Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The antisense method is one of the most promising anti-cancer methods, however, the design of antisense oligonucleotides is difficult because many factors affecting their activitiy and stability must be considered. Recently, the oligonucleotide stabilities related to the antisense effects were quantitatively investigated based on nearest-neighbor parameters. We demonstrated that DeltaG(o) (37, hyb), a free energy change for the hybridization of antisense oligodeoxynucleotides (ODNs) with target RNAs is related to the RNase H cleavage of TAg (SV40 large T antigen) mRNA, the expression of a rabbit globin mRNA, and the protein function encoded by hMDR1 (human multidrug resistance-1) mRNA, while DeltaG(o) (37, hp), a free-energy change for hairpin formations of the antisense ODNs significantly affected the arrest efficiency of the DHFR (dihydrofolate reductase) mRNA transcription, the expression of the proalpha1(I) chain of human, and the hybridization extent for HIV-1 alpha-1. For ras RNA (Ha-ras mRNA), DeltaG(o) (37, sc), a free energy change for the conformational change of the mRNA required for antisense ODN binding showed the best correlation with the equilibrium constants for the hybridization with their target RNA. On the other hand, the antisense effects ifor the HSV-1 IE5 (herpes simplex virus type 1 immediate early pre-mRNA5) showed less of a relationship to the hybridization stability of the antisense ODNs with the target pre-mRNA, because the antisense ODNs targeting the pre-mRNA must collapse its secondary structure around the splicing site to cancel out the expected antisense effects. Based on these results, we illustrate a new concept for the design of antisense ODNs based on DeltaG(o) (37, hyb), DeltaG(o) (37, hp), and DeltaG(o) (37, sc).
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PMID:A new concept for the design of antisense oligonucleotides based on nucleic acid thermostability. 1267 72

The gene (US4) coding for herpes simplex virus type 2 (HSV-2) glycoprotein G (gG-2) was cloned and constitutively expressed in Chinese hamster ovary (CHO) cells. The expression vector containing the dihydrofolate reductase (dhfr) gene, and the HSV-2 US4 gene under the control of the Simian virus 40 early promoter (SV40 EP), was transfected into dhfr-deficient CHO cells. The transfected cells were selected and amplified using methotrexate (MTX). To demonstrate that the gG-2 produced in these transformed cells had antigenic determinants in common with the native glycoprotein, CHO cells expressing gG-2 were used in an immunofluorescent assay (IFA) for the detection of HSV-2 type-specific antibodies in human serum samples. Seven of eight serum samples from adults with prior episodes of culture proven HSV-2 infections were found to be positive by the IFA method whereas none of seven serum samples from young children with culture documented HSV-1 infections were positive by IFA. Thus the recombinant CHO : gG-2 cells have diagnostic utility in an HSV-2 specific serologic assay.
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PMID:Detection of antibodies to herpes simplex virus type 2 with a mammalian cell line expressing glycoprotein gG-2. 1556 16

Sleeping Beauty (SB) is a gene-insertion system reconstructed from transposon sequences found in teleost fish and is capable of mediating the transposition of DNA sequences from transfected plasmids into the chromosomes of vertebrate cell populations. The SB system consists of a transposon, made up of a gene of interest flanked by transposon inverted repeats, and a source of transposase. Here we carried out a series of studies to further characterize SB-mediated transposition as a tool for gene transfer to chromosomes and ultimately for human gene therapy. Transfection of mouse 3T3 cells, HeLa cells, and human A549 lung carcinoma cells with a transposon containing the neomycin phosphotransferase (NEO) gene resulted in a several-fold increase in drug-resistant colony formation when co-transfected with a plasmid expressing the SB transposase. A transposon containing a methotrexate-resistant dihydrofolate reductase gene was also found to confer an increased frequency of methotrexate-resistant colony formation when co-transfected with SB transposase-encoding plasmid. A plasmid containing a herpes simplex virus thymidine kinase gene as well as a transposon containing a NEO gene was used for counterselection against random recombinants (NEO+TK+) in medium containing G418 plus ganciclovir. Effective counterselection required a recovery period of 5 days after transfection before shifting into medium containing ganciclovir to allow time for transiently expressed thymidine kinase activity to subside in cells not stably transfected. Southern analysis of clonal isolates indicated a shift from random recombination events toward transposition events when clones were isolated in medium containing ganciclovir as well as G418. We found that including both transposon and transposase functions on the same plasmid substantially increased the stable gene transfer frequency in Huh7 human hepatoma cells. The results from these experiments contribute technical and conceptual insight into the process of transposition in mammalian cells, and into the optimal provision of transposon and transposase functions that may be applicable to gene therapy studies.
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PMID:Counterselection and co-delivery of transposon and transposase functions for Sleeping Beauty-mediated transposition in cultured mammalian cells. 1615 96

The consumption of green tea (Camellia sinensis) has been shown to have many physiological and pharmacological health benefits. In the past two decades several studies have reported that epigallocatechin-3-gallate (EGCG), the main constituent of green tea, has anti-infective properties. Antiviral activities of EGCG with different modes of action have been demonstrated on diverse families of viruses, such as Retroviridae, Orthomyxoviridae and Flaviviridae and include important human pathogens like human immunodeficiency virus, influenza A virus and the hepatitis C virus. Furthermore, the molecule interferes with the replication cycle of DNA viruses like hepatitis B virus, herpes simplex virus and adenovirus. Most of these studies demonstrated antiviral properties within physiological concentrations of EGCG in vitro. In contrast, the minimum inhibitory concentrations against bacteria were 10-100-fold higher. Nevertheless, the antibacterial effects of EGCG alone and in combination with different antibiotics have been intensively analysed against a number of bacteria including multidrug-resistant strains such as methicillin-resistant Staphylococcus aureus or Stenotrophomonas maltophilia. Furthermore, the catechin EGCG has antifungal activity against human-pathogenic yeasts like Candida albicans. Although the mechanistic effects of EGCG are not fully understood, there are results indicating that EGCG binds to lipid membranes and affects the folic acid metabolism of bacteria and fungi by inhibiting the cytoplasmic enzyme dihydrofolate reductase. This review summarizes the current knowledge and future perspectives on the antibacterial, antifungal and antiviral effects of the green tea constituent EGCG.
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PMID:Anti-infective properties of epigallocatechin-3-gallate (EGCG), a component of green tea. 2307 20

Tc toxins are bacterial protein complexes that inject cytotoxic enzymes into target cells using a syringe-like mechanism. Tc toxins are composed of a membrane translocator and a cocoon that encapsulates a toxic enzyme. The toxic enzyme varies between Tc toxins from different species and is not conserved. Here, we investigate whether the toxic enzyme can be replaced by other small proteins of different origin and properties, namely Cdc42, herpes simplex virus ICP47, Arabidopsis thaliana iLOV, Escherichia coli DHFR, Ras-binding domain of CRAF kinase, and TEV protease. Using a combination of electron microscopy, X-ray crystallography and in vitro translocation assays, we demonstrate that it is possible to turn Tc toxins into customizable molecular syringes for delivering proteins of interest across membranes. We also infer the guidelines that protein cargos must obey in terms of size, charge, and fold in order to apply Tc toxins as a universal protein translocation system.
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PMID:Towards the application of Tc toxins as a universal protein translocation system. 3174 51


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