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.

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.

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.

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.

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.

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.

Multifunctional effect of epigallocatechin-3-gallate (EGCG) in downregulation of gelatinase-A (MMP-2) in human breast cancer cell line MCF-7.

AIMS: The tumor inhibiting property of green tea polyphenol epigallocatechin-3-gallate (EGCG) is well documented. Studies reveal that matrix-metalloproteinases (MMPs) play pivotal roles in tumor invasion through degradation of basement membranes and extracellular matrix (ECM). We studied the effect of EGCG on matrixmetalloproteinases-2 (MMP-2), the factors involved in activation, secretion and signaling molecules that might be involved in the regulation of MMP-2 in human breast cancer cell line, MCF-7. MAIN METHODS: MCF-7 was treated with EGCG (20 muM, 24 h), the effect of EGCG on MMP-2 expression, activity and its regulatory molecules were studied by gelatin zymography, Western blot, quantitative and semi-quantitative real time RT-PCR, immunoflourescence and cell adhesion assay. KEY FINDINGS: EGCG treatment reduced the activity, protein expression and mRNA expression level of MMP-2. EGCG treatment reduced the expression of focal adhesion kinase (FAK), membrane type-1-matrix metalloproteinase (MT1-MMP), nuclear factor-kappa B (NF-kB), vascular endothelial growth factor (VEGF) and reduced the adhesion of MCF-7 cells to ECM, fibronectin and vitronectin. Real time RT-PCR revealed a reduced expression of integrin receptors alpha5, beta1, alphav and beta3 due to EGCG treatment. SIGNIFICANCE: Down regulation of expression of MT1-MMP, NF-kB, VEGF and disruption of functional status of integrin receptors may indicate decreased MMP-2 activation; low levels of FAK expression might indicate disruption in FAK-induced MMP-2 secretion and decrease in activation of phosphatidyl-inositol-3-kinase (PI-3K), extracellular regulated kinase (ERK) indicates probable hindrance in MMP-2 regulation and induction. We propose EGCG as potential inhibitor of expression and activity of pro-MMP-2 by a process involving multiple regulatory molecules in MCF-7.

Rapid expression and activation of MMP-2 and MMP-9 upon exposure of human breast cancer cells (MCF-7) to fibronectin in serum free medium.

Interactions between tumour cells and the extracellular matrix (ECM) strongly influence tumour development, affecting cell survival, proliferation and migration. Many of these interactions are mediated through a family of cell surface receptors named integrins. Fibronectin and its integrin receptors play important roles in tumour development. The alpha5beta 1 integrin interacts with the central cell adhesive region of fibronectin and requires both the RGD and synergy sites for maximal binding. Matrix metalloproteinases (MMPs) are a family of zinc dependent endopeptidases. They are capable of digesting the different components of the ECM and basement membrane. The ECM gives structural support to cells and plays a central role in cell adhesion, differentiation, proliferation and migration. Binding of ECM to integrins modulates expression and activity of the different MMPs. Our experimental findings demonstrate that cultivation of human breast cancer cells, MCF-7, in serum free medium in the presence of fibronectin upregulates the activity of MMP-2 and MMP-9. Blocking of alpha5beta 1 integrin with anti-alpha5 monoclonal antibody inhibits the fibronectin-induced MMP activation response appreciably. This strongly indicates alpha5beta 1 mediated signalling events in activation of MMP-2 and MMP-9. Phosphorylation of FAK and PI-3 kinase and the nuclear translocation of ERK and NF-kappaB upon fibronectin binding demonstrate possible participation of the FAK/PI-3K/ERK signalling pathways in the regulation of MMP-2 activity.

Culture of human A375 melanoma cells in the presence of fibronectin causes expression of MMP-9 and activation of MMP-2 in culture supernatants.

UNLABELLED: Interactions between tumor cell surface integrin receptors and extracellular matrix (ECM) ligands play an important role in tumor development, affecting cell survival, proliferation, and migration. Integrin-ECM ligand interaction leads to phosphorylation of focal adhesion kinase (FAK) and activation of mitogen-activated protein kinase (MAPK) pathways. It has been reported that integrins also regulate expression and function of matrix metalloproteinases (MMPs). In this present work, we cultured human A375 melanoma cells in the presence of fibronectin to study fibronectin-integrin mediated modulation of MMP activity. METHODS: A375 cells were cultured in serum-free culture medium (SFCM) in the presence of fibronectin (25 microg/0.75 ml), SFCM was collected and gelatin zymography was performed. Western blot and RT-PCR were performed with A375 cells cultured in the presence of fibronectin. RESULTS: Culture of A375 cells in the presence of fibronectin led to expression of MMP-9 and activation of MMP-2 within 2 h. When cells were treated with ERK inhibitor (PD98059) or PI-3K inhibitor (LY294002) and grown in the presence of fibronectin, MMP-9 expression and MMP-2 activation was inhibited. Tyrosine phosphorylation of FAK and ERK were increased in A375 cells grown in the presence of fibronectin. Increased MMP-9 mRNA expression and processing of MT1-MMP were also observed in A375 cells grown in the presence of fibronectin. CONCLUSIONS: Our findings indicate culture of A375 cells in SFCM in the presence of fibronectin perhaps generates a signaling cascade that leads to expression of MMP-9 and activation of MMP-2 in culture supernatants within 2 h. The signaling pathway activated is probably the FAK/ERK pathway.

Hybrid multiresolution Slantlet transform and fuzzy c-means clustering approach for normal-pathological brain MR image segregation.

The paper presents a new approach for automated segregation of brain MR images, using an improved orthogonal discrete wavelet transform (DWT), known as the Slantlet transform (ST), and a fuzzy c-means (FCM) clustering approach. ST has excellent time-frequency resolution characteristics and these can be achieved with shorter supports for the filter, compared to DWT employed for identical situations. FCM clustering, on the other hand, can provide efficient classification results, if it is implemented for well-processed input feature vectors. Thus, by combining both the ST and the FCM clustering approaches, a hybrid scheme has been developed that can segregate brain MR images. This automated tool when developed can infer whether the input image is that of a normal brain or a pathological brain. The proposed technique has been applied to several benchmark brain MR images and the results reveal excellent accuracy in characterizing human brain MR imaging.

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.

GnT-V expression and metastatic phenotypes in macrophage-melanoma fusion hybrids is down-regulated by 5-Aza-dC: evidence for methylation sensitive, extragenic regulation of GnT-V transcription.

Beta1,6-acetylglucosaminyltransferase V (GnT-V) forms beta1,6 branching on the trimannosyl terminus of N-glycans, allowing for the production of beta1,6Glc-NAc-bearing oligosaccharides. These are used by healthy myeloid cells and cancer cells alike for systemic migration. GnT-V has multiple glycoprotein substrates and thereby exerts global effects on cancer progression, characteristic of a master regulator of metastasis. Yet little is known of the regulation of GnT-V expression by tumor cells. It was previously reported that fusion of macrophages with Cloudman S91 mouse melanoma cells produced macrophage-melanoma hybrids with up-regulated GnT-V expression regarding mRNA and enzymatic activity. Majority of these hybrids showed increased chemotactic motility in vitro and elevated metastatic potential in vivo. Here we attempted to understand this at the molecular genetic level focusing on DNA hypermethylation as a potentially key step. Treatment of cells with 5-Aza-dC, an inhibitor of DNA methylation, resulted in decreased expression of GnT-V mRNA and beta1,6-branched oligosaccharides along with reduced glycosylation of LAMP-1, a major substrate for GnT-V. This was accompanied by reduced chemotactic motility of the cells. The results suggested that DNA hypermethylation in some fashion stimulated GnT-V expression. We thus investigated the promoter region of the GnT-V gene for hypermethylation of CpG islands, comparing macrophage-melanoma hybrids of low and high metastatic potential with the parental melanoma cell line. Genomic DNA after bisulfite modification amplified from this region showed identical sequences between the cell lines. The findings indicated that differential methylation of the promoter region of GnT-V gene was not responsible for its transcriptional control, rather, appeared to be controlled through a negative regulator, nm23, whose own expression was regulated by hypermethylation. Although our studies involved a highly experimental system, the results further suggest that by whatever mechanism, reduction of GnT-V activity through 5-Aza-dC treatment might provide a new approach towards prevention of metastatic progression.

Culture of human cervical cancer cells, SiHa, in the presence of fibronectin activates MMP-2.

PURPOSE: Several studies indicate that integrin receptors are involved in the regulation of matrix metalloproteinase (MMP) expression. Integrin-ECM ligand interaction leads to phosphorylation of focal adhesion kinase (FAK) and activation of mitogen activated protein kinase pathways. In this present communication, we cultured human cervical cancer cells, SiHa, in the presence of fibronectin to study fibronectin-integrin mediated modulation of MMP activity. METHODS: SiHa cells were cultured in serum-free medium (SFCM) in the presence of fibronectin, SFCM was collected and gelatin zymography was performed. Western blot, RT-PCR and immunocytochemistry were performed with SiHa cells cultured in the presence of fibronectin. RESULTS: The culture of SiHa cells in the presence of 50 microg/1.5 ml fibronectin led to expression of pro-MMP-9 and activation of MMP-2 within 2 h. When cells were treated with ERK inhibitor (PD98059) and grown in the presence of fibronectin MMP-2 activation was partially inhibited, but when cells were treated with PI-3K inhibitor (LY294002) and grown in the presence of fibronectin MMP-2 activation was appreciably reduced. Tyrosine phosphorylation of FAK, PI-3K and ERK and nuclear trafficking of ERK were increased in SiHa cells grown in the presence of fibronectin. Increased MT1-MMP mRNA expression and processing of MT1-MMP were also observed in SiHa cells grown in the presence of fibronectin. CONCLUSIONS: Our findings indicate that the culture of SiHa cells in SFCM in the presence of fibronectin perhaps generates a signalling cascade which leads to the expression of pro-MMP-9 and the activation of MMP-2 within 2 h. The signalling pathways activated seem to be the FAK/ERK/PI-3K pathway.

Cell membrane-associated MT1-MMP dependent activation of MMP-2 in SiHa (human cervical cancer) cells.

UNLABELLED: Among the soluble MMPs, MMP-2 (gelatinase A) is particularly important in the invasive property of tumor cells. Cell membrane-associated MMP-2 activation is one of the challenging areas in tumor biology. In the present communication, we studied the membrane dependent activation of MMP-2 in SiHa cells. METHODS: Activation of pro-MMP-2 by membrane fraction, membrane extract, and live SiHa cells was studied by gelatin zymography. The role of MT1-MMP in MMP-2 activation was studied by incubating SiHa cells and cell membrane fractions with anti-MT1-MMP antibody. RESULTS: Activation of purified pro-MMP-2 by membrane fraction isolated from SiHa cells, by SiHa cell membrane extract and by SiHa cells, pro-MMP-2 from Con A treated HT-1080 conditioned medium by SiHa cells, and pro-MMP-2 from serum free culture medium of SiHa cells and cervical tissue homogenate by SiHa cell membrane fraction was shown by gelatin zymography. SiHa membrane fraction activated only pro-MMP-2 from purified MMP-9/MMP-2 mixture, indicating that the activation is specific for MMP-2. Inhibition of MMP-2 activation in the presence of anti-MT1-MMP antibody strongly indicated that the cell membrane mediated MMP-2 activation is MT1-MMP dependent. Immunocytochemistry of SiHa cells demonstrated expression of MT1-MMP at focal points. Invasion assay showed that invasiveness of anti-MT1-MMP antibody treated SiHa cells through Matrigel was drastically reduced compared to control SiHa cells. CONCLUSIONS: Our findings furnish an example of the cell membrane-associated MT1-MMP mediated MMP-2 activation in SiHa cells and suggest that this MT1-MMP mediated MMP-2 activation is of importance in tumor invasion and metastasis. This MT1-MMP mediated MMP-2 activation on tumor cell surface could be a realistic target for managing metastatic diseases.

Culture of human fibrosarcoma HT-1080 cells in presence of fibronectin activates MMP-2.

UNLABELLED: The importance of tumor cell surface integrin receptors in regulation of matrix metalloproteinase (MMP) expression and function has been reported. Integrin-ECM ligand interaction leads to phosphorylation of focal adhesion kinase (FAK) and activation of mitogen activated protein kinase (MAPK) pathways. In this present study, we cultured human fibrosarcoma cells, HT-1080, in presence of fibronectin to study fibronectin-integrin mediated modulation of MMP activity. METHODS: HT-1080 cells were cultured in serum free medium (SFCM) in presence of fibronectin, SFCM was collected, and gelatin zymography was performed. Western blot and immunocytochemistry were performed with HT-1080 cells cultured in presence of fibronectin. RESULTS: Culture of HT-1080 cells in presence of 50 microg/1.5 ml fibronectin led to expression of pro-MMP-9 and activation of MMP-2 within 1 hr. When HT-1080 cells were treated with PI-3K inhibitor (LY294002) and grown in presence of fibronectin, MMP-2 activation was partially inhibited, but when cells were treated with ERK inhibitor (PD98059) and grown in presence of fibronectin, MMP-2 activation was almost completely inhibited. Tyrosine phosphorylation of FAK and ERK were increased in HT-1080 cells grown in presence of fibronectin. Processing of MT1-MMP was also observed in HT-1080 cells grown in presence of fibronectin. The reorganization of actin filaments in fibronectin treated HT-1080 cells was also noticeable. CONCLUSIONS: Our findings indicate that culture of HT-1080 cells in SFCM in presence of fibronectin perhaps generates a signaling cascade that leads to expression of pro-MMP-9 and activation of MMP-2 within 1 hr. The signaling pathway activated seems to be the FAK/ERK pathway.

Curcumin, a potential inhibitor of MMP-2 in human laryngeal squamous carcinoma cells HEp2.

UNLABELLED: Curcumin (diferuloylmethane) has been widely studied for its tumor inhibiting and anticarcino-genic properties. In the present communication, we studied the effect of curcumin on matrix-metalloproteinase-2 (MMP-2), integrin receptors, and focal adhesion kinase (FAK) in human laryngeal cancer cells, HEp2. METHODS: HEp2 cells were treated with curcumin (5 microM) for 30 days and then grown without curcumin for 28 days. Effect of curcumin on MMP-2 expression and activity and on membrane type matrixmetalloproteinase-1 (MT1-MMP), FAK, and integrin receptors was studied by zymography, Western blot, ELISA, RT-PCR, and cell adhesion assay. RESULTS: Treatment of HEp2 cells with curcumin downregulated MMP-2 expression and activity and expression of integrin receptors, FAK, and MT1-MMP to almost background levels. MMP-2 (but not MMP-9) mRNA expression was abolished on curcumin treatment, indicating specific inhibition of MMP-2. Invasive potential of HEp2 cells was also significantly reduced. After drug withdrawal, expression of MMP-2, integrin receptors, MT1-MMP, and FAK returned to control levels. However, MMP-2 activity in serum free medium remained low. CONCLUSIONS: Downregulation of integrin receptors and low levels of FAK may hinder integrin-mediated signal transduction, preventing upregulation of MMP-2 activity. Reduction of MMP-2 activity and inhibition of HEp2 cell invasion by curcumin strongly indicate the potential of curcumin as an inhibitor of tumor cell invasion and metastasis.

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.

An input-delay neural-network-based approach for piecewise ECG signal compression.

We propose an input delay neural network (IDNN) based time series prediction algorithm for compressing electrocardiogram (ECG) signals. Our algorithm has been tested and successfully compared vis-à-vis other popular techniques for its compression efficiency and reconstruction capability.

Cell membrane-associated MT1-MMP-dependent activation of pro-MMP-2 in A375 melanoma cells.

UNLABELLED: Matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases, can degrade extracellular matrix components under physiological conditions and during cancer invasion and metastasis. Among the MMPs, the 72 kDa type IV collagenase MMP-2 (gelatinase A) is activated in a membrane-associated manner by an activation complex composed of membrane type 1 matrix metalloproteinase (MT1-MMP), tissue inhibitor of matrixmetalloproteinase-2 (TIMP-2), and pro-MMP-2 in the presence of alphavbeta3 integrin receptor. The activation of pro-MMP-2 correlates with increased occurrence of metastases. Increased MMP-2 activity has been demonstrated in many human tumors. In the present communication, we studied cell surface-associated activation of MMP-2 (72 kDa collagenase type IV) in the moderately metastatic human melanoma cell line A375. RESULTS: Activation of purified 72 kDa collagenase type IV, pro-MMP-2 from cervical cancer tissue homogenate and from serum-free culture medium of HT1080 cells grown in presence of concanavalin A, by A375 cells, was shown by gelatin zymography. A375 cells activated only pro-MMP-2 from purified MMP-9/MMP-2 mixture indicating that the activation is specific for MMP-2. Activation of MMP-2 and purified collagenase type IV by A375 membrane fraction and membrane extract was also demonstrated by gelatin zymography. When A375 cells were first incubated with anti-MT1-MMP polyclonal antibody, activation of collagenase type IV was significantly decreased, indicating that membrane-associated MMP-2 activation is MT1-MMP-mediated. Immunocytochemistry showed MT1-MMP localization at focal adhesion sites. The presence of the components of activation complex-MT1-MMP and integrin alphavbeta3-were confirmed by Western blot, cell adhesion assay, and integrin subunit assay. CONCLUSION: Our experimental findings furnish another example of the unique membrane-associated MMP-2 activation mechanism in A375 melanoma cells and clearly indicate the role of MT1-MMP in MMP-2 activation. The information could help in developing new therapies designed to interfere with MMP activation and management of cancer and metastases.

Effect of curcumin on gelatinase A (MMP-2) activity in B16F10 melanoma cells.

Treatment of highly metastatic murine melanoma cells B16F10 with curcumin (15 microM) for 15 days significantly inhibited matrixmetalloproteinase-2 (MMP-2) activity. Expression of membrane type-1 matrix metalloproteinase (MT1-MMP) and focal adhesion kinase (FAK), an important component of the intracellular signalling pathway, were also reduced to almost background levels. MMP-2, MT1-MMP and FAK did not return to control levels even after 28 days of drug withdrawal. However, effect of curcumin on ligand binding property of integrin receptors was reversible. Downregulation of FAK (which would impair integrin mediated signal transduction cascade) and reduction of MMP-2 activity could be important reasons for anti-metastatic property of curcumin.