Regulatory properties of AMP deaminases from rat tissues

1. 1. Phosphoeelluse column chromatography under double gradient conditions (phosphate and KC1) revealed two forms of AMP deaminase in rat heart and brain and a single form in the liver and skeletal muscle.2. 2. Kinetically all purified AMP deaminases were classified into two categories: those, which elute from the column at lower KCl and Pi concentrations, display low S0.5 value are only moderately affected by MgATP, MgGTP and Pi; and those which elute at higher KCl and Pi concentrations, display high S0.5 values and are strongly regulated by allosteric effectors.3. 3. Physiological significance of the occurrence of two kinetic forms of AMP deaminase in some tissues is discussed.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29663/1/0000752.pd

Visinets: A Web-Based Pathway Modeling and Dynamic Visualization Tool

In this report we describe a novel graphically oriented method for pathway modeling and a software package that allows for both modeling and visualization of biological networks in a user-friendly format. The Visinets mathematical approach is based on causal mapping (CMAP) that has been fully integrated with graphical interface. Such integration allows for fully graphical and interactive process of modeling, from building the network to simulation of the finished model. To test the performance of Visinets software we have applied it to: a) create executable EGFR-MAPK pathway model using an intuitive graphical way of modeling based on biological data, and b) translate existing ordinary differential equation (ODE) based insulin signaling model into CMAP formalism and compare the results. Our testing fully confirmed the potential of the CMAP method for broad application for pathway modeling and visualization and, additionally, showed significant advantage in computational efficiency. Furthermore, we showed that Visinets web-based graphical platform, along with standardized method of pathway analysis, may offer a novel and attractive alternative for dynamic simulation in real time for broader use in biomedical research. Since Visinets uses graphical elements with mathematical formulas hidden from the users, we believe that this tool may be particularly suited for those who are new to pathway modeling and without the in-depth modeling skills often required when using other software packages

Human Cytosolic 5′-Nucleotidase I: CHARACTERIZATION AND ROLE IN NUCLEOSIDE ANALOG RESISTANCE

Nucleoside analogs are important in the treatment of hematologic malignancies, solid tumors, and viral infections. Their metabolism to the triphosphate form is central to their chemotherapeutic efficacy. Although the nucleoside kinases responsible for the phosphorylation of these compounds have been well described, the nucleotidases that may mediate drug resistance through dephosphorylation remain obscure. We have cloned and characterized a novel human cytosolic 5'-nucleotidase (cN-I) that potentially may have an important role in nucleoside analog metabolism. It is expressed at a high level in skeletal and heart muscle, at an intermediate level in pancreas and brain, and at a low level in kidney, testis, and uterus. The recombinant cN-I showed high affinity toward dCMP and lower affinity toward AMP and IMP. ADP was necessary for maximal catalytic activity. Expression of cN-I in Jurkat and HEK 293 cells conferred resistance to 2-chloro-2'-deoxyadenosine, with a 49-fold increase in the IC(50) in HEK 293 and a greater than 400-fold increase in the IC(50) in Jurkat cells. Expression of cN-I also conferred a 22-fold increase in the IC(50) to 2',3'-difluorodeoxycytidine in HEK 293 cells and an 82-fold increase in the IC(50) to 2',3'-dideoxycytidine in Jurkat cells. These data indicate that cN-I may play an important role in the regulation of physiological pyrimidine nucleotide pools and may also alter the therapeutic efficacy of certain nucleoside analogs

Regulation of the Human Inosine Monophosphate Dehydrogenase Type I Gene: UTILIZATION OF ALTERNATIVE PROMOTERS

Catalysis of guanine nucleotide formation from IMP in the de novo purine synthetic pathway is carried out by two isoforms of the enzyme inosine monophosphate dehydrogenase (IMPDH) that are catalytically indistinguishable but are encoded by separate genes. In order to assess the potential for cell type-specific expression of IMPDH activity, we have characterized the IMPDH type I gene and identified three major RNA transcripts that are differentially expressed from three different promoters. A 4.0-kilobase pair (kb) mRNA containing 1.3 kb of 5'-untranslated region is expressed in activated peripheral blood lymphocytes and to a far lesser extent in cultured tumor cell lines. The P1 promoter that regulates the transcription of this mRNA has a high degree of sequence identity to an Alu repetitive sequence. A transcript of 2.7 kb is found in a subset of the tumor cell lines examined, whereas a 2.5-kb mRNA species is universally expressed and is the prevalent mRNA in most cell lines and tissues. The relative strengths of the three promoter regions and the effects of variable extents of 5'-flanking sequence on the P3 promoter differ in Jurkat T, as compared with Raji B lymphoid cell lines, demonstrating a complex cell type-specific transcriptional regulation of IMPDH type I gene expression

Tissue-specific Regulation of the Ecto-5′-nucleotidase Promoter: ROLE OF THE cAMP RESPONSE ELEMENT SITE IN MEDIATING REPRESSION BY THE UPSTREAM REGULATORY REGION

We have isolated the 5' region of the ecto-5'-nucleotidase (low K(m) 5'-NT) gene and established that a 969-base pair (bp) fragment confers cell-specific expression of a CAT reporter gene that correlates with the expression of endogenous ecto-5'-NT mRNA and enzymatic activity. A 768-bp upstream negative regulatory region has been identified that conferred lymphocyte-specific negative regulation in a heterologous system with a 244-bp deoxycytidine kinase core promoter. DNase I footprinting identified several protected areas including Sp1, Sp1/AP-2, and cAMP response element (CRE) binding sites within the 201-bp core promoter region and Sp1, NRE-2a, TCF-1/LEF-1, and Sp1/NF-AT binding sites in the upstream regulatory region. Whereas the CRE site was essential in mediating the negative activity of the upstream regulatory region in Jurkat but not in HeLa cells, mutation of the Sp1/AP-2 site decreased promoter activity in both cell lines. Electrophoretic mobility shift assay analysis of proteins binding to the CRE site identified both ATF-1 and ATF-2 in Jurkat cells. Finally, phorbol 12-myristate 13-acetate increased the activity of both the core and the 969-bp promoter fragments, and this increase was abrogated by mutations at the CRE site. In summary, we have identified a tissue-specific regulatory region 5' of the ecto-5'-NT core promoter that requires the presence of a functional CRE site within the basal promoter for its suppressive activity

Tenascin C interacts with Ecto-5′-nucleotidase (eN) and regulates adenosine generation in cancer cells

Tenascin C is expressed in invasive human solid tumors; however its specific role in cancer biology remains obscure. Previously, we have found that ecto-5'-nucleotidase (eN) is a marker of ER (-) breast carcinoma and elevated expression correlates with invasive mesenchymal cell phenotype. To investigate for the potential relationship between eN and protein components of the extracellular matrix (ECM) we measured adenosine generation from AMP in cells incubated with soluble ECM proteins. We found that tenascin C was the only ECM component that strongly inhibited ecto-5'-nucleotidase (eN) activity in situ and adenosine generation from AMP (75% inhibition, p < 0.01). The inhibition was comparable to that induced by concanavalin A, a well-defined and strong inhibitor of eN. Resin immobilized tenascin C, but not collagen, and only weakly fibronectin, specifically and quantitatively bound cell-extracted eN. We further developed breast cancer cell line with reduced eN expression and tested changes in cell adhesion on different ECM. Breast cancer cells expressing reduced eN attached 56% weaker (p < 0.05) to immobilized tenascin C. This difference was not detected with other ECM proteins. Finally, control breast cancer cells migrated slower on tenascin C when compared with clone with reduced eN expression. These data suggest that eN is a novel and specific receptor for tenascin C and that the interaction between these proteins may influence cell adhesion and migration and also lead to decreased generation of local adenosine

Tenascin C interacts with Ecto-5′-nucleotidase (eN) and regulates adenosine generation in cancer cells

Tenascin C is expressed in invasive human solid tumors; however its specific role in cancer biology remains obscure. Previously, we have found that ecto-5'-nucleotidase (eN) is a marker of ER (-) breast carcinoma and elevated expression correlates with invasive mesenchymal cell phenotype. To investigate for the potential relationship between eN and protein components of the extracellular matrix (ECM) we measured adenosine generation from AMP in cells incubated with soluble ECM proteins. We found that tenascin C was the only ECM component that strongly inhibited ecto-5'-nucleotidase (eN) activity in situ and adenosine generation from AMP (75% inhibition, p < 0.01). The inhibition was comparable to that induced by concanavalin A, a well-defined and strong inhibitor of eN. Resin immobilized tenascin C, but not collagen, and only weakly fibronectin, specifically and quantitatively bound cell-extracted eN. We further developed breast cancer cell line with reduced eN expression and tested changes in cell adhesion on different ECM. Breast cancer cells expressing reduced eN attached 56% weaker (p < 0.05) to immobilized tenascin C. This difference was not detected with other ECM proteins. Finally, control breast cancer cells migrated slower on tenascin C when compared with clone with reduced eN expression. These data suggest that eN is a novel and specific receptor for tenascin C and that the interaction between these proteins may influence cell adhesion and migration and also lead to decreased generation of local adenosine

Visinets: a web-based pathway modeling and dynamic visualization tool.

In this report we describe a novel graphically oriented method for pathway modeling and a software package that allows for both modeling and visualization of biological networks in a user-friendly format. The Visinets mathematical approach is based on causal mapping (CMAP) that has been fully integrated with graphical interface. Such integration allows for fully graphical and interactive process of modeling, from building the network to simulation of the finished model. To test the performance of Visinets software we have applied it to: a) create executable EGFR-MAPK pathway model using an intuitive graphical way of modeling based on biological data, and b) translate existing ordinary differential equation (ODE) based insulin signaling model into CMAP formalism and compare the results. Our testing fully confirmed the potential of the CMAP method for broad application for pathway modeling and visualization and, additionally, showed significant advantage in computational efficiency. Furthermore, we showed that Visinets web-based graphical platform, along with standardized method of pathway analysis, may offer a novel and attractive alternative for dynamic simulation in real time for broader use in biomedical research. Since Visinets uses graphical elements with mathematical formulas hidden from the users, we believe that this tool may be particularly suited for those who are new to pathway modeling and without the in-depth modeling skills often required when using other software packages

Elemental influences in Visinets.

<p>To achieve shown behavior in Visinets, all initial values for species A and P were set as shown and the weights for all influences (arrows) were set at 0.5, except for positive self-influence (E) where the influence of A on P was set at 0.01.</p