Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:2.7.1.21 (thymidine kinase)
7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The gene encoding the major replicative protein, NS1, of minute virus of mice (MVM) was transferred into a recombinant vaccinia virus vector in place of the vaccinia thymidine kinase gene. The NS1 gene was placed under control of a bacteriophage T7 promoter and expressed in cells coinfected with another recombinant vaccinia virus, vTF7-3, which encodes the T7 RNA polymerase. Expression of NS1 was further enhanced by the presence of a 5' untranslated region, derived from encephalomyocarditis virus, which allows efficient cap-independent translation. This system was used to produce and analyze wild-type NS1 and two mutant forms of the protein, NS1K405R and NS1K405M, in which the highly conserved lysine codon located in the putative purine triphosphate binding site of NS1 was changed to arginine and methionine, respectively. Full-length NS1 was expressed efficiently in both human and mouse cells infected with each of the three recombinant viruses, and in each case the NS1 was rapidly and efficiently translocated into the nucleus. Wild-type NS1 expressed in this way was biologically active. It was able to trans-activate an MVM P38 promoter located in a host chromosomal site, whereas the two mutant forms of NS1 showed no significant activity in this assay, and it was capable of resolving palindromic junction fragments cloned from multimeric MVM replicative form DNA molecules. These substrates, representing MVM genomic left-end:left-end and right-end:right-end fusions, were resolved in a DNA synthesis-dependent in vitro reaction supplemented with nuclear extracts containing recombinant wild-type NS1. Neither of the two mutant forms of the polypeptide had any detectable activity in this assay.
...
PMID:Expression of functional parvoviral NS1 from recombinant vaccinia virus: effects of mutations in the nucleotide-binding motif. 141 12

The thymidine kinase encoded by herpes simplex virus type 1 contains an amino acid sequence homologous to a consensus sequence related to the ATP-binding site in many proteins. We have used site-directed mutagenesis to investigate the importance of the five highly conserved amino acids within this segment. When any one of the three glycines was changed to valine the corresponding mutant enzyme was inactive. The mutation of lysine 63 to isoleucine destroyed the enzymatic activity. When threonine 64 was changed to alanine the mutant enzyme lost its activity. However, when this threonine was changed to serine the enzyme was still active but with different apparent Michaelis constants (Km) for thymidine and ATP. The wild-type thymidine kinase has apparent Km's of 0.5 and 20 microM for thymidine and ATP, respectively, while the mutant enzyme displayed Km's of 2.3 and 60 microM for thymidine and ATP. These results indicate that this homologous segment is essential for the function of the thymidine kinase and is involved in the substrate binding domain of the enzyme.
...
PMID:Site-directed mutagenesis of a nucleotide-binding domain in HSV-1 thymidine kinase: effects on catalytic activity. 283 27

Administration of cycloheximide in a single dose of 0.6 mg/kg to 7-day-old rats was used to induce short-term inhibition of protein synthesis at the period of brain 'growth spurt'. Measurement of the rate of [14C]lysine incorporation indicated that the initial inhibition of protein synthesis in the brain (by 75%) was released within about 12 h. The normal rate of protein synthesis was attained by 48 h after cycloheximide administration; there was no sign of protein synthesis stimulation. The estimation of [14C]thymidine incorporation into brain DNA showed that inhibition of DNA synthesis was greater and longer lasting in the forebrain and olfactory bulbs (by about 80%) than in the cerebellum (by about 40%). Similar differential inhibition of thymidine kinase activity was observed in the olfactory bulbs (by 75%) and cerebellum (by 30%) at 24 h after cycloheximide, suggesting that the formation of [14C]thymidine nucleotides may have been impaired. However, a retardation of DNA accumulation was found in the forebrain and cerebellum at 72 h after cycloheximide. Thus, the short-term inhibition of protein synthesis produced prolonged inhibition of DNA synthesis and altered cell proliferation in the developing brain.
...
PMID:Effect of cycloheximide administered to rats in early postnatal life: prolonged inhibition of DNA synthesis in the developing brain. 737 2

The herpes simplex virus type 1 thymidine kinase (HSV-1 TK) is an important pharmacological target of antiviral nucleoside drugs and it uniquely possesses both a thymidine kinase and a thymidylate kinase activity. The structural relationship between these two activities is addressed in this study using a combination of active-site directed photoaffinity analogs, proteases, and tricine-SDS-polyacrylamide gel electrophoresis. For analysis of the thymidylate binding site, the thymidylate analog [32P]5-azido-dUMP was specifically photocrosslinked to the active site of HSV-1 TK. Because the amino acid sequence of HSV-1 TK is known, endoprotease Lys-C, V8 protease, trypsin, or chymotrypsin was used to generate a proteolytic map of photoincorporated peptides by separation on high-resolution tricine-SDS-polyacrylamide gels. Analysis of the resulting peptides indicated that the photoprobe was localized to one region comprising amino acids Ile112-Tyr132. Photolabeling of this region indicates that the thymine base of thymidine and TMP bind at one shared site in HSV-1 TK. In addition, the results reported in this study demonstrate that photolabeling with azidonucleotides can be used to identify photolabeled peptides by proteolytic mapping. This technique bypasses the problems of peptide purification and sequencing and yields rapid results when the primary amino acid structure of the protein of interest is known.
...
PMID:Proteolytic mapping of the thymidine/thymidylate binding site of herpes simplex virus type 1 thymidine kinase: a general photoaffinity labeling method for identifying active-site peptides. 866 May 48

The "Fab immunogene" is a novel gene transfer vehicle in which the Fab fragment of anti-human epidermal growth factor (EGF) receptor antibody B4G7 is conjugated with poly-L-lysine to form an affinity complex with DNA. It was developed to target delivery of therapeutic genes into EGF receptor-hyperproducing tumor cells. Various characteristic features of the immunogene have been documented (Chen et al., 1998). Here we add further evidence to prove that in vitro transfer of beta-galactosidase/Fab immunogene is exclusively to EGF receptor-positive cells and that the herpes simplex virus thymidine kinase (TK)/Fab immunogene induces substantial suicide effects on A431 tumor cells when treated together with ganciclovir. The in vivo specificity of the immunogene transfer was examined using A431 tumor-bearing nude mice. When these nude mice were injected intraperitoneally with the chloramphenicol acetyltransferase (CAT)/Fab immunogene, CAT DNA was detected in the tumors as well as in liver and kidney but not brain, whereas CAT mRNA and enzyme activity were detected only in the tumors. Local and intraperitoneal injection of the TK/Fab immunogene and subsequent administration of ganciclovir effectively suppressed the growth of A431 tumors transplanted on the backs of nude mice. These observations suggest a possible application of the Fab immunogene system in cancer gene therapy.
...
PMID:Targeted in vivo delivery of therapeutic gene into experimental squamous cell carcinomas using anti-epidermal growth factor receptor antibody: immunogene approach. 987 65

We previously developed the "immunogene" approach toward cancer gene therapy using epidermal growth factor receptor (EGFR)-mediated endocytosis. Here, we describe an improved immunogene system, in which the antigen-binding (Fab) fragments of the monoclonal antibody (Ab) B4G7 against the human EGFR were conjugated with poly-L-lysine to form a gene delivery vehicle (designated Fab "immunoporter"). Within 12 hours, the beta-galactosidase beta-gal) gene was transferred via the Fab immunoporter to virtually all of the nuclei of human squamous carcinoma A431 cells that overproduce the EGFR, and the beta-gal enzyme activity was detected within 24 hours and retained for more than 3 days. The beta-gal gene was not transferred into human and mouse cells that were deficient in EGFRs, but it was delivered if those mouse cells were transformed with human EGFR genes. Beta-gal gene transfer via the Fab immunoporter was inhibited by pretreatment with excess amounts of the Fab fragment. The transfer efficiency of the beta-gal gene to A431 cells via the Fab immunoporter was approximately 2%, which is as high as the lipofection method and 20- to 100-fold higher than the whole Ab immunoporter. The transfer of the herpes simplex virus thymidine kinase gene into A431 tumor cells as a form of the thymidine kinase/Fab immunogene was successful, and subsequent treatment with ganciclovir induced remarkable suicide effects which conferred 1000-fold higher drug sensitivity. Thus, the Fab immunogene was substantially improved with regard to the whole Ab immunogene and could be used as a potent gene transfer vehicle for the in vivo targeting of EGFR-hyperproducing tumor cells.
...
PMID:Receptor-mediated gene delivery using the Fab fragments of anti-epidermal growth factor receptor antibodies: improved immunogene approach. 991 90

Crystallographic structures of the ligand-binding domains for the retinoid X (RXR) and estrogen receptors have identified conserved surface residues that participate in dimer formation. Homologous regions have been identified in the human vitamin D receptor (hVDR). Mutating Lys-386 to Ala (K386A) in hVDR significantly reduced binding to glutathione S-transferase-RXRalpha in solution, whereas binding of an I384R/Q385R VDR mutant was almost undetectable. The K386A mutant formed heterodimers with RXRalpha on DR-3 (a direct repeat of AGGTCA spaced by three nucleotides), whereas the I384R/Q385R mutant completely eliminated heterodimer formation. Wild type hVDR effected a 3-fold induction of DR-3-dependent thymidine kinase-luciferase activity in cultured neonatal rat atrial myocytes, an effect that was increased to 8-9-fold by cotransfected hRXRalpha. Induction by K386A, in the presence or absence of RXRalpha, was only slightly lower than that seen with wild type VDR. On the other hand, I384R/Q385R alone displayed no stimulatory activity and less than 2-fold induction in the presence of hRXRalpha. Qualitatively similar findings were observed with the negative regulation of the human atrial natriuretic peptide gene promoter by these mutants. Collectively, these studies identify specific amino acids in hVDR that play a critical role in heterodimer formation and subsequent modulation of gene transcription.
...
PMID:Vitamin D-dependent suppression of human atrial natriuretic peptide gene promoter activity requires heterodimer assembly. 1019 14

The Fab fragment of monoclonal antibody B4G7 against human epidermal growth factor (EGF) receptor was conjugated with cationic poly-L-lysine and the resulting conjugate was further complexed with reporter genes or therapeutic genes. This Fab/DNA complex was designated as "Fab immunogene." The Fab immunogene transfer in vitro was mediated through the EGF receptors in two melanoma cell lines. The frequency of cells expressing beta-galactosidase (beta-Gal) reporter gene was approximately 1%. The induction of suicide effects after Fab immunogene transfer of herpes simplex virus thymidine kinase (TK) or Escherichia coli cytosine deaminase (CD) gene was quite remarkable, and the growth of melanoma cells was inhibited for over 7 days in the presence of ganciclovir (GCV) or 5-fluorocytosine (5-FC). Similarly, when melanoma cells treated in vitro with the Fab immunogene carrying TK or CD were transplanted into the back of nude mouse, subsequent systemic administration of GCV or 5-FC effectively suppressed the growth of tumors, indicating the occurrence of in vivo suicide effects.
...
PMID:Ex vivo delivery of suicide genes into melanoma cells using epidermal growth factor receptor-specific Fab immunogene. 1036 86

A protein of 10,425 Da was purified from the edible mushroom Rozites caperata and shown to inhibit herpes simplex virus types 1 and 2 replication with an IC50 value of < or = 5 microM. The protein designated RC-183 also significantly reduced the severity of HSV-1 induced ocular disease in a murine model of keratitis, indicating in vivo efficacy. HSV mutants lacking ribonucleotide reductase and thymidine kinase were also inhibited, suggesting the mechanism does not involve these viral enzymes. Antiviral activity was also seen against varicella zoster virus, influenza A virus, and respiratory syncytial virus, but not against adenovirus type VI, coxsackie viruses A9 and B5, or human immunodeficiency virus. Characterization of RC-183 by mass spectroscopy, sequencing, and other methods suggests it is composed of a peptide (12 or 13 mer) coupled to ubiquitin via an isopeptide bond between the c-terminal glycine of ubiquitin and the epsilon amino group of a lysine residue in the peptide. The peptide sequence did not match any known sequence. Thus, RC-183 is a novel antiviral that may have clinical utility or serve as a lead compound for further development. Determining the mechanism of action may lead to identification of novel steps in viral replication.
...
PMID:Isolation and partial characterization of an antiviral, RC-183, from the edible mushroom Rozites caperata. 1051 9

The DNA polymerase beta mutant enzyme, which is altered from glutamic acid to lysine at position 249, exhibits a mutator phenotype in primer extension assays and in the herpes simplex virus-thymidine kinase (HSV-tk) forward mutation assay. The basis for this loss of accuracy was investigated by measurement of misincorporation fidelity in single turnover conditions. For the four misincorporation reactions investigated, the fidelity of the E249K mutant was not significantly different from wild type, implying that the mutator phenotype was not caused by a general inability to distinguish between correct and incorrect bases during the incorporation reaction. However, the discrimination between correct and incorrect substrates by the E249K enzyme occurred less during the conformational change and chemical steps and more during the initial binding step, compared with pol beta wild type. This implies that the E249K mutation alters the kinetic mechanism of nucleotide discrimination without reducing misincorporation fidelity. In a missing base primer extension assay, we observed that the mutant enzyme produced mispairs and extended them. This indicates that the altered fidelity of E249K could be due to loss of discrimination against mispaired primer termini. This was supported by the finding that the E249K enzyme extended a G:A mispair 8-fold more efficiently than wild type and a C:T mispair 4-fold more efficiently. These results demonstrate that an enhanced ability to extend mispairs can produce a mutator phenotype and that the Glu-249 side chain of DNA polymerase beta is critical for mispair extension fidelity.
...
PMID:The E249K mutator mutant of DNA polymerase beta extends mispaired termini. 1058 71


1 2 3 Next >>

---