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

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

Polymerase chain reaction (PCR) assays were used to amplify herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) sequences in DNA extracted from formalin-fixed, paraffin embedded corneas of patients undergoing corneal transplantation. PCR reamplification with an internal (nested) set of primers was required for detection in 10 of the 12 positive corneas indicating very low abundance of viral sequences. Three of the positive corneal samples were from failed corneal grafts. Overall, TK sequences were detected in 8 of 11 corneas from subjects with a past history of herpes keratitis and in 4 of 11 corneas from subjects with no past history of herpetic eye disease.
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PMID:Detection of herpes simplex virus DNA sequences in corneal transplant recipients by polymerase chain reaction assays. 165 Jun 62

Herpes simplex virus (HSV) latency in sensory ganglion neurons is well documented, but the existence of extraneuronal corneal latency is less well defined. To investigate the possibility of extraneuronal latency during ocular HSV infection, corneal specimens from 18 patients with quiescent herpes simplex keratitis (HSK) were obtained at the time of keratoplasty. Polymerase chain reaction (PCR) amplification followed by southern blot hybridization with a radiolabeled oligonucleotide probe was done to detect the presence of HSV-1 genome in these human corneal samples. Two pairs of oligonucleotides from the region of the HSV thymidine kinase (TK) gene and the latency-associated transcript (LAT) gene were used as primers in the PCR amplification. The DNA sequences from either the TK or the LAT gene were identified in 15 of 18 HSK corneas (83%). These results demonstrate that the HSV genome was retained, at least in part, in human corneas during quiescent HSV infection, giving further support to the concept of corneal extraneuronal latency.
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PMID:Detection of herpes simplex virus thymidine kinase and latency-associated transcript gene sequences in human herpetic corneas by polymerase chain reaction amplification. 185 32

Polymerase chain reaction (PCR) for detection of bovine herpesvirus-1 (BHV-1) was developed and optimized using 22 bp sense and 20 bp antisense primers in the thymidine kinase (TK) coding region. The amplification product is 183 bp long. The PCR optimization was done using BHV-1 tissue culture supernate (BHV-1TCS), concentrated BHV-1 tissue culture supernate (cBHV-1TCS) and sucrose gradient purified BHV-1 (pBHV-1). The sensitivity of four methods of sample preparation which are standard DNA extraction, modified proteinase K (PK) digestion, GeneReleaserTM + 34 cycles or + 44 cycles, and boiling were compared with virus isolation (VI) using BHV-1TCS. The incorporation of 10% glycerol in the reaction mixture, the incubation in PK for 18 hours and predenaturation of samples and cooling in ice prior to PCR were essential for the amplification of BHV-1 DNA for samples prepared by standard DNA extraction and modified PK digestion. The preparation of samples by Gene-ReleaserTM, a proprietary nucleic acid releasing cocktail, showed 10 to 1,000-fold increase in sensitivity compared to standard DNA extraction and modified PK digestion. No amplification was observed in samples prepared by boiling. The sample preparation of BHV-1 LA strain by GeneReleaserTM showed sensitivity equivalent to virus isolation. The BHV-1 TK PCR using GeneReleaserTM has a detection limit of 1 picogram and 10 fentograms of purified BHV-1 DNA using ethidium bromide stained gel and Southern blot hybridization, respectively. It could detect viral DNA in 1,000 infected cells in a total suspension of 10,000 cells using either ethidium bromide stained gel or Southern blot hybridization.
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PMID:Establishment of conditions for the detection of bovine herpesvirus-1 by polymerase chain reaction using primers in the thymidine kinase region. 764 33

In this study, the growth of locally disseminated breast cancer was modeled using a human breast cancer cell line, MDA-MB-435A, adapted to grow as an ascites tumor in athymic mice. Ex vivo infection of MDA-MB-435A cells with adenovirus containing the herpes simplex virus thymidine kinase gene (HSV-tk) were injected into the intraperitoneal cavity of athymic mice. Ganciclovir (GCV) treatment resulted in prolonged median survival (117 vs. 34 days, p < 0.001) compared to untreated or control animals. Adenovirus containing HSV-tk also demonstrated therapeutic activity after in vivo transduction resulting in prolongation of median survival after GCV treatment (32 vs. 25 days, p < 0.001). However, compared to ex vivo treatment, the effect was modest. In an attempt to increase survival, the viral dose was increased three-fold. Instead of prolonging survival, the increased dose resulted in more toxic deaths. Necropsy demonstrated that the most significant histologic abnormality was marked, diffuse, cytomegalic changes in the liver. Polymerase chain reaction (PCR) analysis of hepatic DNA demonstrated the presence of the virus in the affected tissue. Similar host toxicity and hepatic abnormalities were seen in non-tumor-bearing mice treated with ADV/RSV-tk plus GCV. In conclusion, adenoviral vectors can successfully transfer genes in vivo to cancer cells growing as ascites tumors. Transduction with HSV-tk followed by GCV treatment can prolong survival in this model system of disseminated disease, however toxicity can be substantial. Further refinement in targeting expression of HSV-tk will be required to enhance the therapeutic benefit.
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PMID:Adenovirus-mediated gene transfer of herpes simplex virus thymidine kinase in an ascites model of human breast cancer. 879 49

Previous studies have shown that genetically engineered thymidine kinase (tk)-defective herpes simplex virus type 1 (HSV-1) can effectively and selectively destroy gliomas in animal models. The consequences of viral infection and tumor regression must be characterized before this therapy can be applied in human trials. To study the potential for long-term toxicity, immunocompetent rats harboring 9L gliosarcomas were injected intratumorally with a tk-defective HSV-1, KOS-SB, at titers that previously have been demonstrated to cause tumor regression. In animals surviving 3 months or longer following viral treatment, there was no evidence of persistent infection or inflammation in peritumoral brain tissue or in remote systemic organs studied with routine histological and immunocytochemical analyses. Polymerase chain reaction using primers specific for HSV-1 detected HSV-1 DNA in peritumoral tissue only in animals sacrificed within 3 months of viral injection. There was no evidence of HSV-1 DNA in systemic tissues at any time after treatment. We conclude that stereotactic intratumoral injection of tk-deficient HSV can be attempted for the treatment of brain tumors without risk of systemic infection or significant toxicity to normal brain or remote proliferating tissues.
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PMID:Toxicity studies in thymidine kinase-deficient herpes simplex virus therapy for malignant astrocytoma. 881 71

Polymerase chain reaction (PCR) amplification assays were developed for the detection of thymidine kinase (TK) and protein kinase (PK)-related genes of the channel catfish virus (CCV). Two pairs of primers were constructed based on the published nucleotide sequences of CCV and were used to amplify the expected fragments of 584 and 755 bp for the TK and PK-related genes, respectively. The amplified fragments were shown to be specific for each of the target genes by chemiluminescence Southern blot hybridisation with a digoxigenin-labelled 20-base internal probe. The optimised CCV PCR assay can be used to amplify TK- and PK-related genes in other mammalian and avian herpesviruses. The CCV TK PCR assay also amplified a TK-like gene in some pilchard's gills and tissues obtained in the recent epizootic of pilchard kills in New Zealand.
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PMID:Polymerase chain reaction amplification of the thymidine kinase and protein kinase-related genes of channel catfish virus and a putative pilchard herpesvirus. 888 38

Molecular virology has served to establish bovine herpesvirus 1 (BHV-1) as the prototype member of ruminant herpesviruses. Based on the genomic sequence of the virus, we aim to identify and characterize virus-specified components, to explain their concerted action, and to predict how the chain of events during the lytic and latent phases of the viral life cycle may be interrupted. The nucleotide sequence of the BHV-1 genome (136 kb) has just been completed by international cooperation (July 1995; except for a small gap in UL36). It comprises 67 unique genes and 2 genes, both duplicated, in the inverted repeats. In general, these genes exhibit strong homology at the amino acid sequence level to those of other alphaherpesviruses (HSV-1, VZV, EHV-1) and are arranged in similar order. A few genes are peculiar to only one or two herpesviruses, e.g. in BHV-1 the circ, UL0.5, UL3.5 and US1.5 genes. Not long ago, the repertoire of BHV-1 proteins under study was restricted to the three major glycoproteins (gB, gC, and gD) and thymidine kinase. The repertoire is now growing rapidly and includes 7 additional glycoproteins (gE, gI, gH, gL, gG, gK and gM), a number of enzymes (e.g. ribonucleotide reductase, DNA Polymerase, dUTPase), and a group of regulatory proteins (BICPO, 4, 22, and 27, alpha TIF). Investigations into the functions of these proteins and comparison with their counterparts in other herpesviruses should reveal which are useful targets for diagnosis, prevention or antiviral treatment. Recombinant viruses containing deletions or replacements of individual genes are being created, aiming at vaccine development and insights into pathogenesis, notably latency, neurotropism, and interference with host functions. Molecular analysis of other ruminant herpesviruses is much less advanced. Over a dozen virus species have been described; most share basic properties with BHV-1 and may be classified as alphaherpesviruses. The gammaherpesviruses are represented by the proposed agent of malignant catarrhal fever, alcelaphine herpesvirus 1, and by bovine herpesvirus 4, whose partial sequences exhibit similarity to herpesvirus saimiri.
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PMID:Molecular virology of ruminant herpesviruses. 901 Sep 95

Skin from acute and healed herpes simplex virus or herpes simplex virus-associated erythema multiforme (HAEM) lesions was examined by polymerase chain reaction with primers for DNA polymerase, ICP8, thymidine kinase (5' end of herpes simplex virus genome), and ICP27 (3' end of herpes simplex virus genome). The primers were herpes simplex virus specific and equally sensitive. The four herpes simplex virus genes were seen in acute herpes simplex virus lesions, but except for one patient, only polymerase (or polymerase and ICP8) were seen in 7-d healed lesional skin. Herpes simplex virus DNA was not seen 1-1.5 mo after healing. HAEM skins from 18 of 24 patients (75%) were positive for polymerase DNA and four of 24 (17%) were also positive for ICP8 or thymidine kinase DNA. Only one tissue (4%) was positive for polymerase, ICP8, and ICP27 DNA. Skin from healed HAEM lesions was still polymerase DNA positive 1-3 mo after lesion resolution. The polymerase DNA signal was in the basal and spinous cell layers of the epidermis and in the outer root sheath of the hair follicle. Polymerase RNA was identified by reverse transcriptase polymerase chain reaction in skin from acute, but not healed polymerase DNA positive HAEM lesions, suggesting that polymerase expression is associated with HAEM lesion development.
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PMID:Expression of herpes simplex virus DNA fragments located in epidermal keratinocytes and germinative cells is associated with the development of erythema multiforme lesions. 932 89

Drug-resistance of herpes simplex virus (HSV) is caused most frequently by mutation of the viral thymidine kinase (TK) gene. To elucidate the significance of detecting nucleotide changes of the TK gene for screening drug-resistant viruses, the frequency and variation of the genetic polymorphisms in the whole coding region of the TK gene were studied in 14 acyclovir-susceptible HSV type 1 (HSV-1) clinical isolates from 14 patients with epithelial herpetic keratitis. Two reference HSV-1 laboratory strains, McKrae and PH, and two acyclovir-resistant variants of the PH strain were also studied as controls. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and direct sequencing detected nucleotide differences at 24 positions, and amino acid substitutions at 12 codons in the TK gene of the examined viruses. Nucleotide diversity of 0.0029 per base (the average number of nucleotide substitutions of 3.3 per 1,131 base pairs) in the TK gene in the clinical isolates was comparable to 0.0037 per base of the whole HSV-1 genome in Japanese isolates reported previously. PCR-SSCP analysis of the acyclovir-resistant strains easily detected aberrantly shifted bands by comparing them with those of the parental strain, followed by the quick determination of mutated sequences. These results suggest that detection of nucleotide changes of the TK gene is useful for serial observation of persistent or recurrent HSV infection as observed in immunocompromised hosts, but that it is not useful for screening drug-resistant viruses from nonepidemic clinical isolates because of the comparable genetic polymorphisms in the TK gene as in the whole HSV-1 genome.
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PMID:Polymorphisms of thymidine kinase gene in herpes simplex virus type 1: analysis of clinical isolates from herpetic keratitis patients and laboratory strains. 974 72

We have characterized the susceptibility and genetic stability of varicella-zoster viruses (VZV) isolated from skin lesions of three patients with herpes zoster and six patients with varicella treated with conventional short-term acyclovir (ACV) administration. The susceptibilities to ACV of the serial isolates from the patients were examined, and there was no significant difference in the susceptibility to ACV among the isolates before and during the ACV treatment, indicating that conventional short-term ACV treatment did not generate ACV-resistant VZV infections. Polymerase chain reaction (PCR) analyses of these as well as seven thymidine kinase-deficient VZV strains derived from in vitro ACV treatment were carried out to examine their genomic stability. Five regions containing tandem direct reiterations (R1-R5) were amplified by PCR and compared, and the region containing the Pst I-site was also examined. PCR analyses demonstrated that the R1, R5 and the Pst I-sites were stable and useful in epidemiological studies even after ACV treatment. The R2, R3 and R4 sites were far less stable in these experimental conditions. In this paper we discuss the results of the PCR analyses with regard to the dynamics of VZV population in patients with VZV infection treated with conventional short-term ACV administration.
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PMID:Characterization of acyclovir susceptibility and genetic stability of varicella-zoster viruses isolated during acyclovir therapy. 1069 66


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