MMP9, MMP10, TIMP1, and total protein in the CSF of boys with cALD compared to controls. Levels of MMP10, TIMP1, and total protein in CSF showed significant correlation [p<0.05 for each with pre-transplant MRI Loes Loes scores (R(2) = 0.34, 0.20, 0.55 respectively). Levels of TIMP1 and total protein in CSF significantly correlated with pre-transplant neurologic functional scores (R(2) = 0.22 and 0.48 respectively), and levels of MMP10 and total protein in CSF significantly correlated with one-year post-transplant functional scores (R(2) = 0.38 and 0.69). There was a significant elevation of MMP9 levels in plasma compared to control, but did not correlate with the MRI or neurologic function scores. MMPs were found to be elevated in the CSF of boys with cALD and may mechanistically contribute to the breakdown of the blood-brain-barrier. MMP concentrations directly correlate to radiographic and clinical neurologic severity. Interestingly, increased total protein levels showed superior correlation to MRI score and neurologic function score before and at one year after transplant.

  20. Adipose-derived mesenchymal stem cells promote cell proliferation and invasion of epithelial ovarian cancer

    Energy Technology Data Exchange (ETDEWEB)

    Chu, Yijing; Tang, Huijuan; Guo, Yan; Guo, Jing; Huang, Bangxing; Fang, Fang; Cai, Jing, E-mail: caijingmmm@hotmail.com; Wang, Zehua, E-mail: zehuawang@163.net

    2015-09-10

    Adipose-derived mesenchymal stem cell (ADSC) is an important component of tumor microenvironment. However, whether ADSCs have a hand in ovarian cancer progression remains unclear. In this study, we investigated the impact of human ADSCs derived from the omentum of normal donors on human epithelial ovarian cancer (EOC) cells in vitro and in vivo. Direct and indirect co-culture models including ADSCs and human EOC cell lines were established and the effects of ADSCs on EOC cell proliferation were evaluated by EdU incorporation and flow cytometry. Transwell migration assays and detection of MMPs were performed to assess the invasion activity of EOC cells in vitro. Mouse models were established by intraperitoneal injection of EOC cells with or without concomitant ADSCs to investigate the role of ADSCs in tumor progression in vivo. We found that ADSCs significantly promoted proliferation and invasion of EOC cells in both direct and indirect co-culture assays. In addition, after co-culture with ADSCs, EOC cells secreted higher levels of matrix metalloproteinases (MMPs), and inhibition of MMP2 and MMP9 partially relieved the tumor-promoting effects of ADSCs in vitro. In mouse xenograft models, we confirmed that ADSCs promoted EOC growth and metastasis and elevated the expression of MMP2 and MMP9. Our findings indicate that omental ADSCs play a promotive role during ovarian cancer progression. - Highlights: • Omental adipose derived stem cells enhanced growth and invasion properties of ovarian cancer cells. • Adipose derived stem cells promoted the growth and metastasis of ovarian cancer in mice models. • Adipose derived stem cells promoted MMPs expression and secretion of ovarian cancer cells. • Elevated MMPs mediated the tumor promoting effects of ADSCs.