A Fractional-Order Variational Framework for Retinex: Fractional-Order Partial Differential Equation-Based Formulation for Multi-Scale Nonlocal Contrast Enhancement with Texture Preserving.

This paper discusses a novel conceptual formulation of the fractional-order variational framework for retinex, which is a fractional-order partial differential equation (FPDE) formulation of retinex for the multi-scale nonlocal contrast enhancement with texture preserving. The well-known shortcomings of traditional integer-order computation-based contrast-enhancement algorithms, such as ringing artefacts and staircase effects, are still in great need of special research attention. Fractional calculus has potentially received prominence in applications in the domain of signal processing and image processing mainly because of its strengths like long-term memory, nonlocality, and weak singularity, and because of the ability of a fractional differential to enhance the complex textural details of an image in a nonlinear manner. Therefore, in an attempt to address the aforementioned problems associated with traditional integer-order computation-based contrast-enhancement algorithms, we have studied here, as an interesting theoretical problem, whether it will be possible to hybridize the capabilities of preserving the edges and the textural details of fractional calculus with texture image multi-scale nonlocal contrast enhancement. Motivated by this need, in this paper, we introduce a novel conceptual formulation of the fractional-order variational framework for retinex. First, we implement the FPDE by means of the fractional-order steepest descent method. Second, we discuss the implementation of the restrictive fractional-order optimization algorithm and the fractional-order Courant-Friedrichs-Lewy condition. Third, we perform experiments to analyze the capability of the FPDE to preserve edges and textural details, while enhancing the contrast. The capability of the FPDE to preserve edges and textural details is a fundamental important advantage, which makes our proposed algorithm superior to the traditional integer-order computation-based contrast enhancement algorithms, especially for images rich in textural details.

Microbes as Engines of Ecosystem Function: When Does Community Structure Enhance Predictions of Ecosystem Processes?

Microorganisms are vital in mediating the earth's biogeochemical cycles; yet, despite our rapidly increasing ability to explore complex environmental microbial communities, the relationship between microbial community structure and ecosystem processes remains poorly understood. Here, we address a fundamental and unanswered question in microbial ecology: 'When do we need to understand microbial community structure to accurately predict function?' We present a statistical analysis investigating the value of environmental data and microbial community structure independently and in combination for explaining rates of carbon and nitrogen cycling processes within 82 global datasets. Environmental variables were the strongest predictors of process rates but left 44% of variation unexplained on average, suggesting the potential for microbial data to increase model accuracy. Although only 29% of our datasets were significantly improved by adding information on microbial community structure, we observed improvement in models of processes mediated by narrow phylogenetic guilds via functional gene data, and conversely, improvement in models of facultative microbial processes via community diversity metrics. Our results also suggest that microbial diversity can strengthen predictions of respiration rates beyond microbial biomass parameters, as 53% of models were improved by incorporating both sets of predictors compared to 35% by microbial biomass alone. Our analysis represents the first comprehensive analysis of research examining links between microbial community structure and ecosystem function. Taken together, our results indicate that a greater understanding of microbial communities informed by ecological principles may enhance our ability to predict ecosystem process rates relative to assessments based on environmental variables and microbial physiology.

Extracellular matrix protein laminin induces matrix metalloproteinase-9 in human breast cancer cell line mcf-7.

Studies on interaction of tumor cells with extracellular matrix (ECM) components showed increased extracellular protease activity mediated by the family of matrix metalloproteinases (MMPs). Here we studied the effect of human breast cancer cell line MCF-7-laminin (LM) interaction on MMPs and the underlying signaling pathways. Culturing of MCF-7 cells on LM coated surface upregulated MMP-9 expression as well as reduced tissue inhibitor of metalloproteinases-1 (TIMP-1) expression. LM induced MMP-9 expression is abrogated by the blockade of α2 integrin. Inhibitor studies indicate possible involvement of phosphatidyl-inositol-3-kinase (PI3K), extracellular signal regulated kinase (ERK) and nuclear factor-kappaB (NF-κB) in LM induced signaling. LM treatment also enhanced phosphorylation of FAK (focal adhesion kinase), PI3K, ERK; nuclear translocation of ERK, pERK, NF-κB and cell migration. Our findings indicate that, binding of MCF-7 cells to LM, possibly via α2ß1 integrin, induces signaling involving FAK, PI3K, ERK, NF-κB followed by upregulation of MMP-9 and cell migration.

Matrix metalloproteinase-9 as a potential tumor marker in breast cancer.

This study aimed to detect the comparative expression and activity of matrix metalloproteinase-9 (MMP-9) and its correlation with known pathological parameters such as estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2) in 81 malignant breast tumors and adjacent normal breast tissues and in blood sera of these patients from different clinical TNM stages (ductal carcinoma in situ to T4) of breast cancer. MMP-9 was highly expressed in node-positive tumors and the preoperative blood serum of patients, but MMP-9 activity was appreciably inhibited in blood serum samples collected after surgery. The mature form of MMP-9 (84 kD) was expressed only in clinical stage III tumors (T2-4). Appreciable reduction of tissue inhibitor of metalloproteinase 1, phosphorylation of epidermal growth factor receptor, and translocation of nuclear factor-κΒ suggested their possible role in MMP-9 activation in HER2-positive breast cancer Overexpression and activation of MMP-9 predicted a higher stage of hormone-sensitive ductal breast carcinoma. Downregulation of the endogenous inhibitor of MMP-9, tissue inhibitor of metalloproteinase 1, and translocation of the transcription factor nuclear factor-κΒ in tumors may have an appreciable role in the overexpression of MMP-9. However, MMP-9 activation was not correlated with expression of estrogen and progesterone receptors. Evaluation of MMP-9 expression may provide valuable information about breast cancer treatment.

Extracellular matrix protein fibronectin induces matrix metalloproteinases in human prostate adenocarcinoma cells PC-3.

Studies on interaction of tumor cells with ECM components showed increased extracellular protease activity mediated by the family of matrix metalloproteinases (MMPs). Here we studied the effect of human prostate adenocarcinoma PC-3 cells-fibronectin (FN) interaction on MMPs and the underlying signaling pathways. Culturing of PC-3 cells on FN-coated surface upregulated MMP-9 and MMP-1. This response is abrogated by the blockade of α5 integrin. siRNA and inhibitor studies indicate possible involvement of phosphatidyl-inositol-3-kinase (PI-3K), focal adhesion kinase (FAK) and nuclear factor-kappaB (NF-κB) in FN-induced upregulation of MMPs. FN treatment also enhanced phosphorylation of FAK, PI3K, protein kinase B (PKB or Akt), nuclear translocation of NF-κB, surface expression of CD-44, and cell migration. Our findings indicate that, binding of PC-3 cells to FN, possibly via α5ß1 integrin, induces signaling involving FAK, PI-3K, Akt, NF-κB followed by upregulation of MMP-9 and MMP-1. CD-44 may have role in modulating MMP-9 activity.

Integrins and metastasis.

Metastasis is a combination of biological events that makes the difference between cancer and other diseases. Metastasis requires flow of erroneous but precisely coordinated basic cellular activities like cell migration-invasion, cell survival-apoptosis, cell proliferation, etc. All of these processes require efficient regulation of cell attachment and detachment, which recruit integrin receptors in this flow of events. World literatures show several aspects of interrelation of integrins and metastasis. Integrin molecules are being used as prime target to battle metastasis. In this review we are collating the observations showing importance of integrin biology in regulation of metastasis and the strategies where integrin receptors are being used as targets to regulate metastasis.

Soil metabolic pulses: water, substrate, and biological regulation.

Pulses of metabolic activity are a common ecological response to intermittently available resources, and in soils these pulses often occur in response to wetting. To better understand variation in soil pulses, we conducted a distributed field experiment at seven sites along a 2200-m elevation transect in southern California, USA. Treatments included both water and water + substrate additions and two measurements of soil respiration within one hour. These experiments were repeated 11 times throughout 2009-2010. Additions of substrate led to consistently higher pulse fluxes, exceeding 10 micromol CO2 x m(-2( x s(-1), than additions of water alone. These results support a sequential limitation by two resources where an initial limiting resource acts as a switch and, after activation, processes are regulated by a second resource. In contrast to general expectations of increasing pulses with higher soil organic matter (SOM), pulses exhibited strong scale dependencies. Pulses during the summer period and SOM were correlated positively within sites and negatively between sites. This cross-scale divergence implies that, at low elevations, the proportion of SOM available for pulse metabolism was a much larger fraction than at higher elevations. With expected climate changes leading to more frequent drying-wetting cycles, regulation of metabolic pulses will increasingly influence long-term biogeochemical dynamics.

Modulation of MMPs by cell surface integrin receptor α5ß1.

MMPs are a family of Zn dependent endopeptidases, which can mediate degradation of ECM components during various physiological and pathological processes including cancer. Some ECM components, through interaction with integrin receptor and modulation of downstream signaling, are capable of regulating expression and activity of several MMPs. α5ß1 integrin is the universally accepted receptor for the ECM component fibronectin (FN). The present review deals with the downstream signaling involved in the α5ß1 integrin mediated modulation of expression and activity of MMPs and their effector responses in different cellular system.

Fibronectin-integrin (alpha5beta1) modulates migration and invasion of murine melanoma cell line B16F10 by involving MMP-9.

Cell adhesion to extracellular matrix (ECM) initiates signaling cascade regulated by cell surface integrin receptors, which affects the proliferation and invasion of cells. Cells cultured in the presence of ECM ligand fibronectin (FN) stimulate secretion of matrix metalloproteinases (MMPs), facilitating cancer cell invasion and metastasis. Among all the members of the MMP family, MMP-9 is of crucial importance in tumor invasion and metastasis. The present study aims at studying the effects of integrin receptor alpha5beta1 and its ligand FN on expression of MMP-9 in murine melanoma cell line B16F10 and understanding the molecular mechanism(s) involved. The main experimental methods performed in the study were gelatin zymography, immunoblot, real-time RT-PCR, immunocytochemistry, enzyme linked immunosorbent assay (ELISA), transwell chamber assay, and in vivo metastasis assay in syngenic (C57BL6J) mice. The study reports that FN induces the activity, mRNA, and protein expression of MMP-9 and initiates its proteolytic activation in B16F10 cells. Blockage of the alpha5 receptor abrogated the FN-mediated stimulatory response on MMP-9 in B16F10 cells. Inhibitor studies and immunoblot analysis strongly suggest the involvement of focal adhesion kinase (FAK), extracellular regulated kinase (ERK), and phosphatidylinositol-3-kinase (PI-3K) in the FN-mediated responses. Immunocytochemical analysis showed the nuclear localization of nuclear factor-kappaB (NF-kappaB) might lead to activation of MMP-9 gene upon FN treatment. This study demonstrates that integrin receptor alpha5beta1 and FN interaction induces the invasive potential of B16F10 cells and MMP-9 induction is the downstream effectors in the process. This system serves as a novel model system to understand the molecular mechanism of melanoma growth and invasion.

Culture of human breast cancer cell line (MDA-MB-231) on fibronectin-coated surface induces pro-matrix metalloproteinase-9 expression and activity.

Interaction between cell surface integrin receptors with extracellular matrix (ECM) plays an important role in cell survival, proliferation, and migration including tumor development and invasion. Binding of ECM to integrins initiates intracellular signaling cascades, modulating expression and activity of different matrix metalloproteinases (MMPs) which is important in ECM degradation. The present study investigates fibronectin-integrin-mediated signaling and thereby modulation of MMPs expression and activity in human breast cancer cell line, MDA-MB-231. Culture of MDA-MB-231 cells on fibronectin (FN) induced expression and activity of pro-matrixmetalloproteinase-9 (MMP-9). Appreciable reduction of FN-induced pro-MMP-9 activity was observed in anti-α5 antibody treated cells. Inhibitor studies revealed that inhibitors of phosphatidyl inositiol-3-kinase (PI-3K), and nuclear factor kappa B (NF-κB) inhibited FN-induced pro-MMP-9 activity. FN increased tyrosine phosphorylation of focal adhesion kinase (FAK), integrin linked kinase (ILK), and PI-3K in MDA-MB-231 cells. FN-induced the transactivation of MMP-9 promoter by enhancing DNA binding activity of NF-κB and Sp1. Wound healing assay showed faster migration of MDA-MB-231cells grown on fibronectin-coated as surface as compared to control. Our findings indicated that culture of MDA-MB-231 on fibronectin perhaps send signals via fibronectin-integrin-mediated signaling pathways recruiting FAK, PI-3K, ILK, NF-κB, and modulate expression and activation of pro-MMP-9. These observations may enrich fundamental aspects of cancer biology especially role of α5ß1 integrin in regulation of MMPs expression and activity.

Laminin induces matrix metalloproteinase-9 expression and activation in human cervical cancer cell line (SiHa).

PURPOSE: Interaction between cell surface integrin receptors and extracellular matrix (ECM) components plays an important role in cell survival, proliferation and migration including tumor development and invasion. Matrix metalloproteinases (MMP) are a family of metalloproteinases capable of digesting ECM and facilitate cell migration. Binding of ECM to integrins initiates signaling cascades modulating expression and activity of different MMPs. The present study investigates whether laminin-mediated signaling modulates matrix metalloproteinases (MMP) expression and activity in human cervical cancer cell (SiHa). METHODS: Western blot, immunocytochemistry, ELISA, zymography, RT-PCR, EMSA and wound-healing assay were used. RESULTS: Culture of SiHa cells on laminin (LN)-coated surface induces MMP-9 expression and activation. Wound-healing assay showed that SiHa cells migrate much faster on laminin-coated surface than that of control. LN-induced MMP-9 expression and activation was appreciably reduced with treatment of extracellular signal-regulated kinase (ERK) inhibitor, phosphatidylinositol-3-kinase (PI-3K) inhibitor and anti-α2 antibody. Phosphorylation of focal adhesion kinase (FAK), ERK, and PI-3K was increased upon LN stimulation. LN induces nuclear translocation of PI-3K and nuclear factor kappa B (NF-κB). LN increases DNA-binding activity of NF-κB and activator protein-1 (AP-1) to MMP-9 promoter. CONCLUSIONS: Our findings indicate laminin-induced MMP-9 expression and activation possibly via α2ß1 integrin-mediated signaling involving FAK, PI-3K, ERK followed by transcriptional upregulation of MMP-9.

Epigallocatechin-3-gallate (EGCG) downregulates EGF-induced MMP-9 in breast cancer cells: involvement of integrin receptor α5ß1 in the process.

PURPOSE: Epidermal growth factor receptor (EGFR/ErbB1) is a transmembrane protein with tyrosine kinase activity activated mainly by ligand, EGF. Matrix metalloproteinases (MMPs) are a family of proteinases that catalyses the destruction of ECM, among which MMP-9 has important role in tumor cell invasion. Secretion of MMP-9 is stimulated by a variety of factors, EGFR being significant. Epigallocatechin-3-gallate (EGCG) is a major polyphenol of green tea that inhibits cell proliferation and invasion. Here, we study the effect of EGFR alone and in collaboration with fibronectin on the status of MMP-9 in human breast cancer cell MDA-MB-231 and its molecular mechanism; study the role of EGCG on the induced MMP-9; and elucidate the signaling molecules involved in the process. METHODS: We performed zymography, immunoblots, real-time RT-PCR, cell adhesion assay, siRNA studies, and electrophoretic mobility shift assay to demonstrate the findings. RESULT: EGF induces MMP-9 activity and expression; FAK, PI3 K, and ERK are mainly involved in the process. EGF also causes the transactivation of MMP-9 gene by increasing the DNA binding activity of the transcription factors. EGCG downregulates EGF-induced MMP-9 expression by inhibiting the involved regulatory kinases. EGF collaborates with fibronectin to create a synergistic response, and EGCG inhibits the synergistic response in MDA-MB-231. CONCLUSION: The study demonstrates the requirement of cross talk between cell matrix adhesion molecules and growth factor receptors to improve biological responses and shows FAK/ERK as the pivotal point of this convergence in human breast carcinoma cell line MDA-MB-231. We also establish EGCG as the potential anti-tumor agent in human breast carcinoma.

Correlation technique and least square support vector machine combine for frequency domain based ECG beat classification.

The present work proposes the development of an automated medical diagnostic tool that can classify ECG beats. This is considered an important problem as accurate, timely detection of cardiac arrhythmia can help to provide proper medical attention to cure/reduce the ailment. The proposed scheme utilizes a cross-correlation based approach where the cross-spectral density information in frequency domain is used to extract suitable features. A least square support vector machine (LS-SVM) classifier is developed utilizing the features so that the ECG beats are classified into three categories: normal beats, PVC beats and other beats. This three-class classification scheme is developed utilizing a small training dataset and tested with an enormous testing dataset to show the generalization capability of the scheme. The scheme, when employed for 40 files in the MIT/BIH arrhythmia database, could produce high classification accuracy in the range 95.51-96.12% and could outperform several competing algorithms.

Fibronectin induces matrix metalloproteinase-9 (MMP-9) in human laryngeal carcinoma cells by involving multiple signaling pathways.

Cell adhesion to extracellular matrix initiates intracellular signaling cascade regulated by integrin family of receptors. Evidences show that cultured cells in presence of extracellular matrix adhesion molecule Fibronectin (FN) stimulates secretion of matrix metalloproteinases (MMPs), facilitating cancer cell invasion. Amongst all MMPs, MMP-9 is often reported to play crucial role in tumor cell growth and metastasis. The present study aims at examining the effects of FN on MMP-9 in laryngeal carcinoma cell line, HEp-2, and understand the molecular mechanism(s) involved. The study reports that FN induces the gelatinolytic activity, mRNA and protein expression of MMP-9 in HEp-2 cells. This effect appears to be mediated mainly by integrin receptor α5ß1, since, the blockade of α5 abrogated the FN-mediated stimulatory response on MMP-9. siRNA and inhibitor studies suggested involvement of Focal adhesion kinase (FAK), Phosphatidyl-inositol-3-kinase (PI-3K), Extracellular regulated kinase (ERK) and nuclear factor-kappaB (NFκB) in FN-mediated MMP-9 induction. Immunocytochemical analysis demonstrated the nuclear localization of ERK, PI-3K and NFκB; immunoblot showed enhanced expression of p-FAK, p-PI-3K, p-ERK and nuclear-NF-κB and indicated involvement of ILK in the FN-mediated response. FN-induced transactivation of MMP-9 gene by enhanced DNA binding activity of transcription factors NFκB, Activator protein-1 (AP-1) and Specificity protein-1 (Sp1) to the MMP-9 promoter. Thus, this study suggests that extracellular matrix protein FN induces MMP-9 in HEp-2 cells mainly by involving integrin receptor α5ß1 and involves activation of multiple signaling pathways which independently or in "cross-talk" to each other finally leads to the transactivation of the MMP-9 gene.

Black tea polyphenol (theaflavin) downregulates MMP-2 in human melanoma cell line A375 by involving multiple regulatory molecules.

The tumor-inhibiting property of black tea polyphenol, theaflavin, is well documented. Matrix metalloproteinases (MMPs) play a pivotal role in tumor invasion through degradation of extracellular matrix (ECM). In the present study, we observed the effect of theaflavin on MMP-2, which is upregulated in most tumor types, and its regulatory molecules, in human melanoma cell line, A375. The treatment of theaflavin downregulated the gelatinolytic activity, mRNA and protein expression of MMP-2. It reduced the mRNA and protein expression of membrane type-1 MMP (MT1-MMP) and induced mRNA and protein expression of tissue inhibitor of MMP-2 (TIMP-2), suggesting theaflavin's inhibitory effect on MMP-2 activation. Theaflavin reduced the binding of A375 cell to ECM ligands demonstrating that theaflavin treatment hinders cell-ECM adhesion, cell motility, and integrin-mediated MMP-2 activation. Theaflavin treatment inhibited the protein expression FAK EGFR and ERK, suggesting that, theaflavin treatment downregulates the molecules participating in MMP-2 secretion and regulation. The downregulation of NFchiB suggests downregulation of MMP-2 transactivation. Theaflavin also reduced the tumor volume in syngenic black mice. Thus, we report that theaflavin causes an inhibition of the expression and activity of pro-MMP-2 by a process involving multiple regulatory molecules in human melanoma cells, A375.

Epigallocatechin-3-gallate (EGCG) downregulates gelatinase-B (MMP-9) by involvement of FAK/ERK/NFkappaB and AP-1 in the human breast cancer cell line MDA-MB-231.

Epigallocatechin-3-gallate (EGCG) is effective against the initiation, progression, and invasion of carcinogenesis.Matrix-metalloproteinases (MMPs) are a family of endopeptidases that hydrolyze the majority of extracellular proteins. MMP-9 is one of the most important members of the family and we observed the effect of EGCG on MMP-9 in the human breast cancer cell line, MDA-MB-231.The effect of EGCG on MMP-9 was studied by gelatin zymography, western blot, quantitative and semiquantitative real-time RT-PCR, immunoflourescence, cell adhesion assay, enzyme-linked immunosorbent assay,and electrophoretic mobility shift assay. EGCG treatment reduced the activity, protein, and mRNA expression ofMMP-9 and enhanced the expression of the tissue inhibitor of MMP 1 (TIMP-1). EGCG downregulated the activation of focal adhesion kinase (FAK) and extracellular regulated kinase (ERK), reduced the adhesion of MDA-MB-231 cells to fibronectin and vitronectin, and reduced the mRNA expression of the integrin receptors alpha5beta1 and alphavbeta3. The expression of the nuclear factor kappa B (NFjB), and the DNA binding activity of NFjB and activator protein 1 (AP1)to MMP-9 promoter were noticeably reduced on EGCG treatment. Upregulation of TIMP-1 and disruption of the functional status of integrin receptors may indicate decreased MMP-9 activation; inhibition of FAK andERK activation might indicate disruption in the FAK/ERK-induced MMP-9 secretion and induction. Decreased DNA binding activity of NFjB and AP1 to MMP-9 promoter might indicate transcriptional deregulation of MMP-9 gene on EGCG treatment. We propose EGCG as a potential inhibitor of the expression and activity of MMP-9 by a process involving FAK/ERK and transcription factorsin MDA-MB-231.

All-trans retinoic acid (ATRA) downregulates MMP-9 by modulating its regulatory molecules.

UNLABELLED: The vitamin A derivative all-trans retinoic acid (ATRA) is considered as a potent chemotherapeutic drug for its capability of regulating cell growth and differentiation. We aimed to study the effect of ATRA on MMP-9 in MDA-MB-231, human breast cancer cells and the probable molecular mechanisms through which ATRA exerts its effect. RESULTS: Our experimental findings demonstrate that ATRA enters into the nucleus and regulates various signaling pathways viz. Integrin, FAK, ERK, PI-3K, NF-κB and also EGFR and down regulates pro-MMP-9 activity as well as its expression. As a result MDA-MB-231 cell migration on fibronectin medium gets retarded in presence of ATRA. ATRA up regulates TIMP-1 expression. Our study may help to understand the role of ATRA as a regulator of MMP-9 and the possible signaling pathways which are involved in this ATRA mediated down regulation of MMP-9.

Culture of K562 human myeloid leukemia cells in presence of fibronectin expresses and secretes MMP-9 in serum-free culture medium.

The interaction of cells with adhesion proteins in the extracellular matrix (ECM) provides signals which affect the morphology, motility, gene expression and survival of adherent cells. In the present communication we cultured K562 cells in presence of fibronectin to study the fibronectin-integrin mediated signalling and modulation of MMP expression. Our experimental findings demonstrate that exposure of K562 cells in serum free medium in presence of fibronectin up-regulates the expression of pro-MMP-9 within 2 hrs. Phosphorylation of focal adhesion kinase (FAK), ERK, PI-3K and nuclear translocation of EGFR and NF-kB upon FN binding demonstrate possible involvement of FAK/PI-3K/ERK signalling pathways in the fibronectin-integrin mediated up regulation of MMP-9 expression.

Fibronectin-integrin mediated signaling in human cervical cancer cells (SiHa).

Interaction between cell surface integrin receptors and extracellular matrix (ECM) components plays an important role in cell survival, proliferation, and migration, including tumor development and invasion of tumor cells. Matrix metalloproteinases (MMPs) are a family of metalloproteinases capable of digesting ECM components and are important molecules for cell migration. Binding of ECM to integrins initiates cascades of cell signaling events modulating expression and activity of different MMPs. The aim of this study is to investigate fibronectin-integrin-mediated signaling and modulation of MMPs. Our findings indicated that culture of human cervical cancer cell (SiHa) on fibronectin-coated surface perhaps sends signals via fibronectin-integrin-mediated signaling pathways recruiting focal adhesion kinase (FAK) extracellular signal regulated kinase (ERK), phosphatidyl inositol 3 kinase (PI-3K), integrin-linked kinase (ILK), nuclear factor-kappa B (NF-kappaB), and modulates expression and activation of mainly pro-MMP-9, and moderately pro-MMP-2 in serum-free culture medium.