Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the absence of a growth factor or an appropriate extracellular matrix (ECM), cells are arrested in the G0/G1 phase. In this report, we demonstrate the evidence that TNF-alpha induced DNA synthesis of primary mouse hepatocytes in vitro by activating two distinct pathways. TNF-alpha induced drastic spreading of hepatocytes on hydrophobic plastic, while the adhesion was not influenced. The effect was time and dose dependent. The cell spreading was accompanied by the phosphorylation of paxillin, indicating the stimulation of focal adhesion molecules. TNF-alpha-induced spreading of hepatocytes was not transient, and kinetic analysis and morphologic observation suggest that the effect was different from epidermal growth factor- or hepatocyte growth factor-induced transient hepatocyte spreading. TNF-alpha-induced hepatocyte spreading was blocked by cytochalasin D, Arg-Gly-Asp peptides, cycloheximide, or anti-integrin beta1 Ab. Results of competitive PCR for ECM proteins demonstrated that TNF-alpha increased the expression of laminin alpha3 and gamma1 chains in hepatocytes. These data suggested that TNF-alpha induced cell anchorage for hepatocytes by up-regulating ECM production. More importantly, TNF-alpha, but neither epidermal growth factor nor hepatocyte growth factor, induced DNA synthesis following the spreading in primary hepatocytes on hydrophobic plastic, while mere cell spreading on collagen did not induce DNA synthesis. The DNA synthesis was blocked by the inhibition of either cell spreading or DNA polymerase, demonstrating that TNF-alpha induced DNA synthesis in primary hepatocytes by activating two distinct pathways, i.e., forming the scaffold and inducing growth signals. Taken together, TNF-alpha bifunctionally regulates the proliferation of primary hepatocytes, serving as both an ECM inducer and a growth factor.
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PMID:TNF-alpha bifunctionally induces proliferation in primary hepatocytes: role of cell anchorage and spreading. 936 9

It is well known that EBV has developed strategies to evade immune surveillance. Previously, EBV was shown to bind specifically to monocytes and regulate expression of proinflammatory mediators such as IL-1, IL-6, TNF-alpha, and leukotrienes. EBV was also found to affect phagocytosis of monocytes. In this study, we show that in addition to these effects, EBV suppresses the biosynthesis of PGE2, a pleiotropic immunomodulatory molecule that is synthesized by the dioxygenation of arachidonic acid via the cyclooxygenase (COX) pathway. This down-regulation of PGE2 formation involved the inhibition of the inducible COX-2 isoform expression both at the transcriptional and translational levels, whereas expression of the constitutive COX-1 isoform was unaltered. Furthermore, exposure of monocytes to EBV was found to impact on the NF-kappaB activation pathway, which plays an essential role in the induction of COX-2 in monocytes. The inhibition of PGE2 biosynthesis was relieved when the experiments were conducted in presence of phosphonoacetic acid, an inhibitor of herpesviruses DNA polymerase, indicating that viral replication and/or neosynthesized viral proteins were involved in this process. Thus, inhibition of PGE2 biosynthesis in monocytes may represent an additional mechanism underlying EBV pathogenicity.
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PMID:EBV suppresses prostaglandin E2 biosynthesis in human monocytes. 1084 3

Here we present a novel methodology to quantitate bovine cytokines and growth factors contributing to immunity against bacterial infections of the mammary gland in cattle. Real-time TaqMan PCR systems were developed to overcome limitations of conventional quantitative PCR methods. The TaqMan method is based on the cleavage of fluorescent dye-labeled probes by the 5'-3' exonuclease activity of the Taq DNA polymerase during PCR and measurement of fluorescence intensity by an automated spectrophotometer integrated in a sequence detection system (Applied Biosystems, Foster City, CA). The bovine-specific TaqMan probes were designed to encompass an intron, thus allowing differentiation between complementary DNA (cDNA) and genomic DNA (gDNA) amplification products. Quantitative analysis of cytokine cDNA was performed in comparison to bovine glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Messenger RNA (mRNA) from the universally expressed housekeeping gene GAPDH proved to be useful as an amplification control and allowed for correction of variations in different numbers of cells in the starting material, in the efficiencies of RNA extraction and reverse transcription. With this method, high-throughput analysis of large numbers of samples was possible within a short time. In addition, decreasing the numbers of working steps shortened the time for analysis and increased accuracy. Profiles of cytokines (interleukin (IL)-2, IL-6, IL-8, IL-12 p40, TNF-alpha, IFN-gamma) and granulocyte-macrophage colony stimulating factor (GM-CSF) were established in normal lactating cattle. Differences of cytokine profiles obtained with the real-time TaqMan PCR system and conventional methods are discussed.
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PMID:Quantitation of bovine cytokine mRNA in milk cells of healthy cattle by real-time TaqMan polymerase chain reaction. 1113 25

Rapidly increasing information about the human genome requires a fast and simple method for the detection of single-nucleotide polymorphisms (SNPs). To date, the conventional SNP detection technologies have been unable to identify all possible SNPs and needed further development in cost, speed, and sensitivity. Here we describe a novel method to discriminate and code all possible combinations. SNPs were coded by monitoring the changes in the electrochemical signal of the monobase-modified colloidal gold (Au) nanoparticles. First, a chitosan layer was formed on the alkanethiol self-assembled monolayer-modified Au nanoparticle. The monobases were then attached onto the chitosan-coated Au nanoparticles through their 5' phosphate group via the formation of a phosphoramidate bond with the free amino groups of chitosan. The size of the surface-modified Au nanoparticle was found to be 8.46 +/- 1.53 nm by using atomic force microscopy. If there is a SNP in DNA and the mismatched bases are complementary to the monobase, Au nanoparticles accumulate on the electrode surface in the presence of DNA polymerase I (Klenow fragment), thus resulting in a significant change in the Au oxide wave. In this report, monobase-modified Au nanoparticles show not only the presence of a SNP, but also identify which bases are involved within the pair. Especially, the identification of a transversion SNP, which contains a couple of the same pyrimidine or purine bases, is greatly simplified. A model study was performed by using a synthetic 21-base DNA probe related to tumor necrosis factor (TNF-alpha) along with its all possible mutant combinations. This versatile nanoparticle-based electrochemical protocol is a promising candidate for coding all mutational changes.
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PMID:Electrochemical coding of single-nucleotide polymorphisms by monobase-modified gold nanoparticles. 1505 47

The importance of the ligand presentation format for the production of protein capture microarrays was evaluated using different Affibody molecules, produced either as single 6 kDa monomers or genetically linked head-to-tail multimers containing up to four domains. The performances in terms of selectivity and sensitivity of the monomeric and the multidomain Affibody molecules were compared by immobilization of the ligands on microarray slides, followed by incubation with fluorescent-labeled target protein. An increase in signal intensities for the multimers was demonstrated, with the most pronounced difference observed between monomers and dimers. A protein microarray containing six different dimeric Affibody ligands with specificity for IgA, IgE, IgG, TNF-alpha, insulin, or Taq DNA polymerase was characterized for direct detection of fluorescent-labeled analytes. No cross-reactivity was observed and the limits of detection were 600 fM for IgA, 20 pM for IgE, 70 fM for IgG, 20 pM for TNF-alpha, 60 pM for insulin, and 10 pM for Taq DNA polymerase. Also, different sandwich formats for detection of unlabeled protein were evaluated and used for selective detection of IgA or TNF-alpha in human serum or plasma samples, respectively. Finally, the presence of IgA was determined using detection of directly Cy5-labeled normal or IgA-deficient serum samples.
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PMID:Affibody molecules in protein capture microarrays: evaluation of multidomain ligands and different detection formats. 1720 61

KCTD10 is a TNF-alpha inducible protein that can interact with the small subunit of DNA polymerase a and PCNA. In order to study the function of KCTD10, we prepared the rabbit anti-mouse KCTD10 polyclonal antibody by using the His-tagged recombinant mouse KCTD10 protein to immune New Zealand white rabbit. Mouse KCTD10 shares significant similarity with PDIP1 (polymerase delta-interacting protein 1) and TNFAIP1 (tumor necrosis factor alpha-induced protein 1) protein,and then KCTD10 polyclonal antiserum possesses cross-reactivity with PDIP1 protein and TNFAIP1 protein. The partially digested fragments of homogeneous proteins PDIP1 and TNFAIP1 were mixed and incubated with anti-KCTD10 antiserum at 4 degrees C for 3 h to deplete unspecific antibodies. Through this method, we removed successfully the cross-reactivity of anti-KCTD10 antibody with PDIP1 and TNFAIP1 and obtained specific anti-KCTD10 antibody. Then, the anti-KCTD10 antibody was used in immunohistochemistry experiments of mouse. The results of immunohistochemistry on whole-mount embryo and paraffin section demonstrated that KCTD10 is highly expressed in neuroepithelium of neural tube and dorsal root ganglion of 12.5 d embryos. These results suggest that KCTD10 may play roles in the development of neuroepithelium of neural tube and dorsal root ganglion.
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PMID:[Preparation of mouse KCTD10 antibody and expression analysis of KCTD10 in neuroepithelium of neural tube and dorsal root ganglion]. 1825 28