Gene conversion is strongly induced in human cells by double-strand breaks and is modulated by the expression of BCL-x(L)

NASA Technical Reports Server (NTRS)

Wiese, Claudia; Pierce, Andrew J.; Gauny, Stacey S.; Jasin, Maria; Kronenberg, Amy; Chatterjee, A. (Principal Investigator)

2002-01-01

Homology-directed repair (HDR) of DNA double-strand breaks (DSBs) contributes to the maintenance of genomic stability in rodent cells, and it has been assumed that HDR is of similar importance in DSB repair in human cells. However, some outcomes of homologous recombination can be deleterious, suggesting that factors exist to regulate HDR. We demonstrated previously that overexpression of BCL-2 or BCL-x(L) enhanced the frequency of X-ray-induced TK1 mutations, including loss of heterozygosity events presumed to arise by mitotic recombination. The present study was designed to test whether HDR is a prominent DSB repair pathway in human cells and to determine whether ectopic expression of BCL-x(L) affects HDR. Using TK6-neo cells, we find that a single DSB in an integrated HDR reporter stimulates gene conversion 40-50-fold, demonstrating efficient DSB repair by gene conversion in human cells. Significantly, DSB-induced gene conversion events are 3-4-fold more frequent in TK6 cells that stably overexpress the antiapoptotic protein BCL-X(L). Thus, HDR plays an important role in maintaining genomic integrity in human cells, and ectopic expression of BCL-x(L) enhances HDR of DSBs. This is the first study to highlight a function for BCL-x(L) in modulating DSB repair in human cells.

Mutagenesis in human cells with accelerated H and Fe ions

NASA Technical Reports Server (NTRS)

Kronenberg, Amy

1994-01-01

The overall goals of this research were to determine the risks of mutation induction and the spectra of mutations induced by energetic protons and iron ions at two loci in human lymphoid cells. During the three year grant period the research has focused in three major areas: (1) the acquisition of sufficient statistics for human TK6 cell mutation experiments using Fe ions (400 MeV/amu), Fe ions (600 MeV/amu) and protons (250 MeV/amu); (2) collection of thymidine kinase- deficient (tk) mutants or hypoxanthine phosphoribosyltransferase deficient (hprt) mutants induced by either Fe 400 MeV/amu, Fe 600 MeV/amu, or H 250 MeV/amu for subsequent molecular analysis; and (3) molecular characterization of mutants isolated after exposure to Fe ions (600 MeV/amu). As a result of the shutdown of the BEVALAC heavy ion accelerator in December 1992, efforts were rearranged somewhat in time to complete our dose-response studies and to complete mutant collections in particular for the Fe ion beams prior to the shutdown. These goals have been achieved. A major effort was placed on collection, re-screening, and archiving of 3 different series of mutants for the various particle beam exposures: tk-ng mutants, tk-sg mutants, and hprt-deficient mutants. Where possible, groups of mutants were isolated for several particle fluences. Comparative analysis of mutation spectra has occured with characterization of the mutation spectrum for hprt-deficient mutants obtained after exposure of TK6 cells to Fe ions (600 MeV/amu) and a series of spontaneous mutants.

Genomic instability in human lymphoid cells exposed to 1 GeV/amu Fe ions

NASA Technical Reports Server (NTRS)

Grosovsky, A.; Bethel, H.; Parks, K.; Ritter, L.; Giver, C.; Gauny, S.; Wiese, C.; Kronenberg, A.

2001-01-01

The goal of this study was to assess whether charged particle radiations of importance to spaceflight elicit genomic instability in human TK6 lymphoblasts. The incidence of genomic instability in TK6 cells was assessed 21 days after exposure to 2, 4, or 6 Fe ions (1 GeV/amu, LET= 146 keV/micrometers). Three indices of instability were used: intraclonal karyotypic heterogeneity, mutation rate analysis at the thymidine kinase (TK1) locus, and re-cloning efficiency. Fifteen of sixty clones demonstrated karyotypic heterogeneity. Five clones had multiple indicators of karyotypic change. One clone was markedly hypomutable and polyploid. Six clones were hypomutable, while 21 clones were mutators. Of these, seven were karyotypically unstable. Six clones had low re-cloning efficiencies, one of which was a mutator. All had normal karyotypes. In summary, many clones that survived exposure to a low fluence of Fe ions manifested one or more forms of genomic instability that may hasten the development of neoplasia through deletion or by recombination.

Genomic instability in human lymphoid cells exposed to 1 GeV/amu Fe ions.

PubMed

Grosovsky, A; Bethel, H; Parks, K; Ritter, L; Giver, C; Gauny, S; Wiese, C; Kronenberg, A

2001-01-01

The goal of this study was to assess whether charged particle radiations of importance to spaceflight elicit genomic instability in human TK6 lymphoblasts. The incidence of genomic instability in TK6 cells was assessed ~21 days after exposure to 2, 4, or 6 Fe ions (1 GeV/amu, LET= 146 keV/micrometers). Three indices of instability were used: intraclonal karyotypic heterogeneity, mutation rate analysis at the thymidine kinase (TK1) locus, and re-cloning efficiency. Fifteen of sixty clones demonstrated karyotypic heterogeneity. Five clones had multiple indicators of karyotypic change. One clone was markedly hypomutable and polyploid. Six clones were hypomutable, while 21 clones were mutators. Of these, seven were karyotypically unstable. Six clones had low re-cloning efficiencies, one of which was a mutator. All had normal karyotypes. In summary, many clones that survived exposure to a low fluence of Fe ions manifested one or more forms of genomic instability that may hasten the development of neoplasia through deletion or by recombination.

Immunomodulation of glioma cells after gene therapy: induction of major histocompatibility complex class I but not class II antigen in vitro.

PubMed

Parsa, A T; Chi, J H; Hurley, P T; Jeyapalan, S A; Bruce, J N

2001-09-01

Acquired immunity has been demonstrated in Fischer rats bearing syngeneic 9L tumors after herpes simplex virus (HSV) thymidine kinase (TK) gene transfection and ganciclovir treatment. The nature of this immunity in rats and its relevance to the HSV TK/ganciclovir protocol for human subjects remain to be determined. In this study, levels of major histocompatibility complex (MHC) Class I and II antigen expression were measured before and after HSV TK transfection, in an effort to document immunomodulatory changes caused by gene therapy. Tumor cells from the 9L gliosarcoma cell line, three primary human glioma cultures, and the human glioma cell line U87 MG were transduced with HSV TK vector-containing supernatant from fibroblast-producing cells (titer of 5 x 10(6) colony-forming units/ml) and selected in G418 medium for neomycin resistance. Clones were pooled or individually selected for cell-killing assays with ganciclovir, to confirm TK expression (10(3) cells/well in a 96-well dish). Northern analyses using MHC Class I and Class II complementary deoxyribonucleic acid probes were performed on blots containing total ribonucleic acid from wild-type tumor cells and HSV TK transfectants. A beta-actin complementary deoxyribonucleic acid probe served as an internal control. Cell surface expression was confirmed with flow cytometry. The induction of MHC Class I was tested for cycloheximide and genistein sensitivity. All cell cultures exhibited increases in MHC Class I but not MHC Class II expression, as determined by Northern analysis densitometry and flow cytometry. Cycloheximide treatment did not diminish the up-regulation of MHC Class I after retroviral transfection, implicating a signal transduction pathway that does not require ongoing protein synthesis. Genistein pretreatment of cell cultures did diminish the up-regulation of MHC Class I, implicating a tyrosine kinase in the signaling cascade. Induction of MHC Class I in rat and human glioma cells after HSV TK retroviral gene therapy is a primary effect that is dependent on tyrosine kinase activity. Specific immune responses generated after transfection may represent an important general side effect of gene therapy protocols. Elucidation of the mechanism of immunomodulation after gene therapy will likely yield safer and more effective clinical protocols.

Evidence for an involvement of thymidine kinase in the excision repair of ultraviolet-irradiated herpes simplex virus in human cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Intine, R.V.; Rainbow, A.J.

1990-01-01

A wild-type strain of herpes simplex virus type 1 (HSV-1:KOS) encoding a functional thymidine kinase (tk+) and a tk- mutant strain (HSV-1:PTK3B) were used to study the role of the viral tk in the repair of UV-irradiated HSV-1 in human cells. UV survival of HSV-1:PTK3B was substantially reduced compared with that of HSV-1:KOS when infecting normal human cells. In contrast, the UV survival of HSV-1:PTK3B was similar to that of HSV-1:KOS when infecting excision repair-deficient cells from a xeroderma pigmentosum patient from complementation group A. These results suggest that the repair of UV-irradiated HSV-1 in human cells depends, in partmore » at least, on expression of the viral tk and that the repair process influenced by tk activity is excision repair or a process dependent on excision repair.« less

Monitoring of tumor growth and metastasis potential in MDA-MB-435s/ tk-luc human breast cancer xenografts

NASA Astrophysics Data System (ADS)

Chang, Ya-Fang; Lin, Yi-Yu; Wang, Hsin-Ell; Liu, Ren-Shen; Pang, Fei; Hwang, Jeng-Jong

2007-02-01

Molecular imaging of reporter gene expression provides a rapid, sensitive and non-invasive monitoring of tumor behaviors. In this study, we reported the establishment of a novel animal model for longitudinal examination of tumor growth kinetics and metastatic spreading in vivo. The highly metastatic human breast carcinoma MDA-MB-435s cell line was engineered to stably express herpes simplex virus type 1 thymidine kinase (HSV-1- tk) and luciferase ( luc). Both 131I-FIAU and D-luciferin were used as reporter probes. For orthotopic tumor formation, MDA-MB-435s/ tk-luc cells were implanted into the first nipple of 6-week-old female NOD/SCID mice. For metastatic study, cells were injected via the lateral tail vein. Mice-bearing MDA-MB-435s/ tk-luc tumors were scanned for tumor growth and metastatsis using Xenogen IVIS50 system. Gamma scintigraphy and whole-body autoradiography were also applied to confirm the tumor localization. The results of bioluminescence imaging as well as histopathological finding showed that tumors could be detected in femur, spine, ovary, lungs, kidney, adrenal gland, lymph nodes and muscle at 16 weeks post i.v. injection, and correlated photons could be quantified. This MDA-MB-435s/ tk-luc human breast carcinoma-bearing mouse model combined with multimodalities of molecular imaging may facilitate studies on the molecular mechanisms of cancer invasion and metastasis.

[Hypoxia responsive element regulated herpes simplex virus-thymidine kinase system enhances killing effect of gancyclovir on Ewing's sarcoma cell line under hypoxic condition].

PubMed

Si, Ying-jian; Guang, Li-xia; Yuan, Fa-huan; Zhang, Ke-bin

2006-08-01

To find out a possible approach to improve the effectiveness of radiotherapy and chemotherapy for Ewing's sarcoma by constructing a eukaryotic expression vector expressing herpes simplex virus-thymidine kinase (HSV-TK) regulated by hypoxia responsive element (HRE) under hypoxia and to evaluate the effects of this HRE regulated HSV-TK system on killing effect of gancyclovir (GCV) on Ewing's sarcoma cell line SK-ES under hypoxic condition. The HRE was synthesized according to the literature and cloned into the enhancer site of pIRES(2)-EGFP vector to obtain the pHRE recombinant plasmid. The HSV-TK was amplified by PCR and cloned into the multiple clone site of pIRES(2)-EGFP and pHRE to obtain pTK and pHRE-TK recombinant plasmid. The human Ewing's sarcoma cell line SK-ES was transfected by pTK or pHRE-TK recombinant plasmid with liposome and then was exposed to normoxic (21% oxygen) or hypoxic (3% oxygen) condition. The expression of enhanced green fluorescent protein (EGFP) was monitored by fluorescent microscopy. The sensitivity of human Ewing's sarcoma cell line SK-ES transfected with pTK or pHRE-TK recombinant plasmid to the anti-tumour drug GCV was determined with the method of tetrazolium (MTT) after treating with GCV for five days. (1) The result of sequencing showed that the recombinant plasmid pHRE contained HRE, and that the recombinant plasmid pTK and pHRE-TK contained HSV-TK gene in the sense direction. (2) Comparison of fluorescent optical density (FOD) showed that (1) the EGFP FOD value of pHRE and pHRE-TK group cells exposed to hypoxia was significantly higher than those exposed to normoxia (P < 0.01); (2) when the cells were exposed to hypoxia, the EGFP FOD value of pHRE and pHRE-TK group cells was significantly higher than that of pTK and empty vector group (P < 0.01); (3) there was no significant difference among the four groups of cells when they were exposed to normoxia (P > 0.05). (3) Comparison of the sensitivity of four groups of cells to GCV showed that (1) the cells in pHRE-TK and pTK groups were much more sensitive to GCV than the cells in pHRE group under hypoxia condition (P < 0.01), the higher the GCV concentration, the greater the difference; (2) the cells of pHRE-TK group were more sensitive to GCV than those in pTK group under hypoxic condition (P < 0.01), but was almost equally sensitive under normoxic condition (P > 0.05); (3) the pHRE-TK group cells had higher sensitivity to GCV under hypoxia than normoxia (P < 0.01) while the pTK group cells had almost the same sensitivity to GCV under hypoxia and normoxia (P > 0.05). (1) The eukaryotic expression vector expressing herpes simplex virus-thymidine kinase (HSV-TK) regulated by hypoxia responsive element (HRE) under hypoxia was constructed successfully. (2) HRE could up-regulate expression of EGFP by SK-ES cells under hypoxia condition. (3) HRE could enhance the killing effect of HSV-TK/GCV system on human Ewing's sarcoma cell line SK-ES under hypoxic condition.

Integration of metabolic activation with a predictive toxicogenomics signature to classify genotoxic versus nongenotoxic chemicals in human TK6 cells

PubMed Central

Buick, Julie K.; Moffat, Ivy; Williams, Andrew; Swartz, Carol D.; Recio, Leslie; Hyduke, Daniel R.; Li, Heng‐Hong; Fornace, Albert J.; Aubrecht, Jiri

2015-01-01

The use of integrated approaches in genetic toxicology, including the incorporation of gene expression data to determine the molecular pathways involved in the response, is becoming more common. In a companion article, a genomic biomarker was developed in human TK6 cells to classify chemicals as genotoxic or nongenotoxic. Because TK6 cells are not metabolically competent, we set out to broaden the utility of the biomarker for use with chemicals requiring metabolic activation. Specifically, chemical exposures were conducted in the presence of rat liver S9. The ability of the biomarker to classify genotoxic (benzo[a]pyrene, BaP; aflatoxin B1, AFB1) and nongenotoxic (dexamethasone, DEX; phenobarbital, PB) agents correctly was evaluated. Cells were exposed to increasing chemical concentrations for 4 hr and collected 0 hr, 4 hr, and 20 hr postexposure. Relative survival, apoptosis, and micronucleus frequency were measured at 24 hr. Transcriptome profiles were measured with Agilent microarrays. Statistical modeling and bioinformatics tools were applied to classify each chemical using the genomic biomarker. BaP and AFB1 were correctly classified as genotoxic at the mid‐ and high concentrations at all three time points, whereas DEX was correctly classified as nongenotoxic at all concentrations and time points. The high concentration of PB was misclassified at 24 hr, suggesting that cytotoxicity at later time points may cause misclassification. The data suggest that the use of S9 does not impair the ability of the biomarker to classify genotoxicity in TK6 cells. Finally, we demonstrate that the biomarker is also able to accurately classify genotoxicity using a publicly available dataset derived from human HepaRG cells. Environ. Mol. Mutagen. 56:520–534, 2015. © 2015 The Authors. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc. PMID:25733247

Neural stem cell-based dual suicide gene delivery for metastatic brain tumors.

PubMed

Wang, C; Natsume, A; Lee, H J; Motomura, K; Nishimira, Y; Ohno, M; Ito, M; Kinjo, S; Momota, H; Iwami, K; Ohka, F; Wakabayashi, T; Kim, S U

2012-11-01

In our previous works, we demonstrated that human neural stem cells (NSCs) transduced with the cytosine deaminase (CD) gene showed remarkable 'bystander killer effect' on glioma and medulloblastoma cells after administration of the prodrug 5-fluorocytosine (5-FC). In addition, herpes simplex virus thymidine kinase (TK) is a widely studied enzyme used for suicide gene strategies, for which the prodrug is ganciclovir (GCV). To apply this strategy to brain metastasis treatment, we established here a human NSC line (F3.CD-TK) expressing the dual suicide genes CD and TK. We examined whether F3.CD-TK cells intensified the antitumor effect on lung cancer brain metastases. In vitro studies showed that F3.CD-TK cells exerted a marked bystander effect on human lung cancer cells after treatment with 5-FC and GCV. In a novel experimental brain metastases model, intravenously administered F3 cells migrated near lung cancer metastatic lesions, which were induced by the injection of lung cancer cells via the intracarotid artery. More importantly, F3.CD-TK cells in the presence of prodrugs 5-FC and GCV decreased tumor size and considerably prolonged animal survival. The results of the present study indicate that the dual suicide gene-engineered, NSC-based treatment strategy might offer a new promising therapeutic modality for brain metastases.

A New Sandwich ELISA for Quantification of Thymidine Kinase 1 Protein Levels in Sera from Dogs with Different Malignancies Can Aid in Disease Management

PubMed Central

Jagarlamudi, Kiran Kumar; Moreau, Laura; Westberg, Sara; Rönnberg, Henrik; Eriksson, Staffan

2015-01-01

Thymidine kinase 1 (TK1) is a DNA precursor enzyme whose expression is closely correlated with cell proliferation and cell turnover. Sensitive serum TK1 activity assays have been used for monitoring and prognosis of hematological malignancies in both humans and dogs. Here we describe the development of a specific sandwich TK1-ELISA for the quantification of TK1 protein levels in sera from dogs with different malignancies. A combination of rabbit polyclonal anti-dog TK1 antibody and a mouse monoclonal anti-human TK1 antibody was used. Different concentrations of recombinant canine TK1 was used as standard. Clinical evaluation of the ELISA was done by using sera from 42 healthy dogs, 43 dogs with hematological tumors and 55 with solid tumors. An established [3H]-dThd phosphorylation assay was used to determine the TK1 activity levels in the same sera. The mean TK1 activities in dogs with hematological tumors were significantly higher than those found in healthy dogs. In agreement with earlier studies, no significant difference was observed in serum TK1 activities between healthy dogs and dogs with solid tumors. However, the mean TK1 protein levels determined by new TK1-ELISA were significantly higher not only in hematological tumors but also in solid tumors compared to healthy dogs (mean ± SD = 1.30 ± 1.16, 0.67 ± 0.55 and 0.27± 0.10 ng/mL, respectively). Moreover, TK1-ELISA had significantly higher ability to distinguish lymphoma cases from healthy based on receiver operating characteristic analyses (area under the curve, AUC, of 0.96) to that of the activity assay (AUC, 0.84). Furthermore, fluctuations in TK1 protein levels during the course of chemotherapy in dogs with lymphoma closely associated with clinical outcome. Overall, the TK1-ELISA showed significant linear correlation with the TK1 activity assay (r s = 0.6, p<0.0001). Thus, the new TK1-ELISA has sufficient sensitivity and specificity for routine clinical use in veterinary oncology. PMID:26366881

Effect of caffeine on radiation-induced apoptosis in TK6 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhen, W.; Vaughan, A.T.M.

1995-02-01

Apoptosis has been measured in cells of the human TK6 lymphoblastoid cell line by recording the release of endonuclease-digested DNA from affected cells using flow cytometry. In asynchronously dividing cells, DNA degradation characteristic of apoptosis was first seen 12 h after irradiation as a defined DNA fluorescent peak of sub-G{sub 1}-phase content, reaching a maximum of 30-50% of the population by 24-72 h. Treating cells with 2 mM caffeine either before or up to 3 h after irradiation eliminated the degradation of DNA entirely. In addition, the percentage of cells in which apoptosis could be detected microscopically decreased from 62.4more » {+-} 0.95% to 16.7 {+-} 1.5% 72 h after caffeine treatment. Delaying caffeine treatment for 12 h after irradiation reduced DNA degradation by approximately 50% compared to cells receiving radiation alone. DNA degradation induced by serum deprivation was unaffected by caffeine treatment. These data support the contention that irradiation of TK6 cells produces a long-lived cellular signal which triggers apoptosis. Apoptosis produced by serum deprivation does not operate through the same pathway. 36 refs., 5 figs.« less

Enhancement of expression of survivin promoter-driven CD/TK double suicide genes by the nuclear matrix attachment region in transgenic gastric cancer cells.

PubMed

Niu, Ying; Li, Jian-Sheng; Luo, Xian-Run

2014-01-25

This work aimed to study a novel transgenic expression system of the CD/TK double suicide genes enhanced by the nuclear matrix attachment region (MAR) for gene therapy. The recombinant vector pMS-CD/TK containing the MAR-survivin promoter-CD/TK cassette was developed and transfected into human gastric cancer SGC-7901 cells. Expression of the CD/TK genes was detected by quantitative real-time PCR (qPCR) and Western blot. Cell viability and apoptosis were measured using the methyl thiazolyl tetrazolium (MTT) assay and flow cytometry. When the MAR fragment was inserted into the upstream of the survivin promoter, the qPCR result showed that the expression of the CD/TK genes significantly increased 7.7-fold in the transgenic SGC-7901 cells with plasmid pMS-CD/TK compared with that without MAR. MTT and flow cytometry analyses indicated that treatment with the prodrugs (5-FC+GCV) significantly decreased the cellular survival rate and enhanced the cellular apoptosis in the SGC-7901 cells. The expression of the CD/TK double suicide genes driven by the survivin promoter can be enhanced by the MAR fragment in human gastric cancer cells. Copyright © 2013 Elsevier B.V. All rights reserved.

Different DNA damage response of cis and trans isomers of commonly used UV filter after the exposure on adult human liver stem cells and human lymphoblastoid cells.

PubMed

Sharma, Anežka; Bányiová, Katarína; Babica, Pavel; El Yamani, Naouale; Collins, Andrew Richard; Čupr, Pavel

2017-09-01

2-ethylhexyl 4-methoxycinnamate (EHMC), used in many categories of personal care products (PCPs), is one of the most discussed ultraviolet filters because of its endocrine-disrupting effects. EHMC is unstable in sunlight and can be transformed from trans-EHMC to emergent cis-EHMC. Toxicological studies are focusing only on trans-EHMC; thus the toxicological data for cis-EHMC are missing. In this study, the in vitro genotoxic effects of trans- and cis-EHMC on adult human liver stem cells HL1-hT1 and human-derived lymphoblastoid cells TK-6 using a high-throughput comet assay were studied. TK-6 cells treated with cis-EHMC showed a high level of DNA damage when compared to untreated cells in concentrations 1.56 to 25μgmL -1 . trans-EHMC showed genotoxicity after exposure to the two highest concentrations 12.5 and 25μgmL -1 . The increase in DNA damage on HL1-hT1 cells induced by cis-EHMC and trans-EHMC was detected at the concentration 25μgmL -1 . The No observed adverse effect level (NOAEL, mg kg -1 bwday -1 ) was determined using a Quantitative in vitro to in vivo extrapolation (QIVIVE) approach: NOAEL trans-EHMC =3.07, NOAEL cis-EHMC =0.30 for TK-6 and NOAEL trans-EHMC =26.46, NOAEL cis-EHMC =20.36 for HL1-hT1. The hazard index (HI) was evaluated by comparing the reference dose (RfD, mgkg -1 bwday -1 ) obtained from our experimental data with the chronic daily intake (CDI) of the female population. Using comet assay experimental data with the more sensitive TK-6 cells, HI cis-EHMC was 7 times higher than HI trans-EHMC . In terms of CDI, relative contributions were; dermal exposure route>oral>inhalation. According to our results we recommend the RfD trans-EHMC =0.20 and RfD cis-EHMC =0.02 for trans-EHMC and cis-EHMC, respectively, to use for human health risk assessment. The significant difference in trans-EHMC and cis-EHMC response points to the need for toxicological reevaluation and application reassessment of both isomers in PCPs. Copyright © 2017 Elsevier B.V. All rights reserved.

Gamma camera dual imaging with a somatostatin receptor and thymidine kinase after gene transfer with a bicistronic adenovirus in mice.

PubMed

Zinn, Kurt R; Chaudhuri, Tandra R; Krasnykh, Victor N; Buchsbaum, Donald J; Belousova, Natalya; Grizzle, William E; Curiel, David T; Rogers, Buck E

2002-05-01

To compare two systems for assessing gene transfer to cancer cells and xenograft tumors with noninvasive gamma camera imaging. A replication-incompetent adenovirus encoding the human type 2 somatostatin receptor (hSSTr2) and the herpes simplex virus thymidine kinase (TK) enzyme (Ad-hSSTr2-TK) was constructed. A-427 human lung cancer cells were infected in vitro and mixed with uninfected cells at different ratios. A-427 tumors in nude mice (n = 23) were injected with 1 x 10(6) to 5 x 10(8) plaque-forming units (pfu) of Ad-hSSTr2-TK. The expressed hSSTr2 and TK proteins were imaged owing to internally bound, or trapped, technetium 99m ((99m)Tc)-labeled hSSTr2-binding peptide (P2045) and radioiodinated 2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl-5-iodouracil (FIAU), respectively. Iodine 125 ((125)I)-labeled FIAU was used in vitro and iodine 131 ((131)I)-labeled FIAU, in vivo. The (99m)Tc-labeled P2045 and (125)I- or (131)I-labeled FIAU were imaged simultaneously with different window settings with an Anger gamma camera. Treatment effects were tested with analysis of variance. Infected cells in culture trapped (125)I-labeled FIAU and (99m)Tc-labeled P2045; uptake correlated with the percentage of Ad-hSSTr2-TK-positive cells. For 100% of infected cells, 24% +/- 0.4 (mean +/- SD) of the added (99m)Tc-labeled P2045 was trapped, which is significantly lower (P <.05) than the 40% +/- 2 of (125)I-labeled FIAU that was trapped. For the highest Ad-hSSTr2-TK tumor dose (5 x 10(8) pfu), the uptake of (99m)Tc-labeled P2045 was 11.1% +/- 2.9 of injected dose per gram of tumor (thereafter, dose per gram), significantly higher (P <.05) than the uptake of (131)I-labeled FIAU at 1.6% +/- 0.4 dose per gram. (99m)Tc-labeled P2045 imaging consistently depicted hSSTr2 gene transfer in tumors at all adenovirus doses. Tumor uptake of (99m)Tc-labeled P2045 positively correlated with Ad-hSSTr2-TK dose; (131)I-labeled FIAU tumor uptake did not correlate with vector dose. The hSSTr2 and TK proteins were simultaneously imaged following dual gene transfer with an adenovirus vector. Copyright RSNA, 2002

miR-7-5p overexpression suppresses cell proliferation and promotes apoptosis through inhibiting the ability of DNA damage repair of PARP-1 and BRCA1 in TK6 cells exposed to hydroquinone.

PubMed

Luo, Hao; Liang, Hairong; Chen, Yuting; Chen, Shaoyun; Xu, Yongchun; Xu, Longmei; Liu, Jiaxian; Zhou, Kairu; Peng, Jucheng; Guo, Guoqiang; Lai, Bei; Song, Li; Yang, Hui; Liu, Linhua; Peng, Jianming; Liu, Zhidong; Tang, Lin; Chen, Wen; Tang, Huanwen

2018-03-01

Hydroquinone (HQ), one of the major metabolic products of benzene, is a carcinogen, which induces apoptosis and inhibit proliferation in lymphoma cells. microRNA-7-5p (miR-7-5p), a tumor suppressor, participates in various biological processes including cell proliferation and apoptosis regulation by repressing expression of specific oncogenic target genes. To explore whether miR-7-5p is involved in HQ-induced cell proliferation and apoptosis, we assessed the effect of miR-7-5p overexpression on induction of apoptosis analyzed by FACSCalibur flow cytometer in transfection of TK6 cells with miR-7-5p mimic (TK6- miR-7-5p). We observed an increased apoptosis by 25.43% and decreased proliferation by 28.30% in TK6-miR-7-5p cells compared to those negative control cells (TK6-shNC) in response to HQ treatment. Furthermore, HQ might active the apoptotic pathway via partly downregulation the expression of BRCA1 and PARP-1, followed by p53 activation, in TK6-miR-7-5p cells. In contrast, attenuated p53 and BRCA1 expression was observed in shPARP-1 cells than in NC cells after HQ treatment. Therefore, we conclude that HQ may activate apoptotic signals via inhibiting the tumor suppressive effects of miR-7-5p, which may be mediated partly by upregulating the expression of PARP-1 and BRCA1 in control cells. The increase of miR-7-5p expression further intensified downregulation of PARP-1 and BRCA1 in TK6-miR-7-5p cells, resulting in an increase of apoptosis and proliferation inhibited. Copyright © 2018 Elsevier B.V. All rights reserved.

Tissue kallikrein induces SH-SY5Y cell proliferation via epidermal growth factor receptor and extracellular signal-regulated kinase1/2 pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Zhengyu; Department of Neurology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200437; Yang, Qi

2014-03-28

Highlights: • TK promotes EGFR phosphorylation in SH-SY5Y cells. • TK activates ERK1/2 and p38 phosphorylation in SH-SY5Y cells. • TK mediates SH-SY5Y cell proliferation via EGFR and ERK1/2 pathway. - Abstract: Tissue kallikrein (TK) is well known to take most of its biological functions through bradykinin receptors. In the present study, we found a novel signaling pathway mediated by TK through epidermal growth factor receptor (EGFR) in human SH-SY5Y cells. We discovered that TK facilitated the activation of EGFR, extracellular signal-regulated kinase (ERK) 1/2 and p38 cascade. Interestingly, not p38 but ERK1/2 phosphorylation was severely compromised in cells depletedmore » of EGFR. Nevertheless, impairment of signaling of ERK1/2 seemed not to be restricted to EGFR phosphorylation. We also observed that TK stimulation could induce SH-SY5Y cell proliferation, which was reduced by EGFR down-regulation or ERK1/2 inhibitor. Overall, our findings provided convincing evidence that TK could mediate cell proliferation via EGFR and ERK1/2 pathway in vitro.« less

Retained sensitivity to cytotoxic pyrimidine nucleoside analogs in thymidine kinase 2 deficient human fibroblasts.

PubMed

Bjerke, Mia; Solaroli, Nicola; Lesko, Nicole; Balzarini, Jan; Johansson, Magnus; Karlsson, Anna

2010-01-01

Thymidine kinase 2 (TK2) is a mitochondrial deoxyribonucleoside kinase that phosphorylates several nucleoside analogs used in anti-viral and anti-cancer therapy. A fibroblast cell line with decreased TK2 activity was investigated in order to obtain insights in the effects of TK2 deficiency on nucleotide metabolism. The role of TK2 for the sensitivity against cytotoxic nucleoside analogs was also investigated. The TK2 deficient cells retained their sensitivity against all pyrimidine nucleoside analogs tested. This study suggests that nucleoside analog phosphorylation mediated by TK2 may be less important, compared to other deoxyribonucleoside kinases, for the cytotoxic effects of these compounds.

The history of N-methanocarbathymidine: the investigation of a conformational concept leads to the discovery of a potent and selective nucleoside antiviral agent.

PubMed

Marquez, Victor E; Hughes, Stephen H; Sei, Shizuko; Agbaria, Riad

2006-09-01

Conformationally locked (North)-methanocarbathymidine (N-MCT) and (South)-methanocarbathymidine (S-MCT) have been used to investigate the conformational preferences of kinases and polymerases. The herpes kinases show a distinct bias for S-MCT, while DNA polymerases almost exclusively incorporate the North 5'-triphosphate (N-MCT-TP). Only N-MCT demonstrated potent antiviral activity against herpes simplex viruses (HSV-1 and 2) and Kaposi's sarcoma-associated herpesvirus (KSHV). The activity of N-MCT depends on its metabolic transformation to N-MCT-TP by the herpes kinases (HSV-tk or KSHV-tk), which catalyze the mono and diphosphorylation steps; cellular kinases generate the triphosphate. N-MCT at a dose of 5.6 mg/kg was totally protective for mice inoculated intranasally with HSV-1. Tumor cells that are not responsive to antiviral therapy became sensitive to N-MCT if the cells expressed HSV-tk. N-MCT given twice daily (100 mg/kg) for 7 days completely inhibited the growth of MC38 tumors derived from cells that express HSV-tk in mice while exhibiting no effect on tumors derived from non-transduced cells. After i.p. administration, N-MCT was rapidly absorbed and distributed in all organs examined with slow penetration into brain and testes. N-MCT-TP was also a potent inhibitor of HIV replication in human osteosarcoma (HOS) cells expressing HSV-tk.

An application of LOH analysis for detecting the genetic influences of space environmental radiation

NASA Astrophysics Data System (ADS)

Yatagai, F.; Umebayashi, Y.; Honma, M.; Abe, T.; Suzuki, H.; Shimazu, T.; Ishioka, N.; Iwaki, M.

To detect the genetic influence of space environmental radiation at the chromosome level we proposed an application of loss of heterozygosity LOH analysis system for the mutations induced in human lymphoblastoid TK6 cells Surprisingly we succeeded the mutation detection in the frozen dells which were exposed to a low-dose 10 cGy of carbon-ion beam irradiation Mutation assays were performed within a few days or after about one month preservation at --80 r C following irradiation The results showed an increase in mutation frequency at the thymidine kinase TK gene locus 1 6-fold 2 5 X 10 -6 to 3 9 X 10 -6 and 2 1-fold 2 5 X 10 -6 to 5 3 X 10 -6 respectively Although the relative distributions of mutation classes were not changed by the radiation exposure in either assay an interesting characteristic was detected using this LOH analysis system two TK locus markers and eleven microsatellite loci spanning chromosome 17 The radiation-specific patterns of interstitial deletions were observed in the hemizygous LOH mutants which were considered as a result of end-joining repair of carbon ion-induced DNA double-strand breaks These results clearly demonstrate that this analysis can be used for the detection of low-dose ionizing radiation effects in the frozen cells In addition we performed so called adaptive response experiments in which TK6 cells were pre-irradiated with low-dose 2 5 sim 10 cGy of X-ray and then exposed to challenging dose 2Gy of X-rays Interestingly the

Targeted impairment of thymidine kinase 2 expression in cells induces mitochondrial DNA depletion and reveals molecular mechanisms of compensation of mitochondrial respiratory activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Villarroya, Joan, E-mail: joanvillarroya@gmail.com; Institut de Recerca l'Hospital de la Santa Creu i Sant Pau, Barcelona; Lara, Mari-Carmen

Highlights: {yields} We impaired TK2 expression in Ost TK1{sup -} cells via siRNA-mediated interference (TK2{sup -}). {yields} TK2 impairment caused severe mitochondrial DNA (mtDNA) depletion in quiescent cells. {yields} Despite mtDNA depletion, TK2{sup -} cells show high cytochrome oxidase activity. {yields} Depletion of mtDNA occurs without imbalance in the mitochondrial dNTP pool. {yields} Nuclear-encoded ENT1, DNA-pol {gamma}, TFAM and TP gene expression is lowered in TK2{sup -} cells. -- Abstract: The mitochondrial DNA (mtDNA) depletion syndrome comprises a clinically heterogeneous group of diseases characterized by reductions of the mtDNA abundance, without associated point mutations or rearrangements. We have developed themore » first in vitro model to study of mtDNA depletion due to reduced mitochondrial thymidine kinase 2 gene (TK2) expression in order to understand the molecular mechanisms involved in mtDNA depletion syndrome due to TK2 mutations. Small interfering RNA targeting TK2 mRNA was used to decrease TK2 expression in Ost TK1{sup -} cells, a cell line devoid of endogenous thymidine kinase 1 (TK1). Stable TK2-deficient cell lines showed a reduction of TK2 levels close to 80%. In quiescent conditions, TK2-deficient cells showed severe mtDNA depletion, also close to 80% the control levels. However, TK2-deficient clones showed increased cytochrome c oxidase activity, higher cytochrome c oxidase subunit I transcript levels and higher subunit II protein expression respect to control cells. No alterations of the deoxynucleotide pools were found, whereas a reduction in the expression of genes involved in nucleoside/nucleotide homeostasis (human equilibrative nucleoside transporter 1, thymidine phosphorylase) and mtDNA maintenance (DNA-polymerase {gamma}, mitochondrial transcription factor A) was observed. Our findings highlight the importance of cellular compensatory mechanisms that enhance the expression of respiratory components to ensure respiratory activity despite profound depletion in mtDNA levels.« less

Production and characterisation of a novel chicken IgY antibody raised against C-terminal peptide from human thymidine kinase 1.

PubMed

Wu, Chuanjing; Yang, Rongjiang; Zhou, Ji; Bao, Shing; Zou, Li; Zhang, Pinggan; Mao, Yongrong; Wu, Jianping; He, Qimin

2003-06-01

Egg yolk is a good source of highly specific antibodies against mammalian antigens because of the phylogenetic distance between birds and mammals. Chicken egg yolk immunoglobulins (IgY) were generated to a synthetic 31-amino acid peptide from the C-terminal of human HeLa thymidine kinase 1 (TK1) enzyme. The anti-TK1 IgY antibody was purified using affinity chromatography against the 31-amino acid peptide. The purified antibody inhibited the catalytic activity of the TK1 enzyme in the CEM TK1(+) cells and recognized the 25-kDa subunit and tetrameric form of TK1, which has a pI value of 8.3. No immunoreaction was observed in CEM TK1(-) cells. Western blot of the serum TK1 (S-TK1) also showed that only a single band was found in the serum of patients with malignancies. No band was seen in healthy serum. Furthermore, dot blots and enhanced chemiluminescence (ECL) detection of S-TK1 performed on sera of preoperative patients with gastric cancer (GC) (n=31) and healthy controls (n=62) showed that the levels of S-TK1 in the sera of cancer patients were significantly different (P<0.01). Using ECL dot blots, 0.1 pg of TK1 in 3 microl sera could be detected. Immunohistostaining of tissues in the 11 advanced-stage cancer patients (four breast carcinomas, three hepatocarcinomas and four thyroid carcinomas) indicated that a strong staining of TK1 enzyme was found in the cytoplasm of malignant cells. No staining or weak staining was seen in normal tissues. We suggest that screening for TK1 using anti-TK1 IgY may be potentially useful for serological and immunohistochemical detection of TK1 as an early prognosis and for monitoring patients undergoing treatment.

A gene delivery system with a human artificial chromosome vector based on migration of mesenchymal stem cells towards human glioblastoma HTB14 cells.

PubMed

Kinoshita, Yusuke; Kamitani, Hideki; Mamun, Mahabub Hasan; Wasita, Brian; Kazuki, Yasuhiro; Hiratsuka, Masaharu; Oshimura, Mitsuo; Watanabe, Takashi

2010-05-01

Mesenchymal stem cells (MSCs) have been expected to become useful gene delivery vehicles against human malignant gliomas when coupled with an appropriate vector system, because they migrate towards the lesion. Human artificial chromosomes (HACs) are non-integrating vectors with several advantages for gene therapy, namely, no limitations on the size and number of genes that can be inserted. We investigated the migration of human immortalized MSCs bearing a HAC vector containing the herpes simplex virus thymidine kinase gene (HAC-tk-hiMSCs) towards malignant gliomas in vivo. Red fluorescence protein-labeled human glioblastoma HTB14 cells were implanted into a subcortical region in nude mice. Four days later, green fluorescence protein-labeled HAC-tk-hiMSCs were injected into a contralateral subcortical region (the HTB14/HAC-tk-hiMSC injection model). Tropism to the glioma mass and the route of migration were visualized by fluorescence microscopy and immunohistochemical staining. HAC-tk-hiMSCs began to migrate toward the HTB14 glioma area via the corpus callosum on day 4, and gathered around the HTB14 glioma mass on day 7. To test whether the delivered gene could effectively treat glioblastoma in vivo, HTB14/HAC-tk-hiMSC injected mice were treated with ganciclovir (GCV) or PBS. The HTB14 glioma mass was significantly reduced by GCV treatment in mice injected with HAC-tk-hiMSCs. It was confirmed that gene delivery by our HAC-hiMSC system was effective after migration of MSCs to the glioma mass in vivo. Therefore, MSCs containing HACs carrying an anticancer gene or genes may provide a new tool for the treatment of malignant gliomas and possibly of other tumor types.

Gene Therapy for Human Lung Adenocarcinoma Using a Suicide Gene Driven by a Lung-Specific Promoter Delivered by JC Virus-Like Particles.

PubMed

Chao, Chun-Nun; Lin, Mien-Chun; Fang, Chiung-Yao; Chen, Pei-Lain; Chang, Deching; Shen, Cheng-Huang; Wang, Meilin

2016-01-01

Lung adenocarcinoma, the most commonly diagnosed type of lung cancer, has a poor prognosis even with combined surgery, chemotherapy, or molecular targeted therapies. Most patients are diagnosed with an in-operable advanced or metastatic disease, both pointing to the necessity of developing effective therapies for lung adenocarcinoma. Surfactant protein B (SP-B) has been found to be overexpressed in lung adenocarcinoma. In addition, it has also been demonstrated that human lung adenocarcinoma cells are susceptible to the JC polyomavirus (JCPyV) infection. Therefore, we designed that the JCPyV virus-like particle (VLP) packaged with an SP-B promoter-driven thymidine kinase suicide gene (pSPB-tk) for possible gene therapy of human lung adenocarcinoma. Plasmids expressing the GFP (pSPB-gfp) or thymidine kinase gene (pSPB-tk) under the control of the human SP-B promoter were constructed. The promoter's tissue specificity was tested by transfection of pSPB-gfp into A549, CH27, and H460 human lung carcinoma cells and non-lung cells. The JCPyV VLP's gene transfer efficiency and the selective cytotoxicity of pSPB-tk combined with ganciclovir (GCV) were tested in vitro and in a xenograft mouse model. In the current study, we found that SP-B promoter-driven GFP was specifically expressed in human lung adenocarcinoma (A549) and large cell carcinoma (H460) cells. JCPyV VLPs were able to deliver a GFP reporter gene into A549 cells for expression. Selective cytotoxicity was observed in A549 but not non-lung cells that were transfected with pSPB-tk or infected with pSPB-tk-carrying JCPyV VLPs. In mice injected with pSPB-tk-carrying JCPyV VLPs through the tail vein and treated with ganciclovir (GCV), a potent 80% inhibition of growth of human lung adenocarcinoma nodules resulted. The JCPyV VLPs combined with the use of SP-B promoter demonstrates effectiveness as a potential gene therapy against human lung adenocarcinoma.

Activation of caspase-3 noninvolved in the bystander effect of the herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) system.

PubMed

Zhang, Zhihong; Lin, Juqiang; Chu, Jun; Ma, Yan; Zeng, Shaoqun; Luo, Qingming

2008-01-01

Use of the herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) system is one of the promising approaches in the rapidly growing area of gene therapy. The "bystander effect," a phenomenon in which HSV-tk+ cells exposed to GCV are toxic to adjacent HSV-tk- cells, was reported to play an important role in suicide gene therapy. However, the mechanism by which HSV-tk/GCV induces the bystander effect is poorly understood. We monitored the activation of caspase-3 in living cells induced by the HSV-tk/GCV system using a genetically encoded fluorescence resonance energy transfer (FRET) probe CD3, , a caspase-3 recognition site fused with a cyan fluorescent protien (CFP) and a red fluorescent protein (DsRed) which we reported and named in a previous paper. Fluorescence protein (FP)-based multicolor cellular labeling, combined with the multichannel fluorescence imaging and FRET imaging techniques, provides a novel and improved approach to directly determine whether the activation of caspase-3 involved in the HSV-tk/GCV system induces cell apoptosis in tk gene-expressing cells and their neighboring cells. FRET ratio images of CD3, and fluorescence images of the fusion protein of thymidine kinase linked with green fluorescent protein (TK-GFP), indicated that HSV-tk/GCV system-induced apoptosis in human adenoid cystic carcinoma (ACC-M) cells was via a caspase-3 pathway, and the activation of caspase-3 was not involved in the bystander effect of HSV-tk/GCV system.

Frozen human cells can record radiation damage accumulated during space flight: mutation induction and radioadaptation.

PubMed

Yatagai, Fumio; Honma, Masamitsu; Takahashi, Akihisa; Omori, Katsunori; Suzuki, Hiromi; Shimazu, Toru; Seki, Masaya; Hashizume, Toko; Ukai, Akiko; Sugasawa, Kaoru; Abe, Tomoko; Dohmae, Naoshi; Enomoto, Shuichi; Ohnishi, Takeo; Gordon, Alasdair; Ishioka, Noriaki

2011-03-01

To estimate the space-radiation effects separately from other space-environmental effects such as microgravity, frozen human lymphoblastoid TK6 cells were sent to the "Kibo" module of the International Space Station (ISS), preserved under frozen condition during the mission and finally recovered to Earth (after a total of 134 days flight, 72 mSv). Biological assays were performed on the cells recovered to Earth. We observed a tendency of increase (2.3-fold) in thymidine kinase deficient (TK(-)) mutations over the ground control. Loss of heterozygosity (LOH) analysis on the mutants also demonstrated a tendency of increase in proportion of the large deletion (beyond the TK locus) events, 6/41 in the in-flight samples and 1/17 in the ground control. Furthermore, in-flight samples exhibited 48% of the ground-control level in TK(-) mutation frequency upon exposure to a subsequent 2 Gy dose of X-rays, suggesting a tendency of radioadaptation when compared with the ground-control samples. The tendency of radioadaptation was also supported by the post-flight assays on DNA double-strand break repair: a 1.8- and 1.7-fold higher efficiency of in-flight samples compared to ground control via non-homologous end-joining and homologous recombination, respectively. These observations suggest that this system can be used as a biodosimeter, because DNA damage generated by space radiation is considered to be accumulated in the cells preserved frozen during the mission, Furthermore, this system is also suggested to be applicable for evaluating various cellular responses to low-dose space radiation, providing a better understanding of biological space-radiation effects as well as estimation of health influences of future space explores. © Springer-Verlag 2010

Gene therapy of uterine leiomyoma: adenovirus-mediated herpes simplex virus thymidine kinase/ganciclovir treatment inhibits growth of human and rat leiomyoma cells in vitro and in a nude mouse model.

PubMed

Salama, S A; Kamel, M; Christman, G; Wang, H Q; Fouad, H M; Al-Hendy, A

2007-01-01

Uterine leiomyomas (LM) affect a high percentage of reproductive-age women. They develop as discrete, well-defined tumors that are easily accessible with imaging techniques--making this disease ideal for localized gene therapy approaches. In this study, we determined the efficacy of adenovirus-mediated herpes simplex virus thymidine kinase gene transfer in combination with ganciclovir (Ad-TK/GCV) as a potential therapy for LM. Rat ELT-3 LM cells and human LM cells were transfected with different multiplicity of infections (10-100 plaque forming units [PFU]/cell) of Ad-TK and treated with GCV (5, 10, or 20 microg/ml) for 5 days. To test the bystander effect, Ad-TK-transfected ELT-3 cells (100 PFU/cell) or LM cells (10 PFU/cell) were cocultured with corresponding nontransfected cells at increasing percentages and treated with GCV followed by cell counting. In ELT-3 cells transfected with Ad-TK/GCV (10, 20, 50, or 100 PFU/cell), the cell count was reduced by 24, 42, 77, and 87%, respectively, compared with the control cells (transfected with Ad-Lac Z/GCV). Similarly, in LM cells transfected with Ad-TK/GCV (10, 50, or 100 PFU/cell), the cell count was reduced by 31, 62, and 82%, respectively, compared with the control. A strong bystander effect was noted in both ELT-3 and LM cells with significant killing (p = 0.001) at a ratio of infected:uninfected cells of only 1:99 and maximal killing at 1:4. This study demonstrates the potential efficacy of the Ad-TK/GCV gene therapy approach as a viable nonsurgical alternative treatment for uterine LM.

Effect of chemical mutagens and carcinogens on gene expression profiles in human TK6 cells.

PubMed

Godderis, Lode; Thomas, Reuben; Hubbard, Alan E; Tabish, Ali M; Hoet, Peter; Zhang, Luoping; Smith, Martyn T; Veulemans, Hendrik; McHale, Cliona M

2012-01-01

Characterization of toxicogenomic signatures of carcinogen exposure holds significant promise for mechanistic and predictive toxicology. In vitro transcriptomic studies allow the comparison of the response to chemicals with diverse mode of actions under controlled experimental conditions. We conducted an in vitro study in TK6 cells to characterize gene expression signatures of exposure to 15 genotoxic carcinogens frequently used in European industries. We also examined the dose-responsive changes in gene expression, and perturbation of biochemical pathways in response to these carcinogens. TK6 cells were exposed at 3 dose levels for 24 h with and without S9 human metabolic mix. Since S9 had an impact on gene expression (885 genes), we analyzed the gene expression data from cells cultures incubated with S9 and without S9 independently. The ribosome pathway was affected by all chemical-dose combinations. However in general, no similar gene expression was observed among carcinogens. Further, pathways, i.e. cell cycle, DNA repair mechanisms, RNA degradation, that were common within sets of chemical-dose combination were suggested by clustergram. Linear trends in dose-response of gene expression were observed for Trichloroethylene, Benz[a]anthracene, Epichlorohydrin, Benzene, and Hydroquinone. The significantly altered genes were involved in the regulation of (anti-) apoptosis, maintenance of cell survival, tumor necrosis factor-related pathways and immune response, in agreement with several other studies. Similarly in S9+ cultures, Benz[a]pyrene, Styrene and Trichloroethylene each modified over 1000 genes at high concentrations. Our findings expand our understanding of the transcriptomic response to genotoxic carcinogens, revealing the alteration of diverse sets of genes and pathways involved in cellular homeostasis and cell cycle control.

Effect of Chemical Mutagens and Carcinogens on Gene Expression Profiles in Human TK6 Cells

PubMed Central

Godderis, Lode; Thomas, Reuben; Hubbard, Alan E.; Tabish, Ali M.; Hoet, Peter; Zhang, Luoping; Smith, Martyn T.; Veulemans, Hendrik; McHale, Cliona M.

2012-01-01

Characterization of toxicogenomic signatures of carcinogen exposure holds significant promise for mechanistic and predictive toxicology. In vitro transcriptomic studies allow the comparison of the response to chemicals with diverse mode of actions under controlled experimental conditions. We conducted an in vitro study in TK6 cells to characterize gene expression signatures of exposure to 15 genotoxic carcinogens frequently used in European industries. We also examined the dose-responsive changes in gene expression, and perturbation of biochemical pathways in response to these carcinogens. TK6 cells were exposed at 3 dose levels for 24 h with and without S9 human metabolic mix. Since S9 had an impact on gene expression (885 genes), we analyzed the gene expression data from cells cultures incubated with S9 and without S9 independently. The ribosome pathway was affected by all chemical-dose combinations. However in general, no similar gene expression was observed among carcinogens. Further, pathways, i.e. cell cycle, DNA repair mechanisms, RNA degradation, that were common within sets of chemical-dose combination were suggested by clustergram. Linear trends in dose–response of gene expression were observed for Trichloroethylene, Benz[a]anthracene, Epichlorohydrin, Benzene, and Hydroquinone. The significantly altered genes were involved in the regulation of (anti-) apoptosis, maintenance of cell survival, tumor necrosis factor-related pathways and immune response, in agreement with several other studies. Similarly in S9+ cultures, Benz[a]pyrene, Styrene and Trichloroethylene each modified over 1000 genes at high concentrations. Our findings expand our understanding of the transcriptomic response to genotoxic carcinogens, revealing the alteration of diverse sets of genes and pathways involved in cellular homeostasis and cell cycle control. PMID:22723965

Inhibition of autophagy enhances Hydroquinone-induced TK6 cell death.

PubMed

Xu, Longmei; Liu, Jiaxian; Chen, Yuting; Yun, Lin; Chen, Shaoyun; Zhou, Kairu; Lai, Bei; Song, Li; Yang, Hui; Liang, Hairong; Tang, Huanwen

2017-06-01

Hydroquinone (HQ), one of the metabolic products of benzene, is a carcinogen. It can induce apoptosis in lymphoma cells. However, whether HQ can induce autophagy and what roles autophagy plays in TK6 cells exposured to HQ remains unclear. In this study, we found that HQ could induce autophagy through techniques of qRT-PCR, Western blot, immunofluorescent assay of LC3 and transmission electron microscope. Furthermore, inhibiting autophagy using 3-methyladenine (3-MA) or chloroquine (CQ) significantly enhanced HQ-induced cell apoptosis, suggesting that autophagy may be a survival mechanism. Our study also showed that HQ activated PARP-1. Moreover, knockdown of PARP-1 strongly exhibited decreased autophagy related genes expression. In contrast, the absence of SIRT1 increased that. Altogether, our data provided evidence that HQ induced autophagy in TK6 cells and autophagy protected TK6 from HQ attack-induced injury in vitro, and the autophagy was partially mediated via activation of the PARP-1-SIRT1 signaling pathway. Copyright © 2017 Elsevier Ltd. All rights reserved.

PARP-1 may be involved in hydroquinone-induced apoptosis by poly ADP-ribosylation of ZO-2

PubMed Central

Liu, Jiaxian; Yuan, Qian; Ling, Xiaoxuan; Tan, Qiang; Liang, Hairong; Chen, Jialong; Lin, Lianzai; Xiao, Yongmei; Chen, Wen; Liu, Linhua; Tang, Huanwen

2017-01-01

Hydroquinone (HQ), a major reactive metabolite of benzene, contributes to benzene-induced leukemia. The molecular mechanisms that underlie this activity remain to be elucidated. Poly ADP-ribosylation (PARylation) is a type of reversible posttranslational modification that is performed by enzymes in the PAR polymerase (PARP) family and mediates different biological processes, including apoptosis. Zona occludens 2 (ZO-2) is a tight junction scaffold protein, which is involved in cell proliferation and apoptosis. The present study investigated the activity and mechanisms regulated by PARP-1 during HQ-induced apoptosis using TK6 lymphoblastoid cells and PARP-1-silenced TK6 cells. The results revealed that exposure to 10 µM HQ for 72 h induced apoptosis in TK6 cells and that apoptosis was attenuated in PARP-1-silenced TK6 cells. In cells treated with HQ, inhibition of PARP-1 increased the expression of B cell leukemia/lymphoma 2 (Bcl-2), increased ATP production and reduced reactive oxygen species (ROS) production relative to the levels observed in cells treated with HQ alone. Co-localization of ZO-2 and PAR (or PARP-1 protein) was determined using immunofluorescence confocal microscopy. The findings of the present study revealed that ZO-2 was PARylated via an interaction with PARP-1, which was consistent with an analysis of protein expression that was performed using western blot analysis, which determined that ZO-2 protein expression was upregulated in HQ-treated control cells and downregulated in HQ-treated PARP-1-silenced TK6 cells. These findings indicated that prolonged exposure to a low dose of HQ induced TK6 cells to undergo apoptosis, whereas inhibiting PARP-1 attenuates cellular apoptosis by activating Bcl-2 and energy-saving processes and reducing ROS. The present study determined that PARP-1 was involved in HQ-induced apoptosis by PARylation of ZO-2. PMID:28983606

Unequal homologous recombination between tandemly arranged sequences stably incorporated into cultured rat cells.

PubMed Central

Stringer, J R; Kuhn, R M; Newman, J L; Meade, J C

1985-01-01

Cultured rat cells deficient in endogenous thymidine kinase activity (tk) were stably transformed with a recombination-indicator DNA substrate constructed in vitro by rearrangement of the herpes simplex virus tk gene sequences into a partially redundant permutation of the functional gene. The recombination-indicator DNA did not express tk, but was designed to allow formation of a functional tk gene via homologous recombination. A clonal cell line (519) was isolated that harbored several permuted herpes simplex virus tk genes. 519 cells spontaneously produced progeny that survived in medium containing hypoxanthine, aminopterin, and thymidine. Acquisition of resistance to hypoxanthine, aminopterin, and thymidine was accompanied by the rearrangement of the defective tk gene to functional configuration. The rearrangement apparently occurred by unequal exchange between one permuted tk gene and a replicated copy of itself. Recombination was between 500-base-pair tracts of DNA sequence homology that were separated by 3.4 kilobases. Exchanges occurred spontaneously at a frequency of approximately 5 X 10(-6) events per cell per generation. Recombination also mediated reversion to the tk- phenotype; however, the predominant mechanism by which cells escaped death in the presence of drugs rendered toxic by thymidine kinase was not recombination, but rather inactivation of the intact tk gene. Images PMID:3016511

In Vitro Antitumor Effects of AHR Ligands Aminoflavone (AFP 464) and Benzothiazole (5F 203) in Human Renal Carcinoma Cells.

PubMed

Luzzani, Gabriela A; Callero, Mariana A; Kuruppu, Anchala I; Trapani, Valentina; Flumian, Carolina; Todaro, Laura; Bradshaw, Tracey D; Loaiza Perez, Andrea I

2017-12-01

We investigated activity and mechanism of action of two AhR ligand antitumor agents, AFP 464 and 5F 203 on human renal cancer cells, specifically examining their effects on cell cycle progression, apoptosis, and migration. TK-10, SN12C, Caki-1, and ACHN human renal cancer cell lines were treated with AFP 464 and 5F 203. We evaluated cytotoxicity by MTS assays, cell cycle arrest, and apoptosis by flow cytometry and corroborated a mechanism of action involving AhR signal transduction activation. Changes in migration properties by wound healing assays were investigated: 5F 203-sensitive cells show decreased migration after treatment, therefore, we measured c-Met phosphorylation by Western blot in these cells. A 5F 203 induced a decrease in cell viability which was more marked than AFP 464. This cytotoxicity was reduced after treatment with the AhR inhibitor α-NF for both compounds indicating AhR signaling activation plays a role in the mechanism of action. A 5F 203 is sequestered by TK-10 cells and induces CYP1A1 expression; 5F 203 potently inhibited migration of TK-10, Caki-1, and SN12C cells, and inhibited c-Met receptor phosphorylation in TK-10 cells. AhR ligand antitumor agents AFP 464 and 5F 203 represent potential new candidates for the treatment of renal cancer. A 5F 203 only inhibited migration of sensitive cells and c-Met receptor phosphorylation in TK-10 cells. c-Met receptor signal transduction is important in migration and metastasis. Therefore, we consider that 5F 203 offers potential for the treatment of metastatic renal carcinoma. J. Cell. Biochem. 118: 4526-4535, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Complete regression of human malignant mesothelioma xenografts following local injection of midkine promoter-driven oncolytic adenovirus

PubMed Central

Kubo, Shuji; Kawasaki, Yoshiko; Yamaoka, Norie; Tagawa, Masatoshi; Kasahara, Noriyuki; Terada, Nobuyuki; Okamura, Haruki

2010-01-01

Background Malignant mesothelioma is a highly aggressive tumor with poor prognosis. Conventional therapies for mesothelioma are generally non-curative, and new treatment paradigms are urgently needed. We hypothesized that the tumor-specific midkine (Mdk) promoter could confer transcriptional targeting to oncolytic adenoviruses for effective treatment of malignant mesothelioma. Methods We analyzed Mdk expression by quantitative RT-PCR in six human mesothelioma cell lines, and tested Mdk promoter activity by luciferase reporter assay. Based on these data, we constructed a replication-selective oncolytic adenovirus, designated AdMdk-E1-iresTK, which contains an Mdk promoter-driven adenoviral E1 gene and HSV-thymidine kinase (TK) suicide gene, and CMV promoter-driven green fluorescent protein (GFP) marker gene. Selectivity of viral replication and cytolysis were characterized in normal vs. mesothelioma cells in vitro, and intratumoral spread and antitumor efficacy were investigated in vivo. Results Mdk promoter activity was restricted in normal cells, but highly activated in mesothelioma cell lines. AdMdk-E1-iresTK was seen to efficiently replicate, produce viral progeny, and spread in multiple mesothelioma cell lines. Lytic spread of AdMdk-E1-iresTK mediated efficient killing of these mesothelioma cells, and its in vitro cytocidal effect was significantly enhanced by treatment with the prodrug, ganciclovir. Intratumoral injection of AdMdk-E1-iresTK caused complete regression of MESO4 and MSTO human mesothelioma xenografts in athymic mice. In vivo fluorescence imaging demonstrated intratumoral spread of AdMdk-E1-iresTK-derived signals, which vanished after tumor eradication. Conclusions These data indicate that transcriptional targeting of viral replication by the Mdk promoter represents a promising general strategy for oncolytic virotherapy of cancers with upregulated Mdk expression, including malignant mesothelioma. PMID:20635326

Tissue kallikrein promotes cardiac neovascularization by enhancing endothelial progenitor cell functional capacity.

PubMed

Yao, Yuyu; Sheng, Zulong; Li, Yefei; Yan, Fengdi; Fu, Cong; Li, Yongjun; Ma, Genshan; Liu, Naifeng; Chao, Julie; Chao, Lee

2012-08-01

Tissue kallikrein (TK) has been demonstrated to improve neovasculogenesis after myocardial infarction (MI). In the present study, we examined the role and underlying mechanisms of TK in peripheral endothelial progenitor cell (EPC) function. Peripheral blood-derived mononuclear cells containing EPCs were isolated from rat. The in vitro effects of TK on EPC differentiation, apoptosis, migration, and vascular tube formation capacity were studied in the presence or absence of TK, kinin B(2) receptor antagonist (icatibant), and phosphatidylinositol-3 kinase inhibitor (LY294002). Apoptosis was evaluated by flow-cytometry analysis using Annexin V-FITC/PI staining, as well as western-blot analysis of Akt phosphorylation and cleaved caspase-3. Using an MI mouse model, we then examined the in vivo effects of human TK gene adenoviral vector (Ad.hTK) administration on the number of CD34(+)Flk-1(+) progenitors in the peripheral circulation, heart tissue, extent of vasculogenesis, and heart function. Administration of TK significantly increased the number of Dil-LDL/UEA-lectin double-positive early EPCs, as well as their migration and tube formation properties in vitro. Transduction of TK in cultured EPCs attenuated apoptosis induced by hypoxia and led to an increase in Akt phosphorylation and a decrease in cleaved caspase-3 levels. The beneficial effects of TK were blocked by pretreatment with icatibant and LY294002. The expression of recombinant human TK in the ischemic mouse heart significantly improved cardiac contractility and reduced infarct size 7 days after gene delivery. Compared with the Ad.Null group, Ad.hTK reduced mortality and preserved left ventricular function by increasing the number of CD34(+)Flk-1(+) EPCs and promoting the growth of capillaries and arterioles in the peri-infarct myocardium. These data provide direct evidence that TK promotes vessel growth by increasing the number of EPCs and enhancing their functional properties through the kinin B(2) receptor-Akt signaling pathway.

Analysis of cellular response by exposure to acute or chronic radiation in human lymphoblastoid TK-6 cells

NASA Astrophysics Data System (ADS)

Ohnishi, T.; Yasumoto, J.; Takahashi, A.; Ohnishi, K.

To clarify the biological effects of low-dose rate radiation on human health for long-term stay in space, we analyzed the induction of apoptosis and apoptosis-related gene expression after irradiation with different dose-rate in human lymphoblastoid TK-6 cells harboring wild-type p53 gene. We irradiated TK-6 cells by X-ray at 1.5 Gy (1 Gy/min) and then sampled at 25 hr after culturing. We also irradiated by gamma-ray at 1.5 Gy (1 mGy/min) and then sampled immediately or 25 hr after irradiation. For DNA ladder analysis, we extracted DNA from these samples and electrophoresed with 2% agarose gel. In addition, we extracted mRNA from these samples for DNA-array analysis. mRNA from non-irradiated cells was used as a control. After labeling the cDNA against mRNA with [α -33P]-dCTP and hybridizing onto DNA array (Human Apoptosis Expression Array, R&D Systems), we scanned the profiles of the spots by a phosphorimager (BAS5000, FUJI FILM) and calculated using a NIH Image program. The data of each DNA-array were normalized with eight kinds of house keeping genes. We analyzed the expression level of apoptosis-related genes such as p53-related, Bcl-2 family, Caspase family and Fas-related genes. DNA ladders were obviously detected in the cells exposed to a high dose-rate radiation. We detected the induction of the gene expression of apoptosis-promotive genes. In contrast, almost no apoptosis was observed in the cells exposed to the chronic radiation at a low dose-rate. In addition, we detected the induction of the gene expression of apoptosis-suppressive genes as compared with apoptosis promotive-genes immediately after chronic irradiation. These results lead the importance of biological meaning of exposure to radiation at low dose-rate from an aspect of carcinogenesis. Finally, the effects of chronic irradiation become a highly important issue in space radiation biology for human health.

Design of a functional cyclic HSV1-TK reporter and its application to PET imaging of apoptosis

PubMed Central

Wang, Zhe; Wang, Fu; Hida, Naoki; Kiesewetter, Dale O; Tian, Jie; Niu, Gang; Chen, Xiaoyuan

2017-01-01

Positron emission tomography (PET) is a sensitive and noninvasive imaging method that is widely used to explore molecular events in living subjects. PET can precisely and quantitatively evaluate cellular apoptosis, which has a crucial role in various physiological and pathological processes. In this protocol, we describe the design and use of an engineered cyclic herpes simplex virus 1–thymidine kinase (HSV1-TK) PET reporter whose kinase activity is specifically switched on by apoptosis. The expression of cyclic TK (cTK) in healthy cells leads to inactive product, whereas the activation of apoptosis through the caspase-3 pathway cleaves cTK, thus restoring its activity and enabling PET imaging. In addition to detailing the design and construction of the cTK plasmid in this protocol, we include assays for evaluating the function and specificity of the cTK reporter in apoptotic cells, such as assays for measuring the cell uptake of PET tracer in apoptotic cells, correlating doxorubicin (Dox)-induced cell apoptosis to cTK function recovery, and in vivo PET imaging of cancer cell apoptosis, and we also include corresponding data acquisition methods. The time to build the entire cTK reporter is ~2–3 weeks. The selection of a stable cancer cell line takes ~4–6 weeks. The time to implement assays regarding cTK function in apoptotic cells and the in vivo imaging varies depending on the experiment. The cyclization strategy described in this protocol can also be adapted to create other reporter systems for broad biomedical applications. PMID:25927390

Tissue kallikrein-modified human endothelial progenitor cell implantation improves cardiac function via enhanced activation of akt and increased angiogenesis.

PubMed

Yao, Yuyu; Sheng, Zulong; Li, YeFei; Fu, Cong; Ma, Genshan; Liu, Naifeng; Chao, Julie; Chao, Lee

2013-05-01

Endothelial progenitor cells (EPCs) have been shown to enhance angiogenesis not only by incorporating into the vasculature but also by secreting cytokines, thereby serving as an ideal vehicle for gene transfer. As tissue kallikrein (TK) has pleiotropic effects in inhibiting apoptosis and oxidative stress, and promoting angiogenesis, we evaluated the salutary potential of kallikrein-modified human EPCs (hEPCs; Ad.hTK-hEPCs) after acute myocardial infarction (MI). We genetically modified hEPCs with a TK gene and evaluated cell survival, engraftment, revascularization, and functional improvement in a nude mouse left anterior descending ligation model. hEPCs were manipulated to overexpress the TK gene. In vitro, the antiapoptotic and paracrine effects were assessed under oxidative stress. TK protects hEPCs from oxidative stress-induced apoptosis via inhibition of activation of caspase-3 and -9, induction of Akt phosphorylation, and secretion of vascular endothelial growth factor. In vivo, the Ad.hTK-hEPCs were transplanted after MI via intracardiac injection. The surviving cells were tracked after transplantation using near-infrared optical imaging. Left ventricular (LV) function was evaluated by transthoracic echocardiography. Capillary density was quantified using immunohistochemical staining. Engrafted Ad.hTK-hEPCs exhibited advanced protection against ischemia by increasing LV ejection fraction. Compared with Ad.Null-hEPCs, transplantation with Ad.hTK-hEPCs significantly decreased cardiomyocyte apoptosis in association with increased retention of transplanted EPCs in the myocardium. Capillary density and arteriolar density in the infarct border zone was significantly higher in Ad.hTK-hEPC-transplanted mice than in Ad.Null-hEPC-treated mice. Transplanted hEPCs were clearly incorporated into CD31(+) capillaries. These results indicate that implantation of kallikrein-modified EPCs in the heart provides advanced benefits in protection against ischemia-induced MI by enhanced angiogenesis and reducing apoptosis.

Monitoring apoptosis of TK-GFP-expressing ACC-M cells induced by ACV using FRET technique

NASA Astrophysics Data System (ADS)

Xiong, Tao; Zhang, Zhihong; Lin, Juqiang; Yang, Jie; Zeng, Shaoqun; Luo, Qingming

2006-05-01

Apoptosis is an evolutionary conserved cellular process that plays an important role during development, but it is also involved in tissue homeostasis and in many diseases. To study the characteristics of suicide gene system of the herpes simplex virus thymidine kinase (HSV-tk) gene in tumor cells and explore the apoptosis phenomena in this system and its effect on the human adenoid cystic carcinoma line ACC-M cell, we detected apoptosis of CD3- (ECFP-CRS-DsRed) and TK-GFP-expressing ACC-M (ACC-M-TK-GFP-CD3) cells induced by acyclovir (ACV) using fluorescence resonance energy transfer (FRET) technique. CD3 is a FRET-based indicator for activity of caspase-3, which is composed of an enhanced cyan fluorescent protein, a caspase-3 sensitive linker, and a red fluorescent protein from Discosoma with efficient maturation property. FRET from ECFP to DsRed could be detected in normal ACC-M-TK-GFP-CD3 cells, and the FRET efficient was remarkably decreased and then disappeared during the cells apoptosis induced by ACV. It was due to the activated caspase-3 cleaved the CD3 fusion protein. In this study, the results suggested that the ACV-induced apoptosis of ACC-M-TK-GFP-CD3 cells was through caspase-3 pathway.

Monitoring apoptosis of TK-GFP-expressing ACC-M cells induced by ACV using FRET technique

NASA Astrophysics Data System (ADS)

Xiong, Tao; Zhang, Zhihong; Lin, Juqiang; Yang, Jie; Zeng, Shaoqun; Luo, Qingming

2006-09-01

Apoptosis is an evolutionary conserved cellular process that plays an important role during development, but it is also involved in tissue homeostasis and in many diseases. To study the characteristics of suicide gene system of the herpes simplex virus thymidine kinase (HSV-tk) gene in tumor cells and explore the apoptosis phenomena in this system and its effect on the human adenoid cystic carcinoma line ACC-M cell, we detected apoptosis of CD3- (ECFP-CRS-DsRed) and TK-GFP-expressing ACC-M (ACC-M-TK-GFP-CD3) cells induced by acyclovir (ACV) using fluorescence resonance energy transfer (FRET) technique. CD3 is a FRET-based indicator for activity of caspase-3, which is composed of an enhanced cyan fluorescent protein, a caspase-3 sensitive linker, and a red fluorescent protein from Discosoma with efficient maturation property. FRET from ECFP to DsRed could be detected in normal ACC-M-TK-GFP-CD3 cells, and the FRET efficient was remarkably decreased and then disappeared during the cells apoptosis induced by ACV. It was due to the activated caspase-3 cleaved the CD3 fusion protein. In this study, the results suggested that the AVC-induced apoptosis of ACC-M-TK-GFP-CD3 cells was through caspase-3 pathway.

[Killing effects of PWZL plasmid-mediated double suicide gene on human lens epithelium cells].

PubMed

Yan, Xiao-ran; Wu, Hong; Yu, Hai-tao; Wang, Xiu; Zhang, Yu

2008-04-01

To investigate the killing efficiency of PWZL plasmid-mediated herpes simplex virus-thymidine kinase (TK) and E. coli cytosine deaminase (CD) on human lens epithelium cells followed by the treatment of prodrugs. PWZL plasmid was used as a vehicle, to transduce double suicide genes into the human lens epithelium in vitro, then the cells were treated with fluorocytosine (5-FC) and/or ganciclovir (GCV) at different concentrations. The cell growth of the lens epithelium cells was observed by light microscope. MTT analysis was used to estimate the cell survival rate and the bystander effect was analyzed simultaneously. The significance of difference between each group was treated by statistical tests. The CD and TK gene could be joined into PWZL plasmid successfully, and did not have any special effect on normal cells. There was no significant difference in cell viability between CD-TK transfected cells and control cells. Cell viability in cells treated with prodrugs was decreased in a time-dependent manner. At the end of the experiment, cell viability was lowest in GCV 10 mg/L +5-FC 60 mg/L group, GCV 10 mg/L + 5-FC 100 mg/L group and GCV 100 mg/L + 5-FC 100 mg/L group. There were no significant differences between these three groups (X2 = 1.25 , P > 0.01). Analysis of bystander effect indicated that the cell viability in GCV 100 mg/L + 5-FC 100 mg/L group and GCV 10 mg/L +5-FC 60 mg/L group was significantly lower than that in the controls (t = 10.26, 13.16; P < 0.01). PWZL plasmid can transfect the CD and TK genes into lens epithelium cells successfully and efficiently. CD and TK genes can be expressed steadily. Transfection of double suicide gene reduces the dosage of prodrugs required for killing cells. The combination of 5-FC with GCV shows the greatest killing effect and also has the bystander effect.

Gene expression deregulation in postnatal skeletal muscle of TK2 deficient mice reveals a lower pool of proliferating myogenic progenitor cells.

PubMed

Paredes, João A; Zhou, Xiaoshan; Höglund, Stefan; Karlsson, Anna

2013-01-01

Loss of thymidine kinase 2 (TK2) causes a heterogeneous myopathic form of mitochondrial DNA (mtDNA) depletion syndrome (MDS) in humans that predominantly affects skeletal muscle tissue. In mice, TK2 deficiency also affects several tissues in addition to skeletal muscle, including brain, heart, adipose tissue, kidneys and causes death about 3 weeks after birth. We analysed skeletal muscle and heart muscle tissues of Tk2 knockout mice at postnatal development phase and observed that TK2 deficient pups grew slower and their skeletal muscles appeared significantly underdeveloped, whereas heart was close to normal in size. Both tissues showed mtDNA depletion and mitochondria with altered ultrastructure, as revealed by transmission electron microscopy. Gene expression microarray analysis showed a strong down-regulation of genes involved in cell cycle and cell proliferation in both tissues, suggesting a lower pool of undifferentiated proliferating cells. Analysis of isolated primary myoblasts from Tk2 knockout mice showed slow proliferation, less ability to differentiate and signs of premature senescence, even in absence of mtDNA depletion. Our data demonstrate that TK2 deficiency disturbs myogenic progenitor cells function in postnatal skeletal muscle and we propose this as one of the causes of underdeveloped phenotype and myopathic characteristic of the TK2 deficient mice, in addition to the progressive mtDNA depletion, mitochondrial damage and respiratory chain deficiency in post-mitotic differentiated tissue.

Gene Expression Deregulation in Postnatal Skeletal Muscle of TK2 Deficient Mice Reveals a Lower Pool of Proliferating Myogenic Progenitor Cells

PubMed Central

Paredes, João A.; Zhou, Xiaoshan; Höglund, Stefan; Karlsson, Anna

2013-01-01

Loss of thymidine kinase 2 (TK2) causes a heterogeneous myopathic form of mitochondrial DNA (mtDNA) depletion syndrome (MDS) in humans that predominantly affects skeletal muscle tissue. In mice, TK2 deficiency also affects several tissues in addition to skeletal muscle, including brain, heart, adipose tissue, kidneys and causes death about 3 weeks after birth. We analysed skeletal muscle and heart muscle tissues of Tk2 knockout mice at postnatal development phase and observed that TK2 deficient pups grew slower and their skeletal muscles appeared significantly underdeveloped, whereas heart was close to normal in size. Both tissues showed mtDNA depletion and mitochondria with altered ultrastructure, as revealed by transmission electron microscopy. Gene expression microarray analysis showed a strong down-regulation of genes involved in cell cycle and cell proliferation in both tissues, suggesting a lower pool of undifferentiated proliferating cells. Analysis of isolated primary myoblasts from Tk2 knockout mice showed slow proliferation, less ability to differentiate and signs of premature senescence, even in absence of mtDNA depletion. Our data demonstrate that TK2 deficiency disturbs myogenic progenitor cells function in postnatal skeletal muscle and we propose this as one of the causes of underdeveloped phenotype and myopathic characteristic of the TK2 deficient mice, in addition to the progressive mtDNA depletion, mitochondrial damage and respiratory chain deficiency in post-mitotic differentiated tissue. PMID:23341978

Adenovirus-mediated suicide gene therapy under the control of Cox-2 promoter for colorectal cancer.

PubMed

Wang, Zhao-Xia; Bian, Hai-Bo; Yang, Jing-Song; De, Wei; Ji, Xiao-Hui

2009-08-01

Colorectal cancer is a most frequent type of gastrointestinal tract cancers. The prognosis of patients with colorectal cancer remains poor despite intensive interventions. Tumor specific promoter-directed gene therapy and adenoviral technology can be promising strategies for such advanced disease. This study was conducted to explore the possible therapeutic approach of Cox-2 promoter-directed suicide gene therapy with herpes simplex virus thymidine kinase (HSV-tk) in combination with adenoviral technology for advanced colorectal cancer. Firstly, the activity of Cox-2 promoter was assessed by dual luciferase and enhanced green fluorescent protein reporter gene assays in colorectal cancer cell lines and normal human intestinal epithelial cell line. Then, the expression of coxsackievirus and adenovirus receptor (CAR) was detected in colorectal cancer cell lines. The Cox-2 promoter-directed HSV-tk/ganciclovir (GCV) system mediated by adenovirus (Ad-Cp-TK) was developed (Ad-CMVp-TK, Ad-null and no Ad as controls). In vitro cytoxicity, colony formation and apoptosis assays were performed using Ad-Cp-TK. An animal study was carried out in which BALB/C nude mice bearing tumors were treated with Ad-Cp-TK and GCV treatments. Results showed that Cox-2 promoter possessed high transcriptional activity in a tumor-specific manner. All colorectal cancer cells were detected CAR-positive. In vitro cytotoxic and colony formation assays showed that colorectal cancer cells infected with Ad-Cp-TK became more sensitive to GCV but the sensitivity of normal cells infected with Ad-Cp-TK to GCV were not altered. Moreover, the Ad-Cp-TK system combined with GCV treatment could significantly induce apoptosis of colorectal cancer cells but not normal intestinal epithelial cells. Furthermore, this system also significantly inhibited the growth of subcutaneous tumors and prolonged survival of mice. Thus, adenovirus primary receptor was positive in colorectal cancer cells and adenovirus-mediated suicide gene therapy under the control of Cox-2 promoter could provide a promising treatment modality for advanced colorectal cancer with tumor specificity.

Human plasma metabolic profiles of benzydamine, a flavin-containing monooxygenase probe substrate, simulated with pharmacokinetic data from control and humanized-liver mice.

PubMed

Yamazaki-Nishioka, Miho; Shimizu, Makiko; Suemizu, Hiroshi; Nishiwaki, Megumi; Mitsui, Marina; Yamazaki, Hiroshi

2018-02-01

1. Benzydamine is used clinically as a nonsteroidal anti-inflammatory drug in oral rinses and is employed in preclinical research as a flavin-containing monooxygenase (FMO) probe substrate. In this study, plasma concentrations of benzydamine and its primary N-oxide and N-demethylated metabolites were investigated in control TK-NOG mice, in humanized-liver mice, and in mice whose liver cells had been ablated with ganciclovir. 2. Following oral administration of benzydamine (10 mg/kg) in humanized-liver TK-NOG mice, plasma concentrations of benzydamine N-oxide were slightly higher than those of demethyl benzydamine. In contrast, in control and ganciclovir-treated TK-NOG mice, concentrations of demethyl benzydamine were slightly higher than those of benzydamine N-oxide. 3. Simulations of human plasma concentrations of benzydamine and its N-oxide were achieved using simplified physiologically based pharmacokinetic models based on data from control TK-NOG mice and from reported benzydamine concentrations after low-dose administration in humans. Estimated clearance rates based on data from humanized-liver and ganciclovir-treated TK-NOG mice were two orders magnitude high. 4. The pharmacokinetic profiles of benzydamine were different for control and humanized-liver TK-NOG mice. Humanized-liver mice are generally accepted human models; however, drug oxidation in mouse kidney might need to be considered when probe substrates undergo FMO-dependent drug oxidation in mouse liver and kidney.

Trichosanthes kirilowii Exerts Androgenic Activity via Regulation of PSA and KLK2 in 22Rv1 Prostate Cancer Cells.

PubMed

Jeong, Soo-Jin; Choi, Ji-Yoon; Dong, Mi-Sook; Seo, Chang-Seob; Shin, Hyeun-Kyoo

2017-01-01

The androgen comprises a group of hormones that play roles in male reproductive activity as well as personal characteristics. We investigated the androgenic activity of various herbal medicines in human prostate cancer 22Rv1 cells. Herbal extracts of Trichosanthes kirilowii (TK), Asarum sieboldii (AS), Sanguisorba officinalis (SO), and Xanthium strumarium (XS) were selected to have androgenic effects based on a preliminary in vitro screening system. TK, AS, SO, and XS enhanced the proliferation of 22Rv1 cells without having cytotoxic effects. All tested herbal extracts increased androgen receptor (AR)-induced transcriptional activity in the absence or presence of dihydrotestosterone (DHT). In an AR-binding assay, TK, but not AS, SO, or XS, produced a significant inhibition of AR binding activity, indicating it has androgenic activity. Additionally, TK treatment positively regulated mRNA expression of the AR-related molecular targets prostate-specific antigen (PSA) and kallikrein 2 (KLK2) compared with untreated control. Taken together, TK-enhanced AR-mediated transcriptional activity might be an attractive candidate drug for treating androgen-related diseases. Trichosantheskirilowii (TK), Asarumsieboldii (AS), Sanguisorbaofficinalis (SO), and Xanthium strumarium (XS) enhanced the proliferation of 22Rv1 cells without having cytotoxic effects.TK, AS, SO, and XS increased androgen receptor (AR)-induced transcriptional activity.TK, but not AS, SO, or XS, produced a significant inhibition against AR-binding activity.TK treatment positively regulated mRNA expression of the AR-related molecular targets prostate-specific antigen and kallikrein 2. Abbreviations used: BPH: benign prostatic hyperplasia; AR: androgen receptor; DHT: dihydrotestosterone; PSA: prostate-specific antigen; TK: Trichosanthes kirilowii; AS: Asarum sieboldii; SO: Sanguisorba officinalis; XS: Xanthium strumarium; ATCC: American Type Culture Collection; FBS: fetal bovine serum; PBS: phosphate-buffered saline; SD: standard deviation; ARE: androgenresponsive element; KLK: kallikrein.

Effects of pre-radiation exposure to LLLT of normal and malignant cells.

PubMed

Barasch, Andrei; Raber-Durlacher, Judith; Epstein, Joel B; Carroll, James

2016-06-01

Low-level laser therapy (LLLT) efficacy for the prevention of cancer treatment-induced oral mucositis (OM) has been amply described. However, potential protection of malignant cells remains a legitimate concern for clinicians. We tested LLLT-induced protection from ionizing radiation killing in both malignant and normal cells. We treated six groups each of normal human lymphoblasts (TK6) and human leukemia cells (HL60) with He-Ne LLLT (632.8 nm, 35 mW, CW, 1 cm(2), 35 mW/cm(2) for 3-343 s, 0.1-12 J/cm(2)) prior to exposure to ionizing radiation (IR). Cells were then incubated and counted daily to determine their survival. Optimization of IR dose and incubation time was established prior to testing the effect of LLLT. Growth curves for both cell lines showed significant declines after exposure to 50-200 cGy IR when compared to controls. Pre-radiation exposure to LLLT (4.0 J/cm(2)) followed by 1-h incubation blocked this decline in TK6 but not in HL60 cells. The latter cells were sensitized to the killing effects of IR in a dose-dependent manner. This study shows that pre-IR LLLT treatment results in a differential response of normal vs. malignant cells, suggesting that LLLT does not confer protection and may even sensitize cancer cells to IR killing.

Caspase-3-independent pathways proceeding in bystander effect of HSV-tk/GCV system

NASA Astrophysics Data System (ADS)

Lin, Juqiang; Ma, Yan; Zeng, Shaoqun; Zhang, Zhihong

2008-02-01

HSV-tk/GCV system, which is the virus-directed enzyme/prodrug therapy of herpes simplex virus (HSV) thymidine kinase (tk) gene / the anti-viral reagent ganciclovir (GCV), is one of the promising approaches in the rapidly growing area of gene therapy. As gene therapy of cancer such as suicide gene therapy has entered the clinic, another therapy effect which is called 'bystander effect' was reported. Bystander effect can lead to killing of non-transduced tumor cells in the immediate vicinity of GCV-treated HSV-TK-positive cells. Now the magnitude of 'bystander effect' is an essential factor for this anti-tumor approach in vivo. However, the mechanism which HSV-tk/ACV brings "bystander effect" is poorly understood. In this study, we monitor the activation of caspase-3 in HSV-tk/GCV system by a FRET probe CD3, a FRET-based indicator for activity of caspase3, which is composed of an enhanced cyan fluorescent protein, a caspase-sensitive linker, and a red fluorescent protein from Discosoma with efficient maturation property. Through application of CD3 we have visualized the activation of caspase-3 in tk gene positive human adenoid cystic carcinoma (ACC-M) cells but not in bystander effect of HSV-tk/GCV system induced by GCV. This finding provides needed information for understanding the mechanisms by which suicide gene approaches actually kill cancer cells, and may prove to be helpful for the clinical treatment of cancers.

Two structurally distinct inhibitors of glycogen synthase kinase 3 induced centromere positive micronuclei in human lymphoblastoid TK6 cells.

PubMed

Mishima, Masayuki; Tanaka, Kenji; Takeiri, Akira; Harada, Asako; Kubo, Chiyomi; Sone, Sachiko; Nishimura, Yoshikazu; Tachibana, Yukako; Okazaki, Makoto

2008-08-25

Glycogen synthase kinase 3 (GSK3) is an attractive novel pharmacological target. Inhibition of GSK3 is recently regarded as one of the viable approaches to therapy for Alzheimer's disease, cancer, diabetes mellitus, osteoporosis, and bipolar mood disorder. Here, we have investigated the aneugenic potential of two potent and highly specific inhibitors of GSK3 by using an in vitro micronucleus test with human lymphoblastoid TK6 cells. One inhibitor was a newly synthesized maleimide derivative and the other was a previously known aminopyrimidine derivative. Both compounds elicited statistically significant and concentration-dependent increases in micronucleated cells. One hundred micronuclei (MN) of each were analyzed using centromeric DNA staining with fluorescence in situ hybridization. Both the two structurally distinct compounds induced centromere-positive micronuclei (CMN). Calculated from the frequency of MN cells and the percentage of CMN, CMN cell incidence after treatment with the maleimide compound at 1.2 microM, 2.4 microM, and 4.8 microM was 11.6, 27.7, and 56.3 per 1000 cells, respectively; the negative control was 4.5. CMN cell incidence after the treatment with the aminopyrimidine compound at 1.8 microM, 3.6 microM, and 5.4 microM was 6.7, 9.8 and 17.2 per 1000 cells, respectively. Both compounds exhibited concentration-dependent increase in the number of mitotic cells. The frequency of CMN cells correlated well with mitotic cell incidence after treatment with either compound. Furthermore, both inhibitors induced abnormal mitotic cells with asymmetric mitotic spindles and lagging anaphase chromosomes. These results lend further support to the hypothesis that the inhibition of GSK3 activity affects microtubule function and exhibits an aneugenic mode of action.

Expression of a suicidal gene under control of the human secreted protein acidic and rich in cysteine (SPARC) promoter in tumor or stromal cells led to the inhibition of tumor cell growth

PubMed Central

Lopez, María V.; Blanco, Patricia; Viale, Diego L.; Cafferata, Eduardo G.; Carbone, Cecilia; Gould, David; Chernajovsky, Yuti; Podhajcer, Osvaldo L.

2009-01-01

The successful use of transcriptional targeting for cancer therapy depends on the activity of a given promoter inside the malignant cell. Because solid human tumors evolve as a “cross-talk” between the different cell types within the tumor, we hypothesized that targeting the entire tumor mass might have better therapeutic effect. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein overexpressed in different human cancers malignant melanomas both in the malignant cells compartment as in the stromal one (fibroblasts and endothelial cells). We have shown that expression of the herpes simplex virus-thymidine kinase (TK) gene driven by the SPARC promoter in combination with ganciclovir inhibited human melanoma cell growth in monolayer as well as in multicellular spheroids. This inhibitory effect was observed both in homotypic spheroids composed of melanoma cells alone as well as in spheroids made of melanoma cells and stromal cells. Expression of the TK gene was also efficient to inhibit the in vivo tumor growth of established melanomas when TK was expressed either by the malignant cells themselves or by coadministered endothelial cells. Our data suggest that the use of therapeutic genes driven by SPARC promoter could be a valuable strategy for cancer therapy aiming to target all the cellular components of the tumor mass. PMID:17041094

Enhancement of in vivo Antitumor Activity of a Novel Antimitotic 1‐Phenylpropenone Derivative, AM‐132, by Tumor Necrosis Factor‐cc or Interleukin‐6

PubMed Central

Tatsumi, Yasuaki; Arioka, Hitoshi; Ikeda, Shun‐ichi; Fukumoto, Hisao; Miyamoto, Ken‐ichi; Fukuoka, Kazuya; Ohe, Yuichiro; Saijo, Nagahiro

2001-01-01

TK5048 and its derivatives, AM‐132, AM‐138, and AM‐97, are recently developed antimitotic (AM) compounds. These 1‐phenylpropenone derivatives induce cell cycle arrest at the G2/M phase of the cell cycle. TK5048 inhibited tubulin polymerization in human lung cancer PC‐14 cells in a concentration‐dependent manner. In a polymerization assay using bovine brain tubulin, AM‐132 and AM‐138 were quite strong, AM‐97 was moderately strong, and TK5048 was a relatively weak inhibitor of tubulin polymerization. A murine leukemia cell line resistant to a sulfonamide antimitotic agent, E7010, which binds to colchicine‐binding sites on tubulin, was cross‐resistant to the in vitro growth‐inhibitory effect of AM compounds. Inhibition of tubulin polymerization is therefore one of the mechanisms of action of these AM compounds against tumor cells. To profile the antitumor effect of AM compounds, the in vivo antitumor effect of AM‐132 was evaluated against cytokine‐secreting Lewis lung carcinoma (LLC). Tumor‐bearing mice were treated with intravenous AM‐132 using three different treatment schedules. LLC tumors expressing tumor necrosis factor‐a (TNF‐α), granulocyte macrophage colony‐stimulating factor (GM‐CSF), or interleukin (TL)‐6 were very sensitive to AM‐132. In particular, LLC tumors expressing IL‐6 were markedly reduced by AM‐132 treatment, and showed coloring of the tumor surface and unusual hemorrhagic necrosis. These results suggest a combined effect of AM‐132 and cytokines on the blood supply to tumors. PMID:11473728

Enhancement of in vivo antitumor activity of a novel antimitotic 1-phenylpropenone derivative, AM-132, by tumor necrosis factor-alpha or interleukin-6.

PubMed

Tatsumi, Y; Arioka, H; Ikeda, S; Fukumoto, H; Miyamoto, K; Fukuoka, K; Ohe, Y; Saijo, N; Nishio, K

2001-07-01

TK5048 and its derivatives, AM-132, AM-138, and AM-97, are recently developed antimitotic (AM) compounds. These 1-phenylpropenone derivatives induce cell cycle arrest at the G2 / M phase of the cell cycle. TK5048 inhibited tubulin polymerization in human lung cancer PC-14 cells in a concentration-dependent manner. In a polymerization assay using bovine brain tubulin, AM-132 and AM-138 were quite strong, AM-97 was moderately strong, and TK5048 was a relatively weak inhibitor of tubulin polymerization. A murine leukemia cell line resistant to a sulfonamide antimitotic agent, E7010, which binds to colchicine-binding sites on tubulin, was cross-resistant to the in vitro growth-inhibitory effect of AM compounds. Inhibition of tubulin polymerization is therefore one of the mechanisms of action of these AM compounds against tumor cells. To profile the antitumor effect of AM compounds, the in vivo antitumor effect of AM-132 was evaluated against cytokine-secreting Lewis lung carcinoma (LLC). Tumor-bearing mice were treated with intravenous AM-132 using three different treatment schedules. LLC tumors expressing tumor necrosis factor-alpha (TNF-alpha), granulocyte macrophage colony-stimulating factor (GM-CSF), or interleukin (IL)-6 were very sensitive to AM-132. In particular, LLC tumors expressing IL-6 were markedly reduced by AM-132 treatment, and showed coloring of the tumor surface and unusual hemorrhagic necrosis. These results suggest a combined effect of AM-132 and cytokines on the blood supply to tumors.

A dual function fusion protein of Herpes simplex virus type 1 thymidine kinase and firefly luciferase for noninvasive in vivo imaging of gene therapy in malignant glioma.

PubMed

Söling, Ariane; Theiss, Christian; Jungmichel, Stephanie; Rainov, Nikolai G

2004-08-04

BACKGROUND: Suicide gene therapy employing the prodrug activating system Herpes simplex virus type 1 thymidine kinase (HSV-TK)/ ganciclovir (GCV) has proven to be effective in killing experimental brain tumors. In contrast, glioma patients treated with HSV-TK/ GCV did not show significant treatment benefit, most likely due to insufficient transgene delivery to tumor cells. Therefore, this study aimed at developing a strategy for real-time noninvasive in vivo monitoring of the activity of a therapeutic gene in brain tumor cells. METHODS: The HSV-TK gene was fused to the firefly luciferase (Luc) gene and the fusion construct HSV-TK-Luc was expressed in U87MG human malignant glioma cells. Nude mice with subcutaneous gliomas stably expressing HSV-TK-Luc were subjected to GCV treatment and tumor response to therapy was monitored in vivo by serial bioluminescence imaging. Bioluminescent signals over time were compared with tumor volumes determined by caliper. RESULTS: Transient and stable expression of the HSV-TK-Luc fusion protein in U87MG glioma cells demonstrated close correlation of both enzyme activities. Serial optical imaging of tumor bearing mice detected in all cases GCV induced death of tumor cells expressing the fusion protein and proved that bioluminescence can be reliably used for repetitive and noninvasive quantification of HSV-TK/ GCV mediated cell kill in vivo. CONCLUSION: This approach may represent a valuable tool for the in vivo evaluation of gene therapy strategies for treatment of malignant disease.

Synthesis and biological evaluation of 5-substituted derivatives of the potent antiherpes agent (north)-methanocarbathymine.

PubMed

Russ, Pamela; Schelling, Pierre; Scapozza, Leonardo; Folkers, Gerd; Clercq, Erik De; Marquez, Victor E

2003-11-06

The conformationally locked nucleoside, (north)-methanocarbathymine (1a), is a potent and selective anti-herpes agent effective against herpes simplex type 1 (HSV1) and type 2 (HSV2) viruses. Hereby, we report on the synthesis and biological evaluation of a small set of 5-substituted pyrimidine nucleosides belonging to the same class of bicyclo[3.1.0]hexane nucleosides. Both the 5-bromovinyl (4) and the 5-bromo analogue (3) appeared to be exclusive substrates of HSV1 thymidine kinase (TK), contrasting with the 5-iodo analogue (2), which was significantly phosphorylated by the human cytosolic TK. The binding affinity constant and catalytic turnover for HSV1 TK were measured to assess the influence of the substitution on these parameters. In the plaque reduction and cytotoxicity assays, the 5-bromo analogue (3) showed good activity against HSV1 and HSV2 with less general toxicity than 1a. Against varicella-zoster virus (VZV), the north-locked 5-bromovinyl analogue (4) proved to be as potent as its conformationally unlocked 2'-deoxyriboside equivalent BVDU. The three compounds were also tested in vitro as prodrugs used in a gene therapy context on three osteosarcoma cell lines, either deficient in TK (TK(-)), nontransduced, or stably transduced with HSV1 TK. The 5-iodo compound (2, CC(50) 25 +/- 7 microM) was more efficient than ganciclovir (GCV, CC(50) 75 +/- 35 microM) in inhibiting growth of HSV1-TK transfected cells and less inhibitory than GCV toward TK(-) cells, whereas compound 3 inhibited transfected and nontransfected cell lines in a relatively similar dose-dependent manner.

Selective elimination of long INterspersed element-1 expressing tumour cells by targeted expression of the HSV-TK suicide gene

PubMed Central

Chendeb, Mariam; Schneider, Robert; Davidson, Irwin; Fadloun, Anas

2017-01-01

In gene therapy, effective and selective suicide gene expression is crucial. We exploited the endogenous Long INterspersed Element-1 (L1) machinery often reactivated in human cancers to integrate the Herpes Simplex Virus Thymidine Kinase (HSV-TK) suicide gene selectively into the genome of cancer cells. We developed a plasmid-based system directing HSV-TK expression only when reverse transcribed and integrated in the host genome via the endogenous L1 ORF1/2 proteins and an Alu element. Delivery of these new constructs into cells followed by Ganciclovir (GCV) treatment selectively induced mortality of L1 ORF1/2 protein expressing cancer cells, but had no effect on primary cells that do not express L1 ORF1/2. This novel strategy for selective targeting of tumour cells provides high tolerability as the HSV-TK gene cannot be expressed without reverse transcription and integration, and high selectivity as these processes take place only in cancer cells expressing high levels of functional L1 ORF1/2. PMID:28415677

Thymidine kinase 1 combined with CEA, CYFRA21-1 and NSE improved its diagnostic value for lung cancer.

PubMed

Jiang, Z F; Wang, M; Xu, J L

2018-02-01

Thymidine kinase 1 (TK1) is a tumor biomarker in human malignancies. The purpose of this study was to evaluate the diagnostic efficiency of this marker for lung cancer using the combined analysis of carcinoembryonic antigen (CEA), cytokeratin-19 fragment (CYFRA21-1), neuron specific enolase (NSE) and TK1. From 2013 to 2014, 147 patients with lung cancer and 228 patients with lung benign diseases who were admitted to our hospital were reviewed. Peripheral blood samples were collected for the detection of TK1, CEA, CYFRA21-1 and NSE. The diagnostic value of each marker was analyzed using receiver operating characteristic (ROC) curves and logistic regression equations. The serum levels of TK1, CEA, CYFRA21-1 and NSE were significantly higher than those in patients with lung benign diseases (all P<0.05). The TK1 concentration was dependent on TNM stage (P=0.005). The ROC curve analyses showed that the diagnostic value of TK1 combined with CEA, CYFRA21-1 and NSE in lung cancer was significantly higher than that of each biomarker alone (all P<0.0001). In addition, TK1 combined with CEA, CYFRA21-1, or NSE could also improve the diagnosis of the squamous cell carcinoma, adenocarcinoma and small cell lung cancer subtypes, respectively. The combined detection of TK1 and the other three markers significantly improved the diagnosis of lung cancer. Furthermore, the detection of TK1 combined with that of CYFRA21-1, CEA or NSE increased the diagnostic value of TK1 for lung squamous cell carcinoma, adenocarcinoma and SCLC, respectively. Copyright © 2017 Elsevier Inc. All rights reserved.

Deletion of the thymidine kinase gene induces complete attenuation of the Georgia isolate of African swine fever virus.

PubMed

Sanford, B; Holinka, L G; O'Donnell, V; Krug, P W; Carlson, J; Alfano, M; Carrillo, C; Wu, Ping; Lowe, Andre; Risatti, G R; Gladue, D P; Borca, M V

2016-02-02

African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal viral disease of domestic pigs. There are no vaccines to control Africa swine fever (ASF). Experimental vaccines have been developed using genetically modified live attenuated ASFVs obtained by specifically deleting virus genes involved in virulence, including the thymidine kinase (TK) gene. TK has been shown to be involved in the virulence of several viruses, including ASFV. Here we report the construction of a recombinant virus (ASFV-G/V-ΔTK) obtained by deleting the TK gene in a virulent strain of ASFV Georgia adapted to replicate in Vero cells (ASFV-G/VP30). ASFV-G/P-ΔTK demonstrated decreased replication both in primary swine macrophage cell cultures and in Vero cells compared with ASFV-G/VP30. In vivo, intramuscular administration of up to 10(6) TCID50 of ASFV-G/V-ΔTK does not result in ASF disease. However, these animals are not protected when challenged with the virulent parental Georgia strain. Published by Elsevier B.V.

Cadmium chloride, benzo[a]pyrene and cyclophosphamide tested in the in vitro mammalian cell micronucleus test (MNvit) in the human lymphoblastoid cell line TK6 at Covance laboratories, Harrogate UK in support of OECD draft Test Guideline 487.

PubMed

Fowler, Paul; Whitwell, James; Jeffrey, Laura; Young, Jamie; Smith, Katie; Kirkland, David

2010-10-29

The following genotoxic chemicals were tested in the in vitro micronucleus assay, at Covance Laboratories, Harrogate, UK in the human lymphoblastoid cell line TK6. Cadmium chloride (an inorganic carcinogen), benzo[a]pyrene (a polycyclic aromatic hydrocarbon requiring metabolic activation) and cyclophosphamide (an alkylating agent requiring metabolic activation) were treated with and without cytokinesis block (by addition of cytochalasin B). This work formed part of a collaborative evaluation of the toxicity measures recommended in the draft OECD Test Guideline 487 for the in vitro micronucleus test. The toxicity measures used, capable of detecting both cytostasis and cell death, were relative population doubling, relative increase in cell counts and relative cell counts for treatments in the absence of cytokinesis block, and replication index or cytokinesis blocked proliferation index in the presence of cytokinesis block. All of the chemicals tested gave significant increases in the percentage of micronucleated cells with and without cytokinesis block at concentrations giving approximately 60% toxicity (cytostasis and cell death) or less by all of the toxicity measures used. The outcomes from this series of tests support the use of relative increase in cell counts and relative population doubling, as well as relative cell counts, as appropriate measures of cytotoxicity for the non-cytokinesis blocked in the in vitro micronucleus assay. Copyright © 2010 Elsevier B.V. All rights reserved.

Trichosanthes kirilowii Exerts Androgenic Activity via Regulation of PSA and KLK2 in 22Rv1 Prostate Cancer Cells

PubMed Central

Jeong, Soo-Jin; Choi, Ji-Yoon; Dong, Mi-Sook; Seo, Chang-Seob; Shin, Hyeun-Kyoo

2017-01-01

Background: The androgen comprises a group of hormones that play roles in male reproductive activity as well as personal characteristics. Objective: We investigated the androgenic activity of various herbal medicines in human prostate cancer 22Rv1 cells. Materials and Methods: Herbal extracts of Trichosanthes kirilowii (TK), Asarum sieboldii (AS), Sanguisorba officinalis (SO), and Xanthium strumarium (XS) were selected to have androgenic effects based on a preliminary in vitro screening system. Results: TK, AS, SO, and XS enhanced the proliferation of 22Rv1 cells without having cytotoxic effects. All tested herbal extracts increased androgen receptor (AR)-induced transcriptional activity in the absence or presence of dihydrotestosterone (DHT). In an AR-binding assay, TK, but not AS, SO, or XS, produced a significant inhibition of AR binding activity, indicating it has androgenic activity. Additionally, TK treatment positively regulated mRNA expression of the AR-related molecular targets prostate-specific antigen (PSA) and kallikrein 2 (KLK2) compared with untreated control. Conclusion: Taken together, TK-enhanced AR-mediated transcriptional activity might be an attractive candidate drug for treating androgen-related diseases. SUMMARY Trichosantheskirilowii (TK), Asarumsieboldii (AS), Sanguisorbaofficinalis (SO), and Xanthium strumarium (XS) enhanced the proliferation of 22Rv1 cells without having cytotoxic effects.TK, AS, SO, and XS increased androgen receptor (AR)-induced transcriptional activity.TK, but not AS, SO, or XS, produced a significant inhibition against AR-binding activity.TK treatment positively regulated mRNA expression of the AR-related molecular targets prostate-specific antigen and kallikrein 2. Abbreviations used: BPH: benign prostatic hyperplasia; AR: androgen receptor; DHT: dihydrotestosterone; PSA: prostate-specific antigen; TK: Trichosanthes kirilowii; AS: Asarum sieboldii; SO: Sanguisorba officinalis; XS: Xanthium strumarium; ATCC: American Type Culture Collection; FBS: fetal bovine serum; PBS: phosphate-buffered saline; SD: standard deviation; ARE: androgenresponsive element; KLK: kallikrein PMID:28216900

Selective enhancement of radiation response of herpes simplex virus thymidine kinase transduced 9L gliosarcoma cells in vitro and in vivo by antiviral agents

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Jae Ho; Kim, Sang Hie; Kolozsvary, A.

1995-11-01

The purpose of this investigation was to demonstrate in a well-characterized tumor model that the radiosensitivity of tumor cells transduced with a herpes simplex virus thymidine kinase gene (HS-tk) would be selectively enhanced by antiviral agents. Rat 9L gliosarcoma cells transduced with a retroviral vector containing an HS-tk gene, 9L-tk cells were exposed to various doses or irradiation under either in vitro or in vivo conditions. The radiation sensitizing potential of two antiviral drugs, bromovinyl deoxyuridine (BVdU) and dihydroxymethyl ethyl methyl guanine (acyclovir), was evaluated in vitro. The radiosensitizing ability of BVdU was also evaluated with a 9L-tk tumor growingmore » in the rat brain. Tumors growing in the right hemisphere of rat brains were irradiated stereotactically with single-dose irradiation. The radiation response of 9L-tk cells was selectively enhanced by antiviral agents relative to nontransduced cells. In the cell culture, when a 24-h drug exposure (20 {mu}g/ml) preceded radiation, the sensitizer enhancement ratio (SER) for BVdU and acyclovir was 1.4 {plus_minus} 0.1 and 1.3 {plus_minus} 0.1, respectively. Exposure of cells to 10 {mu}g/ml acyclovir for two 24-h periods both pre- and postirradiation resulted in a SER of 1.6 {plus_minus} 0.1. In vivo, a significant increase in median survival time of rats with 9L-tk tumors was found when BVdU was administered prior to single-dose irradiation relative to the survival time of similar rats receiving radiation alone. An antiviral agent can enhance cell killing by radiation with selective action in cells transduced with the herpes simplex virus thymidine kinase gene. The results suggest that the three-pronged therapy of HS-tk gene transduction, systemically administered antiviral drug, and stereotactically targeted radiation therapy will improve the effectiveness of radiation therapy for the treatment of radioresistant tumors. 25 refs., 6 figs.« less

Thymidine kinase 2 deficiency-induced mtDNA depletion in mouse liver leads to defect β-oxidation.

PubMed

Zhou, Xiaoshan; Kannisto, Kristina; Curbo, Sophie; von Döbeln, Ulrika; Hultenby, Kjell; Isetun, Sindra; Gåfvels, Mats; Karlsson, Anna

2013-01-01

Thymidine kinase 2 (TK2) deficiency in humans causes mitochondrial DNA (mtDNA) depletion syndrome. To study the molecular mechanisms underlying the disease and search for treatment options, we previously generated and described a TK2 deficient mouse strain (TK2(-/-)) that progressively loses its mtDNA. The TK2(-/-) mouse model displays symptoms similar to humans harboring TK2 deficient infantile fatal encephalomyopathy. Here, we have studied the TK2(-/-) mouse model to clarify the pathological role of progressive mtDNA depletion in liver for the severe outcome of TK2 deficiency. We observed that a gradual depletion of mtDNA in the liver of the TK2(-/-) mice was accompanied by increasingly hypertrophic mitochondria and accumulation of fat vesicles in the liver cells. The levels of cholesterol and nonesterified fatty acids were elevated and there was accumulation of long chain acylcarnitines in plasma of the TK2(-/-) mice. In mice with hepatic mtDNA levels below 20%, the blood sugar and the ketone levels dropped. These mice also exhibited reduced mitochondrial β-oxidation due to decreased transport of long chain acylcarnitines into the mitochondria. The gradual loss of mtDNA in the liver of the TK2(-/-) mice causes impaired mitochondrial function that leads to defect β-oxidation and, as a result, insufficient production of ketone bodies and glucose. This study provides insight into the mechanism of encephalomyopathy caused by TK2 deficiency-induced mtDNA depletion that may be used to explore novel therapeutic strategies.

Thymidine Kinase 2 Deficiency-Induced mtDNA Depletion in Mouse Liver Leads to Defect β-Oxidation

PubMed Central

von Döbeln, Ulrika; Hultenby, Kjell; Isetun, Sindra; Gåfvels, Mats; Karlsson, Anna

2013-01-01

Thymidine kinase 2 (TK2) deficiency in humans causes mitochondrial DNA (mtDNA) depletion syndrome. To study the molecular mechanisms underlying the disease and search for treatment options, we previously generated and described a TK2 deficient mouse strain (TK2−/−) that progressively loses its mtDNA. The TK2−/− mouse model displays symptoms similar to humans harboring TK2 deficient infantile fatal encephalomyopathy. Here, we have studied the TK2−/− mouse model to clarify the pathological role of progressive mtDNA depletion in liver for the severe outcome of TK2 deficiency. We observed that a gradual depletion of mtDNA in the liver of the TK2−/− mice was accompanied by increasingly hypertrophic mitochondria and accumulation of fat vesicles in the liver cells. The levels of cholesterol and nonesterified fatty acids were elevated and there was accumulation of long chain acylcarnitines in plasma of the TK2−/− mice. In mice with hepatic mtDNA levels below 20%, the blood sugar and the ketone levels dropped. These mice also exhibited reduced mitochondrial β-oxidation due to decreased transport of long chain acylcarnitines into the mitochondria. The gradual loss of mtDNA in the liver of the TK2−/− mice causes impaired mitochondrial function that leads to defect β-oxidation and, as a result, insufficient production of ketone bodies and glucose. This study provides insight into the mechanism of encephalomyopathy caused by TK2 deficiency-induced mtDNA depletion that may be used to explore novel therapeutic strategies. PMID:23505564

Plasmodium falciparum full life cycle and Plasmodium ovale liver stages in humanized mice.

PubMed

Soulard, Valérie; Bosson-Vanga, Henriette; Lorthiois, Audrey; Roucher, Clémentine; Franetich, Jean-François; Zanghi, Gigliola; Bordessoulles, Mallaury; Tefit, Maurel; Thellier, Marc; Morosan, Serban; Le Naour, Gilles; Capron, Frédérique; Suemizu, Hiroshi; Snounou, Georges; Moreno-Sabater, Alicia; Mazier, Dominique

2015-07-24

Experimental studies of Plasmodium parasites that infect humans are restricted by their host specificity. Humanized mice offer a means to overcome this and further provide the opportunity to observe the parasites in vivo. Here we improve on previous protocols to achieve efficient double engraftment of TK-NOG mice by human primary hepatocytes and red blood cells. Thus, we obtain the complete hepatic development of P. falciparum, the transition to the erythrocytic stages, their subsequent multiplication, and the appearance of mature gametocytes over an extended period of observation. Furthermore, using sporozoites derived from two P. ovale-infected patients, we show that human hepatocytes engrafted in TK-NOG mice sustain maturation of the liver stages, and the presence of late-developing schizonts indicate the eventual activation of quiescent parasites. Thus, TK-NOG mice are highly suited for in vivo observations on the Plasmodium species of humans.

Using HSV-TK/GCV suicide gene therapy to inhibit lens epithelial cell proliferation for treatment of posterior capsular opacification

PubMed Central

Jiang, Yong-Xiang; Liu, Tian-Jing; Yang, Jin; Chen, Yan; Fang, Yan-Wen

2011-01-01

Purpose To establish a novel, targeted lentivirus-based HSV-tk (herpes simplex virus thymidine kinase)/GCV (ganciclovir) gene therapy system to inhibit lens epithelial cell proliferation for treatment of posterior capsular opacification (PCO) after cataract surgery. Methods An enhanced Cre recombinase (Cre/loxP) system with a lentiviral vector expressing Cre under the control of the lens-specific promoter LEP503 (Lenti-LEP503-HSVtk-Cre [LTKCRE]) was constructed, as well as another lentiviral vector containing a switching unit. The latter vector contains a stuffer sequence encoding EGFP (Lenti-hPGK-Loxp-EGFP-pA-Loxp-HSVtk [PGFPTK]) with a functional polyadenylation signal between two loxP sites, followed by the herpes simplex virus thymidine kinase (HSV-tk) gene, both under the control of the human posphoglycerate kinase (hPGK) promoter. Expression of the downstream gene (HSV-tk) is activated by co-expression of Cre. Human lens epithelial cells (HLECs) or retinal pigmental epithelial cells (RPECs) were co-infected with LTKCRE and PGFPTK. The inhibitory effects on HLECs and RPECs infected by the enhanced specific lentiviral vector combination at the concentration of 20 µg/ml GCV were assayed and compared. Results The specific gene expression of Cre and HSV-tk in HLECs is activated by the LEP503 promoter. LTKCRE and PGFPTK co-infected HLECs, but not RPECs, expressed high levels of the HSV-tk protein. After 96 h of GCV treatment, the percentage of apoptotic HLECs infected by the enhanced specific lentiviral vector combination was 87.23%, whereas that of apoptotic RPECs was only 10.12%. Electron microscopy showed that GCV induced apoptosis and necrosis of the infected HLECs. Conclusions The enhanced specific lentiviral vector combination selectively and effectively expressed HSV-tk in HLECs. A concentration of 20 µg/ml, GCV is effective against the proliferation of HLECs in vitro. This cell-type-specific gene therapy using a Cre/loxP lentivirus system may be a feasible treatment strategy to prevent PCO. PMID:21283526

Simultaneous imaging of temporal changes of NF-κB activity and viable tumor cells in Huh7/NF-κB-tk-luc2/rfp tumor-bearing mice.

PubMed

Wang, Wei-Hsun; Chiang, I-Tsang; Liu, Yu-Chang; Hsu, Fei-Ting; Chen, Hong-Wen; Chen, Chuan-Lin; Lee, Yi-Jang; Lin, Wuu-Jyh; Hwang, Jeng-Jong

2013-01-01

Few studies have reported that the effect of sorafenib on advanced human hepatocellular carcinoma (HCC) is taking place via the inhibition of NF-κB signal transduction. Here we constructed a human HCC Huh7 stable clone with NF-κB-responsive element to drive dual reporter genes, herpes simplex virus thymidine kinase (tk) and firefly luciferase (luc2), and co-transfected with a third red fluorescent protein (rfp) gene, renamed as Huh7/NF-κB-tk-luc2/rfp cells, and combined with bioluminescent imaging (BLI) and red fluorescent protein imaging (RFPI) to monitor the effect of sorafenib on NF-κB activation and tumor inhibition. The results show that sorafenib could suppress the NF-κB-DNA binding activity, and the expression of downstream effector proteins. Notably, the relative photon fluxes obtained from RFPI and BLI, which represent the viable tumor cells and cells with NF-κB activation, decreased after sorafenib treatment by 50 to 65%, and 87.5 to >90%, respectively, suggesting that NF-κB activation is suppressed in viable HCC cells by sorafenib. Simultaneous molecular imaging of the temporal change of NF-κB activity and of viable cells in the same Huh7/NF-κB-tk-luc2/rfp tumors of the animal may reflect the real status of NF-κB activity and the viable tumor cells at the time of imaging.

The pyrimidine nucleotide carrier PNC1 and mitochondrial trafficking of thymidine phosphates in cultured human cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Franzolin, Elisa; Miazzi, Cristina; Frangini, Miriam

2012-10-15

In cycling cells cytosolic de novo synthesis of deoxynucleotides is the main source of precursors for mitochondrial (mt) DNA synthesis. The transfer of deoxynucleotides across the inner mt membrane requires protein carriers. PNC1, a SLC25 family member, exchanges pyrimidine nucleoside triphosphates in liposomes and its downregulation decreases mtUTP concentration in cultured cells. By an isotope-flow protocol we confirmed transport of uridine nucleotides by PNC1 in intact cultured cells and investigated PNC1 involvement in the mt trafficking of thymidine phosphates. Key features of our approach were the manipulation of PNC1 expression by RNA interference or inducible overexpression, the employment of cellsmore » proficient or deficient for cytosolic thymidine kinase (TK1) to distinguish the direction of flow of thymidine nucleotides across the mt membrane during short pulses with [{sup 3}H]-thymidine, the determination of mtdTTP specific radioactivity to quantitate the rate of mtdTTP export to the cytoplasm. Downregulation of PNC1 in TK1{sup -} cells increased labeled dTTP in mitochondria due to a reduced rate of export. Overexpression of PNC1 in TK1{sup +} cells increased mtdTTP pool size and radioactivity, suggesting an involvement in the import of thymidine phosphates. Thus PNC1 is a component of the network regulating the mtdTTP pool in human cells. -- Highlights: Black-Right-Pointing-Pointer Thymidine phosphates exchange between mitochondria and cytosol in mammalian cells. Black-Right-Pointing-Pointer siRNA-downregulation of PNC1 delays mitochondrial dTTP export in TK1{sup -} cells. Black-Right-Pointing-Pointer PNC1 overexpression accumulates dTTP in mitochondria of TK1{sup +} cells. Black-Right-Pointing-Pointer PNC1 exchanges thymidine nucleotides across the mitochondrial inner membrane. Black-Right-Pointing-Pointer PNC1 participates in the regulation of the mtdTTP pool supporting mtDNA synthesis.« less

The benzene metabolite, hydroquinone and etoposide both induce endoreduplication in human lymphoblastoid TK6 cells

PubMed Central

Ji, Zhiying; Zhang, Luoping; Guo, Weihong; McHale, Cliona M.; Smith, Martyn T.

2009-01-01

Both occupational exposure to the leukemogen benzene and in vitro exposure to its metabolite hydroquinone (HQ) lead to the induction of numerical and structural chromosome changes. Several studies have shown that HQ can form DNA adducts, disrupt microtubule assembly and inhibit DNA topoisomerase II (topo II) activity. As these are potential mechanisms underlying endoreduplication (END), a phenomenon that involves DNA amplification without corresponding cell division, we hypothesized that HQ could cause END. We measured END in the human lymphoblastoid cell line, TK6, treated with HQ (0–20 μM) and etoposide (0–0.2 μM) for 48 h. Etoposide was used as a positive control as it is a topo II poison and established human leukemogen that has previously been shown to induce END in Chinese hamster ovary cells. Both HQ and etoposide significantly induced END in a dose-dependent manner (Ptrend < 0.0001 and Ptrend = 0.0003, respectively). Since END may underlie the acquisition of high chromosome numbers by tumour cells, it may play a role in inducing genomic instability and subsequent carcinogenesis from HQ and etoposide. In order to further explore the cytogenetic effects of HQ and etoposide, we also examined specific structural changes. HQ did not induce translocations of chromosome 11 [t(11;?)] but significantly induced translocations of chromosome 21 [t(21;?)] and structural chromosome aberrations (SCA) (Ptrend = 0.0415 and Ptrend < 0.0001, respectively). Etoposide potently induced all these structural changes (Ptrend < 0.0001). The lack of an effect of HQ on t(11;?) and the reduced ability of HQ to induce t(21;?) and SCA, compared with etoposide, further suggests that HQ acts primarily as a topo II catalytic inhibitor rather than as a topo II poison in intact human cells. PMID:19491217

Pre-clinical development of gene modification of haematopoietic stem cells with chimeric antigen receptors for cancer immunotherapy.

PubMed

Larson, Sarah M; Truscott, Laurel C; Chiou, Tzu-Ting; Patel, Amie; Kao, Roy; Tu, Andy; Tyagi, Tulika; Lu, Xiang; Elashoff, David; De Oliveira, Satiro N

2017-05-04

Patients with refractory or recurrent B-lineage hematologic malignancies have less than 50% of chance of cure despite intensive therapy and innovative approaches are needed. We hypothesize that gene modification of haematopoietic stem cells (HSC) with an anti-CD19 chimeric antigen receptor (CAR) will produce a multi-lineage, persistent immunotherapy against B-lineage malignancies that can be controlled by the HSVsr39TK suicide gene. High-titer third-generation self-inactivating lentiviral constructs were developed to deliver a second-generation CD19-specific CAR and the herpes simplex virus thymidine kinase HSVsr39TK to provide a suicide gene to allow ablation of gene-modified cells if necessary. Human HSC were transduced with such lentiviral vectors and evaluated for function of both CAR and HSVsr39TK. Satisfactory transduction efficiency was achieved; the addition of the suicide gene did not impair CAR expression or antigen-specific cytotoxicity, and determined marked cytotoxicity to ganciclovir. NSG mice transplanted with gene-modified human HSC showed CAR expression not significantly different between transduced cells with or without HSVsr39TK, and expression of anti-CD19 CAR conferred anti-tumor survival advantage. Treatment with ganciclovir led to significant ablation of gene-modified cells in mouse tissues. Haematopoietic stem cell transplantation is frequently part of the standard of care for patients with relapsed and refractory B cell malignancies; following HSC collection, a portion of the cells could be modified to express the CD19-specific CAR and give rise to a persistent, multi-cell lineage, HLA-independent immunotherapy, enhancing the graft-versus-malignancy activity.

Retroviral-mediated gene therapy for the treatment of hepatocellular carcinoma: an innovative approach for cancer therapy.

PubMed Central

Huber, B E; Richards, C A; Krenitsky, T A

1991-01-01

An approach involving retroviral-mediated gene therapy for the treatment of neoplastic disease is described. This therapeutic approach is called "virus-directed enzyme/prodrug therapy" (VDEPT). The VDEPT approach exploits the transcriptional differences between normal and neoplastic cells to achieve selective killing of neoplastic cells. We now describe development of the VDEPT approach for the treatment of hepatocellular carcinoma. Replication-defective, amphotrophic retroviruses were constructed containing a chimeric varicella-zoster virus thymidine kinase (VZV TK) gene that is transcriptionally regulated by either the hepatoma-associated alpha-fetoprotein or liver-associated albumin transcriptional regulatory sequences. Subsequent to retroviral infection, expression of VZV TK was limited to either alpha-fetoprotein- or albumin-positive cells, respectively. VZV TK metabolically activated the nontoxic prodrug 6-methoxypurine arabinonucleoside (araM), ultimately leading to the formation of the cytotoxic anabolite adenine arabinonucleoside triphosphate (araATP). Cells that selectively expressed VZV TK became selectively sensitive to araM due to the VZV TK-dependent anabolism of araM to araATP. Hence, these retroviral-delivered chimeric genes generated tissue-specific expression of VZV TK, tissue-specific anabolism of araM to araATP, and tissue-specific cytotoxicity due to araM exposure. By utilizing such retroviral vectors, araM was anabolized to araATP in hepatoma cells, producing a selective cytotoxic effect. Images PMID:1654555

The human phosphotyrosine signaling network: Evolution and hotspots of hijacking in cancer

PubMed Central

Li, Lei; Tibiche, Chabane; Fu, Cong; Kaneko, Tomonori; Moran, Michael F.; Schiller, Martin R.; Li, Shawn Shun-Cheng; Wang, Edwin

2012-01-01

Phosphotyrosine (pTyr) signaling, which plays a central role in cell–cell and cell–environment interactions, has been considered to be an evolutionary innovation in multicellular metazoans. However, neither the emergence nor the evolution of the human pTyr signaling system is currently understood. Tyrosine kinase (TK) circuits, each of which consists of a TK writer, a kinase substrate, and a related reader, such as Src homology (SH) 2 domains and pTyr-binding (PTB) domains, comprise the core machinery of the pTyr signaling network. In this study, we analyzed the evolutionary trajectories of 583 literature-derived and 50,000 computationally predicted human TK circuits in 19 representative eukaryotic species and assigned their evolutionary origins. We found that human TK circuits for intracellular pTyr signaling originated largely from primitive organisms, whereas the inter- or extracellular signaling circuits experienced significant expansion in the bilaterian lineage through the “back-wiring” of newly evolved kinases to primitive substrates and SH2/PTB domains. Conversely, the TK circuits that are involved in tissue-specific signaling evolved mainly in vertebrates by the back-wiring of vertebrate substrates to primitive kinases and SH2/PTB domains. Importantly, we found that cancer signaling preferentially employs the pTyr sites, which are linked to more TK circuits. Our work provides insights into the evolutionary paths of the human pTyr signaling circuits and suggests the use of a network approach for cancer intervention through the targeting of key pTyr sites and their associated signaling hubs in the network. PMID:22194470

Novel selective human mitochondrial kinase inhibitors: design, synthesis and enzymatic activity.

PubMed

Ciliberti, Nunzia; Manfredini, Stefano; Angusti, Angela; Durini, Elisa; Solaroli, Nicola; Vertuani, Silvia; Buzzoni, Lisa; Bonache, Maria Cruz; Ben-Shalom, Efrat; Karlsson, Anna; Saada, Ann; Balzarini, Jan

2007-04-15

Selective and effective TK2 inhibitors can be obtained by introduction of bulky lipophilic chains (acyl or alkyl entities) at the 2' position of araT and BVaraU, nucleoside analogues naturally endowed with a low TK2 affinity. These derivatives showed a competitive inhibitory activity against TK2 in micromolar range. BVaraU nucleoside analogues, modified on the 2'-O-acyl chain with a terminal N-Boc amino-group, conserved or increased the inhibitory activity against TK2 (7l and 7m IC(50): 6.4 and 3.8 microM, respectively). The substitution of an ester for a carboxamide moiety at the 2' position of araT afforded a consistent reduction of the inhibitory activity (25, IC(50): 480 microM). On the contrary, modifications at 2'-OH position of araC and araG, have provided inactive derivatives against TK2 and dGK, respectively. The biological activity of a representative compound, 2'-O-decanoyl-BVaraU, was also investigated in normal human fibroblasts and was found to impair mitochondrial function due to TK2 inhibition.

A development of chimeric VEGFR2 TK inhibitor based on two ligand conformers from PDB: 1Y6A complex--medicinal chemistry consequences of a TKs analysis.

PubMed

Lintnerová, Lucia; García-Caballero, Melissa; Gregáň, Fridrich; Melicherčík, Milan; Quesada, Ana R; Dobiaš, Juraj; Lác, Ján; Sališová, Marta; Boháč, Andrej

2014-01-24

VEGFR2 is an important mediator of angiogenesis and influences fate of some cancer stem cells. Here we analysed all 34 structures of VEGFR2 TK available from PDB database. From them a complex PDB: 1Y6A has an exceptional AAZ ligand bound to TK in form of two conformers (U- and S-shaped). This observation inspired us to develop three chimeric bispyridyl VEGFR2 inhibitors by combining structural features of both AAZ conformers and/or their relative ligand AAX (PDB: 1Y6B). Our most interesting inhibitor 22SYM has an enzymatic VEGFR2 TK activity (IC50: 15.1 nM) comparable or better to the active compounds from clinical drugs Nexavar and Sutent. 22SYM inhibits growth, migration and tube formation of endothelial cells (EC) and selectively induces EC apoptosis. 22SYM also inhibits in vivo angiogenesis in Zebrafish embryo assay. Additionally to the above results, we proved here that tyrosine kinases in an inactive form possessing Type I inhibitors can adopt both a closed or an opened conformation of kinase A-loop independently on their DFG-out arrangement. We proposed here that an activity of certain Type I inhibitors (e.g. 22SYM-like) in complex with DFG-out TK can be negatively influenced by collisions with a dynamically moving TK A-loop. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Genetic analysis of mouse embryonic stem cells bearing Msh3 and Msh2 single and compound mutations.

PubMed

Abuin, A; Zhang, H; Bradley, A

2000-01-01

We have previously described the use of homologous recombination and CRE-loxP-mediated marker recycling to generate mouse embryonic stem (ES) cell lines homozygous for mutations at the Msh3, Msh2, and both Msh3 and Msh2 loci (2). In this study, we describe the analysis of these ES cells with respect to processes known to be affected by DNA mismatch repair. ES cells homozygous for the Msh2 mutation displayed increased resistance to killing by the cytotoxic drug 6-thioguanine (6TG), indicating that the 6TG cytotoxic mechanism is mediated by Msh2. The mutation rate of the herpes simplex virus thymidine kinase 1 (HSV-tk1) gene was unchanged in Msh3-deficient ES cell lines but markedly elevated in Msh2-deficient and Msh3 Msh2 double-mutant cells. Notably, the HSV-tk1 mutation rate was 11-fold higher, on average, than that of the hypoxanthine-guanine phosphoribosyl transferase (Hprt) locus in Msh2-deficient cells. Sequence analysis of HSV-tk1 mutants from these cells indicated the presence of a frameshift hotspot within the HSV-tk1 coding region. Msh3-deficient cells displayed a modest (16-fold) elevation in the instability of a dinucleotide repeat, whereas Msh2-deficient and Msh2 Msh3 double-mutant cells displayed markedly increased levels of repeat instability. Targeting frequencies of nonisogenic vectors were elevated in Msh2-deficient ES cell lines, confirming the role of Msh2 in blocking recombination between diverged sequences (homeologous recombination) in mammalian cells. These results are consistent with accumulating data from other laboratories and support the current model of DNA mismatch repair in mammalian cells.

Genetic Analysis of Mouse Embryonic Stem Cells Bearing Msh3 and Msh2 Single and Compound Mutations

PubMed Central

Abuin, Alejandro; Zhang, HeJu; Bradley, Allan

2000-01-01

We have previously described the use of homologous recombination and CRE-loxP-mediated marker recycling to generate mouse embryonic stem (ES) cell lines homozygous for mutations at the Msh3, Msh2, and both Msh3 and Msh2 loci (2). In this study, we describe the analysis of these ES cells with respect to processes known to be affected by DNA mismatch repair. ES cells homozygous for the Msh2 mutation displayed increased resistance to killing by the cytotoxic drug 6-thioguanine (6TG), indicating that the 6TG cytotoxic mechanism is mediated by Msh2. The mutation rate of the herpes simplex virus thymidine kinase 1 (HSV-tk1) gene was unchanged in Msh3-deficient ES cell lines but markedly elevated in Msh2-deficient and Msh3 Msh2 double-mutant cells. Notably, the HSV-tk1 mutation rate was 11-fold higher, on average, than that of the hypoxanthine-guanine phosphoribosyl transferase (Hprt) locus in Msh2-deficient cells. Sequence analysis of HSV-tk1 mutants from these cells indicated the presence of a frameshift hotspot within the HSV-tk1 coding region. Msh3-deficient cells displayed a modest (16-fold) elevation in the instability of a dinucleotide repeat, whereas Msh2-deficient and Msh2 Msh3 double-mutant cells displayed markedly increased levels of repeat instability. Targeting frequencies of nonisogenic vectors were elevated in Msh2-deficient ES cell lines, confirming the role of Msh2 in blocking recombination between diverged sequences (homeologous recombination) in mammalian cells. These results are consistent with accumulating data from other laboratories and support the current model of DNA mismatch repair in mammalian cells. PMID:10594017

Tissue kallikrein protects neurons from hypoxia/reoxygenation-induced cell injury through Homer1b/c.

PubMed

Su, Jingjing; Tang, Yuping; Zhou, Houguang; Liu, Ling; Dong, Qiang

2012-11-01

Previous studies have demonstrated that human tissue kallikrein (TK) gene delivery protects against mouse cerebral ischemia/reperfusion (I/R) injury through bradykinin B2 receptor (B2R) activation. We have also reported that exogenous TK administration can suppress glutamate- or acidosis-induced neurotoxicity through the extracellular signal-regulated kinase1/2 (ERK1/2) pathway. To further explore the neuroprotection mechanisms of TK, in the present study we performed immunoprecipitation analysis and identified a scaffolding protein Homer1b/c using MALDI-TOF MS analysis. Here, we tested the hypothesis that TK reduces cell injury induced by oxygen and glucose deprivation/reoxygenation (OGD/R) through activating Homer1b/c. We found that TK increased the expression of Homer1b/c in a concentration- and time-dependent manner. Moreover, TK facilitated the translocation of Homer1b/c to the plasma membrane under OGD/R condition by confocal microscope assays. We also observed that overexpression of Homer1b/c showed the neuroprotection against OGD/R-induced cell injury by enhancing cell survival, reducing LDH release, caspase-3 activity and cell apoptosis. However, the knockdown of Homer1b/c by small interfering RNA showed the opposite effects, indicating that Homer1b/c had protective effects against OGD/R-induced neuronal injury. More interestingly, TK exerted its much more significantly neuroprotective effects after Homer1b/c overexpression, whereas it exerted its reduced effects after Homer1b/c knockdown. In addition, TK pretreatment increased the phosphorylation of the ERK1/2 and Akt-GSK3β through Homer1b/c activation. The beneficial effects of Homer1b/c were abolished by the ERK1/2 or PI3K antagonist. Therefore, we propose novel signaling mechanisms involved in the anti-hypoxic function of TK through activation of Homer1b/c-ERK1/2 and Homer1b/c-PI3K-Akt signaling pathways. Copyright © 2012 Elsevier Inc. All rights reserved.

3'-Azido-2',3'-dideoxythymidine induced deficiency of thymidine kinases 1, 2 and deoxycytidine kinase in H9 T-lymphoid cells.

PubMed

Gröschel, Bettina; Kaufmann, Andreas; Höver, Gerold; Cinatl, Jaroslav; Doerr, Hans Wilhelm; Noordhuis, Paul; Loves, Willem J P; Peters, Godefridus J; Cinatl, Jindrich

2002-07-15

Continuous cultivation of T-lymphoid H9 cells in the presence of 3'-azido-2',3'-dideoxythymidine (AZT) resulted in a cell variant cross-resistant to both thymidine and deoxycytidine analogs. Cytotoxic effects of AZT, 2',3'-didehydro-3'-deoxythymidine as well as different deoxycytidine analogs such as 2',3'-dideoxycytidine, 2',2'-difluoro-2'-deoxycytidine (dFdC) and 1-ss-D-arabinofuranosylcytosine (Ara-C) were strongly reduced in H9 cells continuously exposed to AZT when compared to parental cells (>8.3-, >6.6-, >9.1-, 5 x 10(4)-, 5 x 10(3)-fold, respectively). Moreover, anti-HIV-1 effects of AZT, d4T, ddC and 2',3'-dideoxy-3'-thiacytidine (3TC) were significantly diminished (>222-, >25-, >400-, >200-fold, respectively) in AZT-resistant H9 cells. Study of cellular mechanisms responsible for cross-resistance to pyrimidine analogs in AZT-resistant H9 cells revealed decreased mRNA levels of thymidine kinase 1 (TK1) and lack of deoxycytidine kinase (dCK) mRNA expression. The loss of dCK gene expression was confirmed by western blot analysis of dCK protein as well as dCK enzyme activity assay. Moreover, enzyme activity of TK1 and TK2 was reduced in AZT-resistant cells. In order to determine whether lack of dCK affected the formation of the active triphosphate of the deoxycytidine analog dFdC, dFdCTP accumulation and retention was measured in H9 parental and AZT-resistant cells after exposure to 1 and 10 microM dFdC. Parental H9 cells accumulated about 30 and 100 pmol dFdCTP/10(6) cells after 4hr, whereas in AZT-resistant cells no dFdCTP accumulation was detected. These results demonstrate that continuous treatment of H9 cells in the presence of AZT selected for a thymidine analog resistant cell variant with cross-resistance to deoxycytidine analogs, due to deficiency in TK1, TK2, and dCK.

In vitro and in vivo characterization of a dual-function green fluorescent protein--HSV1-thymidine kinase reporter gene driven by the human elongation factor 1 alpha promoter.

PubMed

Luker, Gary D; Luker, Kathryn E; Sharma, Vijay; Pica, Christina M; Dahlheimer, Julie L; Ocheskey, Joe A; Fahrner, Timothy J; Milbrandt, Jeffrey; Piwnica-Worms, David

2002-01-01

Toward the goal of monitoring activity of native mammalian promoters with molecular imaging techniques, we stably transfected DU145 prostate carcinoma cells with a fusion construct of enhanced green fluorescent protein (EGFP) and wild-type herpes simplex virus-1 thymidine kinase (HSV1-TK) as a reporter gene driven by the promoter for human elongation factor 1 alpha (EF-1 alpha-EGFP-TK). Using this model system, expression of EGFP was quantified by flow cytometry and fluorescence microscopy, while the HSV1-TK component of the reporter was quantified with 8-[3H]ganciclovir (8-[3H]GCV). As analyzed by flow cytometry, passage of EGFP-TK-DU145 transfected cells (ETK) in vitro resulted in populations of cells with high and low expression of EGFP over time. High and low ETK cells retained 23-fold and 5-fold more GCV, respectively, than control. While differences in uptake and retention of GCV corresponded to relative expression of the reporter gene in each subpopulation of cells as determined by both flow cytometry (EGFP) and quantitative RT-PCR, the correlation was not linear. Furthermore, in high ETK cells, net retention of various radiolabeled nucleoside analogues varied; the rank order was 8-[3H]GCV < 9-(4-fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG) approximately 8-[3H]penciclovir (8-[3H]PCV) < 2'-fluoro-2'-deoxy-5-iodouracil-beta-D-arabinofuranoside (2-[14C]FIAU). Xenograft tumors of ETK cells in vivo accumulated 2.5-fold more 8-[3H]GCV per gram of tissue and showed greater fluorescence from EGFP than control DU145 cells, demonstrating that the reporter gene functioned in vivo. These data extend previous reports by showing that a human promoter can be detected in vitro and in vivo with a dual-function reporter exploiting optical and radiotracer techniques.

Development and characterization of a new marine fish cell line from turbot (Scophthalmus maximus).

PubMed

Wang, N; Wang, X L; Sha, Z X; Tian, Y S; Chen, S L

2010-12-01

A new marine fish cell line, TK, derived from turbot (Scophthalmus maximus) kidney, was established by the method of trypsin digestion and subcultured for more than 50 passages over a period of 300 days. The TK cells were maintained in Minimum Essential Medium Eagle (MEM) supplemented with HEPES, antibiotics, fetal bovine serum (FBS), 2-Mercaptoethanol (2-Me), and basic fibroblast growth factor (bFGF). The suitable growth temperature for TK cells was 24°C, and microscopically, TK cells were composed of fibroblast-like cells. Chromosome analysis revealed that the TK cell line has a normal diploid karyotype with 2n=44. Two fish viruses LCDV-C (lymphocystis disease virus from China) and TRBIV (turbot reddish body iridovirus) were used to determine the virus susceptibility of TK cell line. The TK cell line was found to be susceptible to TRBIV, and the infection was confirmed by cytopathic effect (CPE) and transmission electron microscopy, which detected the viral particles in the cytoplasm of virus-infected cells. Finally, significant green fluorescent signals were observed when the TK cells were transfected with pEGFP-N3 vector, indicating its potential utility for fish virus study and genetic manipulation.

B cells are critical to T-cell-mediated antitumor immunity induced by a combined immune-stimulatory/conditionally cytotoxic therapy for glioblastoma.

PubMed

Candolfi, Marianela; Curtin, James F; Yagiz, Kader; Assi, Hikmat; Wibowo, Mia K; Alzadeh, Gabrielle E; Foulad, David; Muhammad, A K M G; Salehi, Sofia; Keech, Naomi; Puntel, Mariana; Liu, Chunyan; Sanderson, Nicholas R; Kroeger, Kurt M; Dunn, Robert; Martins, Gislaine; Lowenstein, Pedro R; Castro, Maria G

2011-10-01

We have demonstrated that modifying the tumor microenvironment through intratumoral administration of adenoviral vectors (Ad) encoding the conditional cytotoxic molecule, i.e., HSV1-TK and the immune-stimulatory cytokine, i.e., fms-like tyrosine kinase 3 ligand (Flt3L) leads to T-cell-dependent tumor regression in rodent models of glioblastoma. We investigated the role of B cells during immune-mediated glioblastoma multiforme regression. Although treatment with Ad-TK+Ad-Flt3L induced tumor regression in 60% of wild-type (WT) mice, it completely failed in B-cell-deficient Igh6(-/-) mice. Tumor-specific T-cell precursors were detected in Ad-TK+Ad-Flt3L-treated WT mice but not in Igh6(-/-) mice. The treatment also failed in WT mice depleted of total B cells or marginal zone B cells. Because we could not detect circulating antibodies against tumor cells and the treatment was equally efficient in WT mice and in mice with B-cell-specific deletion of Prdm 1 (encoding Blimp-1), in which B cells are present but unable to fully differentiate into antibody-secreting plasma cells, tumor regression in this model is not dependent on B cells' production of tumor antigen-specific immunoglobulins. Instead, B cells seem to play a role as antigen-presenting cells (APCs). Treatment with Ad-TK+Ad-Flt3L led to an increase in the number of B cells in the cervical lymph nodes, which stimulated the proliferation of syngeneic T cells and induced clonal expansion of antitumor T cells. Our data show that B cells act as APCs, playing a critical role in clonal expansion of tumor antigen-specific T cells and brain tumor regression.

[BLG gene knockout and hLF gene knock-in at BLG locus in goat by TALENs].

PubMed

Song, Shaozheng; Zhu, Mengmin; Yuan, Yuguo; Rong, Yao; Xu, Sheng; Chen, Si; Mei, Junyan; Cheng, Yong

2016-03-01

To knock out β-lactoglobulin (BLG) gene and insert human lactoferrin (hLF) coding sequence at BLG locus of goat, the transcription activator-like effector nucleases (TALEN) mediated recombination was used to edit the BLG gene of goat fetal fibroblast, then as donor cells for somatic cell nuclear transfer. We designed a pair of specific plasmid TALEN-3-L/R for goat BLG exon III recognition sites, and BLC14-TK vector containing a negative selection gene HSV-TK, was used for the knock in of hLF gene. TALENs plasmids were transfected into the goat fetal fibroblast cells, and the cells were screened three days by 2 μg/mL puromycin. DNA cleavage activities of cells were verified by PCR amplification and DNA production sequencing. Then, targeting vector BLC14-TK and plasmids TALEN-3-L/R were co-transfected into goat fetal fibroblasts, both 700 μg/mL G418 and 2 μg/mL GCV were simultaneously used to screen G418-resistant cells. Detections of integration and recombination were implemented to obtain cells with hLF gene site-specific integration. We chose targeting cells as donor cells for somatic cell nuclear transfer. The mutagenicity of TALEN-3-L/R was between 25% and 30%. A total of 335 reconstructed embryos with 6 BLG-/hLF+ targeting cell lines were transferred into 16 recipient goats. There were 9 pregnancies confirmed by ultrasound on day 30 to 35 (pregnancy rate of 39.1%), and one of 50-day-old fetus with BLG-/hLF+ was achieved. These results provide the basis for hLF gene knock-in at BLG locus of goat and cultivating transgenic goat of low allergens and rich hLF in the milk.

Hydroquinone-induced malignant transformation of TK6 cells by facilitating SIRT1-mediated p53 degradation and up-regulating KRAS.

PubMed

Chen, Yuting; Chen, Jiajia; Yun, Lin; Xu, Longmei; Liu, Jiaxian; Xu, Yongchun; Yang, Hui; Liang, Hairong; Tang, Huanwen

2016-09-30

Hydroquinone (HQ), known as one of the metabolic products of benzene, causes a number of hematologic malignancies. The study evaluated the potential mechanism of Sirtuin 1 (SIRT1) in HQ-induced TK6 cell malignant transformation. The data of our study show that short term exposure of TK6 cells to HQ led to a decrease expression of SIRT1. Knockdown of SIRT1 sensitized to the HQ-induced apoptosis in vitro and increased the expression of p53, p21 and γ-H2AX. Furthermore, chronic HQ-treated (20μM once a week for 19 weeks) caused carcinogenic transformation and was confirmed by abnormal cell proliferation, matrix metalloproteinase 9(MMP9) and subcutaneous tumor formation in nude mice. SIRT1 increased KRAS expression, and decreased H3K9 and H3K18 acetylation, inhibited p53 signaling and the level of caspase-3 in HQ-induced transformation cells. Taken together, these data suggest that SIRT1 is involved in HQ-induced malignant transformation associated with suppressing p53 signaling and activation of KRAS. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Genetically engineered suicide gene in mesenchymal stem cells using a Tet-On system for anaplastic thyroid cancer.

PubMed

Kalimuthu, Senthilkumar; Oh, Ji Min; Gangadaran, Prakash; Zhu, Liya; Lee, Ho Won; Jeon, Yong Hyun; Jeong, Shin Young; Lee, Sang-Woo; Lee, Jaetae; Ahn, Byeong-Cheol

2017-01-01

Anaplastic thyroid cancer (ATC) is the most aggressive malignancy of the thyroid, during which undifferentiated tumors arise from the thyroid follicular epithelium. ATC has a very poor prognosis due to its aggressive behavior and poor response to conventional therapies. Gene-directed enzyme/prodrug therapy using genetically engineered mesenchymal stromal cells (MSC) is a promising therapeutic strategy. The doxycycline (DOX)-controlled Tet inducible system is the most widely utilized regulatory system and could be a useful tool for therapeutic gene-based therapies. For example, use a synthetic "tetracycline-on" switch system to control the expression of the therapeutic gene thymidine kinase, which converts prodrugs to active drugs. The aim of this study was to develop therapeutic MSCs, harboring an inducible suicide gene, and to validate therapeutic gene expression using optical molecular imaging of ATC. We designed the Tet-On system using a retroviral vector expressing herpes simplex virus thymidine kinase (HSV1-sr39TK) with dual reporters (eGFP-Fluc2). Mouse bone marrow-derived mesenchymal stromal cells (BM-MSC) were transduced using this system with (MSC-Tet-TK/Fluc2) or without (MSC-TK/Fluc) the Tet-On system. Transduced cells were screened and characterized. Engineered MSCs were co-cultured with ATC (CAL62/Rluc) cells in the presence of the prodrug ganciclovir (GCV) and stimulated with DOX. The efficiency of cell killing monitored by assessing Rluc (CAL62/Rluc) and Fluc (MSC-Tet-TK/Fluc and MSC-TK/Fluc) activities using IVIS imaging. Fluc activity increased in MSC-Tet-TK/Fluc cells in a dose dependent manner following DOX treatment (R2 = 0.95), whereas no signal was observed in untreated cells. eGFP could also be visualized after induction with DOX, and the HSV1-TK protein could be detected by western blotting. In MSC-TK/Fluc cells, the Fluc activity increased with increasing cell number (R2 = 0.98), and eGFP could be visualized by fluorescence microscopy. The Fluc activity and cell viability of MSC-Tet-TK/Fluc and MSC-TK/Fluc cells decreased significantly following GCV treatment. A bystander effect of the therapeutic cells confirmed in co-cultures of CAL62 cells, an anaplastic thyroid cancer cell line, with either MSC-Tet-TK/Fluc cells or MSC-TK/Fluc cells. The Rluc activity in MSC-Tet-TK/Fluc co-cultures, derived from the CAL62/Rluc cells, decreased significantly with GCV treatment of DOX treated cultures, whereas no significant changes were observed in untreated cultures. In addition, the Fluc activity of MSC-Tet-TK/Fluc cells also decreased significantly with DOX treatment whereas no signal was present in untreated cultures. A bystander effect also be demonstrated in co-cultures with MSC-TK/Fluc cells and CAL62/Rluc; both the Rluc activity and the Fluc activity were significantly decreased following GCV treatment. We have successfully developed a Tet-On system of gene-directed enzyme/prodrug delivery using MSCs. We confirmed the therapeutic bystander effect in CAL62/Rluc cells with respect to MSC-Tet-TK/Fluc and MSC-TK/Fluc cells after GCV treatment with and without DOX. Our results confirm the therapeutic efficiency of a suicide gene, with or without the Tet-On system, for ATC therapy. In addition, our findings provide an innovative therapeutic approach for using the Tet-On system to eradicate tumors by simple, repeated administration of MSC-Tet-TK/Fluc cells with DOX and GCV.

Synthesis and mechanistic studies of curcumin analog-based oximes as potential anticancer agents.

PubMed

Qin, Hua-Li; Leng, Jing; Youssif, Bahaa G M; Amjad, Muhammad Wahab; Raja, Maria Abdul Ghafoor; Hussain, Muhammad Ajaz; Hussain, Zahid; Kazmi, Syeda Naveed; Bukhari, Syed Nasir Abbas

2017-09-01

The incidence of cancer can be decreased by chemoprevention using either natural or synthetic agents. Apart from synthetic compounds, numerous natural products have exhibited promising potential to inhibit carcinogenesis in vivo. In this study, α, β-unsaturated carbonyl-based anticancer compounds were used as starting materials to synthesize new oxime analogs. The findings from the antiproliferative assay using seven different human cancer cell lines provided a clear picture of structure-activity relationship. The oxime analogs namely 7a and 8a showed strong antiproliferative activity against the cell lines. The mechanistic effects of compounds on EGFR-TK kinases and tubulin polymerization and BRAF V 600E were investigated. In addition, the efficacy of compounds in reversing the efflux-mediated resistance developed by cancer cells was also studied. The compounds 5a and 6a displayed potent activity on various targets such as BRAF V 600E and EGFR-TK kinases and also exhibited strong antiproliferative activity against different cell lines hence showing potential of multifunctional anticancer agents. © 2017 John Wiley & Sons A/S.

KSHV-TK is a tyrosine kinase that disrupts focal adhesions and induces Rho-mediated cell contraction

PubMed Central

Gill, Michael B; Turner, Rachel; Stevenson, Philip G; Way, Michael

2015-01-01

Paradoxically, the thymidine kinase (TK) encoded by Kaposi sarcoma-associated herpesvirus (KSHV) is an extremely inefficient nucleoside kinase, when compared to TKs from related herpesviruses. We now show that KSHV-TK, in contrast to HSV1-TK, associates with the actin cytoskeleton and induces extensive cell contraction followed by membrane blebbing. These dramatic changes in cell morphology depend on the auto-phosphorylation of tyrosines 65, 85 and 120 in the N-terminus of KSHV-TK. Phosphorylation of tyrosines 65/85 and 120 results in an interaction with Crk family proteins and the p85 regulatory subunit of PI3-Kinase, respectively. The interaction of Crk with KSHV-TK leads to tyrosine phoshorylation of this cellular adaptor. Auto-phosphorylation of KSHV-TK also induces a loss of FAK and paxillin from focal adhesions, resulting in activation of RhoA-ROCK signalling to myosin II and cell contraction. In the absence of FAK or paxillin, KSHV-TK has no effect on focal adhesion integrity or cell morphology. Our observations demonstrate that by acting as a tyrosine kinase, KSHV-TK modulates signalling and cell morphology. PMID:25471072

Combination effect of oncolytic adenovirus therapy and herpes simplex virus thymidine kinase/ganciclovir in hepatic carcinoma animal models

PubMed Central

Zheng, Fei-qun; Xu, Yin; Yang, Ren-jie; Wu, Bin; Tan, Xiao-hua; Qin, Yi-de; Zhang, Qun-wei

2009-01-01

Aim: Oncolytic adenovirus, also called conditionally replicating adenovirus (CRAD), can selectively propagate in tumor cells and cause cell lysis. The released viral progeny can infect neighboring cancer cells, initiating a cascade that can lead to the ultimate destruction of the tumor. Suicide gene therapy using herpes simplex virus thymidine kinase (HSV-TK) and ganciclovir (GCV) offers a potential treatment strategy for cancer and is undergoing preclinical trials for a variety of tumors. We hypothesized that HSV-TK gene therapy combined with oncolytic adenoviral therapy would have an enhanced effect compared with the individual effects of the therapies and is a potential novel therapeutic strategy to treat liver cancer. Methods: To address our hypothesis, a novel CRAD was created, which consisted of a telomerase-dependent oncolytic adenovirus engineered to express E1A and HSV-TK genes (Ad-ETK). The combined effect of Ad-ETK and GCV was assessed both in vitro and in vivo in nude mice bearing HepG2 cell-derived tumors. Expression of the therapeutic genes by the transduced tumor cells was analyzed by RT-PCR and Western blotting. Results: We confirmed that Ad-ETK had antitumorigenic effects on human hepatocellular carcinoma (HCC) both in vitro and in vivo, and the TK/GCV system enhanced oncolytic adenoviral therapy. We confirmed that both E1A and HSV-TK genes were expressed in vivo. Conclusion: The Ad-ETK construct should provide a relatively safe and selective approach to killing cancer cells and should be investigated as an adjuvant therapy for hepatocellular carcinoma. PMID:19363518

Administration of herpes simplex-thymidine kinase-expressing donor T cells with a T-cell-depleted allogeneic marrow graft.

PubMed

Tiberghien, P; Ferrand, C; Lioure, B; Milpied, N; Angonin, R; Deconinck, E; Certoux, J M; Robinet, E; Saas, P; Petracca, B; Juttner, C; Reynolds, C W; Longo, D L; Hervé, P; Cahn, J Y

2001-01-01

Administration of donor T cells expressing the herpes simplex-thymidine kinase (HS-tk) with a hematopoietic stem cell (HSC) transplantation could allow, if graft-versus-host disease (GVHD) was to occur, a selective in vivo depletion of these T cells by the use of ganciclovir (GCV). The study evaluates the feasibility of such an approach. Escalating numbers of donor HS-tk-expressing CD3(+) gene-modified cells (GMCs) are infused with a T-cell-depleted bone marrow transplantation (BMT). Twelve patients with hematological malignancies received 2 x 10(5) (n = 5), 6 x 10(5) (n = 5), or 20 x 10(5) (n = 2) donor CD3(+) GMCs/kg with a BMT from a human leukocyte antigen (HLA)-identical sibling. No acute toxicity was associated with GMC administration. An early increase of circulating GMCs followed by a progressive decrease and long-lasting circulation of GMCs was documented. GCV treatment resulted in significant rapid decrease in circulating GMCs. Three patients developed acute GVHD, with a grade of at least II, while one patient developed chronic GVHD. Treatment with GCV alone was associated with a complete remission (CR) in 2 patients with acute GVHD, while the addition of glucocorticoids was necessary to achieve a CR in the last case. Long-lasting CR occurred with GCV treatment in the patient with chronic GVHD. Unfortunately, Epstein-Barr virus-lymphoproliferative disease occurred in 3 patients. Overall, the administration of low numbers of HS-tk-expressing T cells early following an HLA-identical BMT is associated with no acute toxicity, persistent circulation of the GMCs, and GCV-sensitive GVHD. Such findings open the way to the infusion of higher numbers of gene-modified donor T cells to enhance post-BMT immune competence while preserving GCV-sensitive alloreactivity.

Morphological study of the TK cholangiocarcinoma cell line with three-dimensional cell culture.

PubMed

Akiyoshi, Kohei; Kamada, Minori; Akiyama, Nobutake; Suzuki, Masafumi; Watanabe, Michiko; Fujioka, Kouki; Ikeda, Keiichi; Mizuno, Shuichi; Manome, Yoshinobu

2014-04-01

Cholangiocarcinoma is an intractable carcinoma originating from the bile duct epithelium. To gain an understanding of the cell biology of cholangiocarcinoma, in vitro cell culture is valuable. However, well‑characterized cell lines are limited. In the present study, the morphology of the TK cholangiocarcinoma cell line was analyzed by three‑dimensional culture. Dispersed TK cells were injected into a gelatin mesh scaffold and cultivated for 3‑20 days. The morphology of the TK cells was investigated by phase‑contrast microscopy, optical microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). TK cells were observed to proliferate three-dimensionally in the scaffold. The cells exhibited a globoid structure and attached to the scaffold. The SEM observation demonstrated typical microvilli and plicae on the surface of the structure. Light microscopy and TEM confirmed intercellular and cell‑to‑scaffold attachment in the three‑dimensional mesh. The culture also exhibited the formation of a duct-like structure covered by structured microvilli. In conclusion, three‑dimensional culture of TK cells demonstrated the morphological characteristics of cholangiocarcinoma in vitro. Production of high levels of carbohydrate antigen (CA)19‑9, CA50 and carcinoembryonic antigen was previously confirmed in the TK cell line. As a characteristic morphology was demonstrated in the present study, the TK cholangiocarcinoma cell line may be useful as an experimental model for further study of cholangiocarcinoma.

Unchanged thymidine triphosphate pools and thymidine metabolism in two lines of thymidine kinase 2-mutated fibroblasts.

PubMed

Frangini, Miriam; Rampazzo, Chiara; Franzolin, Elisa; Lara, Mari-Carmen; Vilà, Maya R; Martí, Ramon; Bianchi, Vera

2009-02-01

Mitochondrial thymidine kinase (TK2) catalyzes the phosphorylation of thymidine in mitochondria. Its function becomes essential for dTTP synthesis in noncycling cells, where cytosolic dTTP synthesis via R1/R2 ribonucleotide reductase and thymidine kinase 1 is turned down. Mutations in the nuclear gene for TK2 cause a fatal mtDNA depletion syndrome. Only selected cell types are affected, suggesting that the other cells compensate for the TK2 deficiency by adapting the enzyme network that regulates dTTP synthesis outside S-phase. Here we looked for such metabolic adaptation in quiescent cultures of fibroblasts from two TK2-deficient patients with a slow-progressing syndrome. In cell extracts, we measured the activities of TK2, deoxycytidine kinase, thymidine phosphorylase, deoxynucleotidases and the amounts of the three ribonucleotide reductase subunits. Patient cells contained 40% or 5% TK2 activity and unchanged activities of the other enzymes. However, their mitochondrial and cytosolic dTTP pools were unchanged, and also the overall composition of the dNTP pools was normal. TK2-dependent phosphorylation of [(3)H]thymidine in intact cells and the turnover of the dTTP pool showed that even the fibroblasts with 5% residual TK2 activity synthesized dTTP at an almost normal rate. Normal fibroblasts apparently contain more TK2 than needed to maintain dTTP during quiescence, which would explain why TK2-mutated fibroblasts do not manifest mtDNA depletion despite their reduced TK2 activity.

Transgene expression of Drosophila melanogaster nucleoside kinase reverses mitochondrial thymidine kinase 2 deficiency.

PubMed

Krishnan, Shuba; Zhou, Xiaoshan; Paredes, João A; Kuiper, Raoul V; Curbo, Sophie; Karlsson, Anna

2013-02-15

A strategy to reverse the symptoms of thymidine kinase 2 (TK2) deficiency in a mouse model was investigated. The nucleoside kinase from Drosophila melanogaster (Dm-dNK) was expressed in TK2-deficient mice that have been shown to present with a severe phenotype caused by mitochondrial DNA depletion. The Dm-dNK(+/-) transgenic mice were shown to be able to rescue the TK2-deficient mice. The Dm-dNK(+/-)TK2(-/-) mice were normal as judged by growth and behavior during the observation time of 6 months. The Dm-dNK-expressing mice showed a substantial increase in thymidine-phosphorylating activity in investigated tissues. The Dm-dNK expression also resulted in highly elevated dTTP pools. The dTTP pool alterations did not cause specific mitochondrial DNA mutations or deletions when 6-month-old mice were analyzed. The mitochondrial DNA was also detected at normal levels. In conclusion, the Dm-dNK(+/-)TK2(-/-) mouse model illustrates how dTMP synthesized in the cell nucleus can compensate for loss of intramitochondrial dTMP synthesis in differentiated tissue. The data presented open new possibilities to treat the severe symptoms of TK2 deficiency.

Homologous recombination between overlapping thymidine kinase gene fragments stably inserted into a mouse cell genome.

PubMed

Lin, F L; Sternberg, N

1984-05-01

We have constructed a substrate to study homologous recombination between adjacent segments of chromosomal DNA. This substrate, designated lambda tk2 , consists of one completely defective and one partially defective herpes simplex virus thymidine kinase (tk) gene cloned in bacteriophage lambda DNA. The two genes have homologous 984-base-pair sequences and are separated by 3 kilobases of largely vector DNA. When lambda tk2 DNA was transferred into mouse LMtk- cells by the calcium phosphate method, rare TK+ transformants were obtained that contained many (greater than 40) copies of the unrecombined DNA. Tk- revertants, which had lost most of the copies of unrecombined DNA, were isolated from these TK+-transformed lines. Two of these Tk- lines were further studied by analysis of their reversion back to the Tk+ phenotype. They generated ca. 200 Tk+ revertants per 10(8) cells after growth in nonselecting medium for 5 days. All of these Tk+ revertants have an intact tk gene reconstructed by homologous recombination; they also retain various amounts of unrecombined lambda tk2 DNA. Southern blot analysis suggested that at least some of the recombination events involve unequal sister chromatid exchanges. We also tested three agents, mitomycin C, 12-O-tetradecanoyl-phorbol-13-acetate, and mezerein, that are thought to stimulate recombination to determine whether they affect the reversion from Tk- to Tk+. Only mitomycin C increased the number of Tk+ revertants.

[Experimental research in vitro of TK/GCV system for osteosarcoma MG-63 cell damage].

PubMed

Zhang, Hua-Dong; Lu, Zhi; Feng, Yi; Liu, Xiao-Li; Hou, Hui-Ming

2014-03-01

To study the killing effects of the liposome-mediated thymidine kinase (TK)/ganciclovir (GCV) system on MG-63 osteosarcoma (OS) cells and its bystander effects. Liposome-mediated TK gene transfected into MG-63 OS cells, the efficiency of transfection was analyzed by flow cytometry and observed under inverted fluorescence microscope. Non-transfected osteosarcoma MG-63 cells were divided into three groups,in the experimental group 1 transfected TK/GCV cells cultured in solutiona liquid mixture by supernatant by 1/10,1/7,1/5,1/2 ratio to original broth; in the experimental group 2 transfected cells cultured in solutiona liquid mixture of supernatant filtered through 0.22 microm filter by 1/10,1/7, 1/5, 1/2 ratio to original broth, in control group the transfection cells cultured in original culture solution. Cell growth inhibition rate and osteosarcoma cell sensitivity to TK/GCV system were measured by MTT assay in each group. The TK gene was transfected into MG-63 OS cells successfully by liposome-mediated, flow cytometry instrument detection TK gene transfection cell transfection efficiency can reach 75.5%. Six days later the MTT assay showed that in the experimental group 1 inhibition rate of all concentration ratio of the mixed culture fluid were statistically significant as compared with the control group (P < 0.05), and in the experimental group 2 that of the 1/10 and 1/7 of concentration ratio of mixed culture medium was not statistically significant as compared with the control group (P > 0.05). TK gene transfected MG-63 cells increased with the the GCV concentration,the cell apoptosis rate increased. The experiment demonstrated that the MG-63 OS cells are sensitive to the liposome-mediated TK/GCV system and bystander effects are significant.

Homologous recombination between overlapping thymidine kinase gene fragments stably inserted into a mouse cell genome.

PubMed Central

Lin, F L; Sternberg, N

1984-01-01

We have constructed a substrate to study homologous recombination between adjacent segments of chromosomal DNA. This substrate, designated lambda tk2 , consists of one completely defective and one partially defective herpes simplex virus thymidine kinase (tk) gene cloned in bacteriophage lambda DNA. The two genes have homologous 984-base-pair sequences and are separated by 3 kilobases of largely vector DNA. When lambda tk2 DNA was transferred into mouse LMtk- cells by the calcium phosphate method, rare TK+ transformants were obtained that contained many (greater than 40) copies of the unrecombined DNA. Tk- revertants, which had lost most of the copies of unrecombined DNA, were isolated from these TK+-transformed lines. Two of these Tk- lines were further studied by analysis of their reversion back to the Tk+ phenotype. They generated ca. 200 Tk+ revertants per 10(8) cells after growth in nonselecting medium for 5 days. All of these Tk+ revertants have an intact tk gene reconstructed by homologous recombination; they also retain various amounts of unrecombined lambda tk2 DNA. Southern blot analysis suggested that at least some of the recombination events involve unequal sister chromatid exchanges. We also tested three agents, mitomycin C, 12-O-tetradecanoyl-phorbol-13-acetate, and mezerein, that are thought to stimulate recombination to determine whether they affect the reversion from Tk- to Tk+. Only mitomycin C increased the number of Tk+ revertants. Images PMID:6328272

Transgene Expression of Drosophila melanogaster Nucleoside Kinase Reverses Mitochondrial Thymidine Kinase 2 Deficiency*♦

PubMed Central

Krishnan, Shuba; Zhou, Xiaoshan; Paredes, João A.; Kuiper, Raoul V.; Curbo, Sophie; Karlsson, Anna

2013-01-01

A strategy to reverse the symptoms of thymidine kinase 2 (TK2) deficiency in a mouse model was investigated. The nucleoside kinase from Drosophila melanogaster (Dm-dNK) was expressed in TK2-deficient mice that have been shown to present with a severe phenotype caused by mitochondrial DNA depletion. The Dm-dNK+/− transgenic mice were shown to be able to rescue the TK2-deficient mice. The Dm-dNK+/−TK2−/− mice were normal as judged by growth and behavior during the observation time of 6 months. The Dm-dNK-expressing mice showed a substantial increase in thymidine-phosphorylating activity in investigated tissues. The Dm-dNK expression also resulted in highly elevated dTTP pools. The dTTP pool alterations did not cause specific mitochondrial DNA mutations or deletions when 6-month-old mice were analyzed. The mitochondrial DNA was also detected at normal levels. In conclusion, the Dm-dNK+/−TK2−/− mouse model illustrates how dTMP synthesized in the cell nucleus can compensate for loss of intramitochondrial dTMP synthesis in differentiated tissue. The data presented open new possibilities to treat the severe symptoms of TK2 deficiency. PMID:23288848

Qualitative and quantitative analysis of tachykinin NK2 receptors in chemically defined human colonic neuronal pathways.

PubMed

Jaafari, Nadia; Khomitch-Baud, Alexandra; Gilhodes, Jean-Claude; Hua, Guoqiang; Julé, Yvon

2008-04-01

The involvement of NK2 receptors (NK2r) in the neuroregulation of human colonic motility has been mainly assessed by using pharmacological approaches. The aim of this study was to characterize the intramural neurons and nerve varicosities expressing NK2r in human colonic neuronal pathways. Neuronal coding in the myenteric plexus and external muscle layers was identified on the basis of the patterns of colocalization of tachykinins (TK), vesicular acetylcholine transporter (VAChT), nitric oxide synthase (NOS), glutamate decarboxylase (GAD), and vasoactive intestinal peptide (VIP) with NK2r immunoreactivity. The proportions of chemically defined synaptophysin-immunoreactive nerve varicosities were accurately determined by using specific software. NK2r immunoreactivity was detected in the soma of many myenteric neurons (71.8%). A large proportion of these neurons was immunoreactive to VAChT (39.3%), TK (30%), and GAD (23.5%) and, to a lesser extent, to NOS and VIP. The proportions of nerve varicosities expressing NK2r showed significant regional differences: the highest proportion (59.8%) was located in the myenteric plexus. High proportions of the myenteric nerve varicosities expressing NK2r were immunoreactive to VIP (80.9%) and NOS (77.9%), and lower proportions were recorded with VAChT, TK, and GAD. In the circular and longitudinal muscle layers, the proportions of nerve varicosities expressing NK2r were 49.6% and 45.3%, respectively. The chemically defined intramuscular varicosities were closely apposed to smooth muscle cells expressing NK2r. In conclusion, the data obtained in this study, in which the expression of NK2r was mapped in the human colonic neuronal pathways, confirm that these receptors are involved in the neuroneuronal and neuromuscular processes regulating human colonic motility. Copyright 2008 Wiley-Liss, Inc.

Retrovirus-mediated transfer of a hygromycin phosphotransferase-thymidine kinase fusion gene into human CD34+ bone marrow cells.

PubMed

Akatsuka, Y; Emi, N; Kato, H; Abe, A; Tanimoto, M; Lupton, S D; Saito, H

1994-12-01

Retrovirus-mediated gene transfer into human hematopoietic stem cells has been proposed as a means of therapy for various inherited diseases and as a method of gene marking. The transduction efficiency of an amphotropic retroviral vector (PA317/HyTK) containing a hygromycin phosphotransferase-thymidine kinase fusion gene was examined with human CD34+ bone marrow cells in the presence of interleukin-3 (IL-3), interleukin-6 (IL-6), and stem cell factor. Transduction efficiencies determined from the ability of transduced granulocyte-macrophage colony forming units (CFU-GM) to grow in hygromycin B and from polymerase chain reaction analysis of individual transduced CFU-GM growing in the presence of hygromycin B were 0.3-3.0% (mean +/- S.D., 1.1 +/- 0.9%) and 0.1-1.2% (mean +/- S.D., 0.5 +/- 0.4%), respectively. Ganciclovir at a dose of approximately 1 microM reduced the number of CFU-GM derived from vector-infected CD34+ cells by 50%. These findings demonstrate that human hematopoietic stem cells infected with this retroviral vector are susceptible to ganciclovir, offering the potential to control transduced gene expression in vivo.

The receptor tyrosine kinase MerTK activates phospholipase C γ2 during recognition of apoptotic thymocytes by murine macrophages

PubMed Central

Todt, Jill C.; Hu, Bin; Curtis, Jeffrey L.

2008-01-01

Apoptotic leukocytes must be cleared efficiently by macrophages (Mø). Apoptotic cell phagocytosis by Mø requires the receptor tyrosine kinase (RTK) MerTK (also known as c-Mer and Tyro12), the phosphatidylserine receptor (PS-R), and the classical protein kinase C (PKC) isoform βII, which translocates to Mø membrane and cytoskeletal fractions in a PS-R-dependent fashion. How these molecules cooperate to induce phagocytosis is unknown. Because the phosphatidylinositol-specific phospholipase (PI-PLC) PLC γ2 is downstream of RTKs in some cell types and can activate classical PKCs, we hypothesized that MerTK signals via PLC γ2. To test this hypothesis, we examined the interaction of MerTK and PLC γ2 in resident murine PMø and in the murine Mø cell line J774A.1 (J774) following exposure to apoptotic thymocytes. We found that, as with PMø, J774 phagocytosis of apoptotic thymocytes was inhibited by antibody against MerTK. Western blotting and immunoprecipitation showed that exposure to apoptotic cells produced three time-dependent changes in PMø and J774: (1) tyrosine phosphorylation of MerTK; (2) association of PLC γ2 with MerTK; and (3) tyrosine phosphorylation of PLC γ2. Phosphorylation of PLC γ2 and its association with MerTK was also induced by cross-linking MerTK using antibody. A PI-PLC appears to be required for phagocytosis of apoptotic cells because the PI-PLC inhibitor Et-18-OCH3 and the PLC inhibitor U73122, but not the inactive control U73343, blocked phagocytosis without impairing adhesion. On apoptotic cell adhesion to Mø, MerTK signals at least in part via PLC γ2. PMID:14704368

Thymidine Kinase 1 Loss Confers Trifluridine Resistance without Affecting 5-Fluorouracil Metabolism and Cytotoxicity.

PubMed

Edahiro, Keitaro; Iimori, Makoto; Kobunai, Takashi; Morikawa-Ichinose, Tomomi; Miura, Daisuke; Kataoka, Yuki; Niimi, Shinichiro; Wakasa, Takeshi; Saeki, Hiroshi; Oki, Eiji; Kitao, Hiroyuki; Maehara, Yoshihiko

2018-06-04

Acquired resistance to therapeutic drugs is a serious problem for cancer patients receiving systemic treatment. Experimentally, drug resistance is established in cell lines in vitro by repeated, continuous exposure to escalating concentrations of the drug; however, the precise mechanism underlying the acquired resistance is not always known. Here, it is demonstrated that the human colorectal cancer cell line DLD1 with acquired resistance to trifluridine (FTD), a key component of the novel, orally administered nucleoside analog-type chemotherapeutic drug trifluridine/tipiracil, lacks functional thymidine kinase 1 (TK1) expression because of one nonsense mutation in the coding exon. Targeted disruption of the TK1 gene also conferred severe FTD resistance, indicating that the loss of TK1 protein expression is the primary cause of FTD resistance. Both FTD-resistant DLD1 cells and DLD1-TK1-/- cells exhibited similar 5-fluorouracil (5-FU) sensitivity to that of the parental DLD1 line. The quantity of cellular pyrimidine nucleotides in these cells and the kinetics of thymidylate synthase ternary complex formation in 5-FU-treated cells is similar to DLD1 cells, indicating that 5-FU metabolism and cytotoxicity were unaffected. The present data provide molecular-based evidence that acquired resistance to FTD does not confer 5-FU resistance, implying that 5-FU-based chemotherapy would be effective even in tumors that become refractory to FTD during trifluridine/tipiracil treatment. 5-fluorouracil-based chemotherapy would be effective even in tumors that become refractory to trifluridine during combined trifluridine/tipiracil treatment. Copyright ©2018, American Association for Cancer Research.

TAF1, From a General Transcription Factor to Modulator of Androgen Receptor in Prostate Cancer

DTIC Science & Technology

2008-02-01

or pARR3tk-Luc), and the Renilla luciferase vector (pRL-TK) as an internal control. Cells were then treated with or without R1881 for 24 h prior to...increasing amount of HA tagged full length TAF1 (pCS2+/TAF1), pRL-TK- Renilla (83 ng/well), and pARR3-tk-Luc (167 ng/well) (A) or pPSA-Luc (1ug/well...numbers of pRL-TK- Renilla , as a non androgenic reporter was also enhanced by TAF1 over-expression in both LNCaP and PC3 cells, the ARR3tk-Luc

STAT3/NF-κB-Regulated Lentiviral TK/GCV Suicide Gene Therapy for Cisplatin-Resistant Triple-Negative Breast Cancer

PubMed Central

Kuo, Wei-Ying; Hwu, Luen; Wu, Chun-Yi; Lee, Jhih-Shian; Chang, Chi-Wei; Liu, Ren-Shyan

2017-01-01

Triple-negative breast cancer (TNBC) represents approximately 20% of all breast cancers and appears resistance to conventional cytotoxic chemotherapy, demonstrating a particularly poor prognosis and a significantly worse clinical outcome than other types of cancer. Suicide gene therapy has been used for the in vivo treatment of various solid tumors in recent clinical trials. In tumor microenvironment, STAT3/NF-κB pathways are constitutively activated in stromal cells as well as in cancer stem cells (CSCs). In this study, we have cloned a novel STAT3/NF-κB-based reporter system to drive the expression of herpes simplex virus thymidine kinase (HSV-TK) against breast cancer. Lentiviral vector expressing HSV-TK under the regulation of STAT3/NF-κB fused response element was developed. In this setting, we exploited the constitutive STAT3/NF-κB activation in tumors to achieve higher transgene expression than that driven by a constitutively active CMV promotor in vivo. An orthotropic MDA-MB-231 triple negative breast cancer mouse model was used for evaluating the feasibility of STAT3-NF-κB-TK/GCV suicide gene therapy system. The basal promoter activity of Lenti-CMV-TK and Lenti-STAT3-NF-κB-TK in MDA-MB-231 cells was compared by 3H-FEAU uptake assay. The Lenti-CMV-TK showed ~5 fold higher 3H-FEAU uptake then Lenti -STAT3-NF-κB-TK. In clonogenic assay, cells expressing Lenti-CMV-TK were 2-fold more sensitive to GCV than Lenti-STAT3-NF-κB-TK transduced cells. In vitro effect of STAT3-NF-κB-induced transgene expression was determined by 10ng/mL TNF-α induction and confirmed by western blot analysis and DsRedm fluorescent microscopy. In vivo evaluation of therapeutic effect by bioluminescence and [18F]FHBG microPET imaging indicated that Lenti-STAT3-NF-κB-TK showed more tumor growth retardation than Lenti-CMV-TK when GCV (20 mg/kg) was administered. The invasiveness and expression of cancer stem cell markers were both decreased after STAT3/NF-κB-regulated HSV-TK/GCV therapy. Moreover, STAT3/NF-κB signaling targeting could further sensitize tumor cells to cisplatin. This study successfully established a theranositic approach to treat triple-negative breast cancer via STAT3-NF-κB responsive element-driven suicide gene therapy. This platform may also be an alternative strategy to handle with drug-resistant cancer cells. PMID:28255357

Transcriptomic profiling of TK2 deficient human skeletal muscle suggests a role for the p53 signalling pathway and identifies growth and differentiation factor-15 as a potential novel biomarker for mitochondrial myopathies

PubMed Central

2014-01-01

Background Mutations in the gene encoding thymidine kinase 2 (TK2) result in the myopathic form of mitochondrial DNA depletion syndrome which is a mitochondrial encephalomyopathy presenting in children. In order to unveil some of the mechanisms involved in this pathology and to identify potential biomarkers and therapeutic targets we have investigated the gene expression profile of human skeletal muscle deficient for TK2 using cDNA microarrays. Results We have analysed the whole transcriptome of skeletal muscle from patients with TK2 mutations and compared it to normal muscle and to muscle from patients with other mitochondrial myopathies. We have identified a set of over 700 genes which are differentially expressed in TK2 deficient muscle. Bioinformatics analysis reveals important changes in muscle metabolism, in particular, in glucose and glycogen utilisation, and activation of the starvation response which affects aminoacid and lipid metabolism. We have identified those transcriptional regulators which are likely to be responsible for the observed changes in gene expression. Conclusion Our data point towards the tumor suppressor p53 as the regulator at the centre of a network of genes which are responsible for a coordinated response to TK2 mutations which involves inflammation, activation of muscle cell death by apoptosis and induction of growth and differentiation factor 15 (GDF-15) in muscle and serum. We propose that GDF-15 may represent a potential novel biomarker for mitochondrial dysfunction although further studies are required. PMID:24484525

Both p53-PUMA/NOXA-Bax-mitochondrion and p53-p21cip1 pathways are involved in the CDglyTK-mediated tumor cell suppression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Zhendong, E-mail: zdyu@hotmail.com; Wang, Hao; Zhang, Libin

CDglyTK fusion suicide gene has been well characterized to effectively kill tumor cells. However, the exact mechanism and downstream target genes are not fully understood. In our study, we found that CDglyTK/prodrug treatment works more efficiently in p53 wild-type (HONE1) cells than in p53 mutant (CNE1) cells. We then used adenovirus-mediated gene delivery system to either knockdown or overexpress p53 and its target genes in these cells. Consistent results showed that both p53-PUMA/NOXA/Bcl2-Bax and p53-p21 pathways contribute to the CDglyTK induced tumor cell suppression. Our work for the first time addressed the role of p53 related genes in the CDglyTK/prodrugmore » system.« less

MOLECULAR ANALYSIS OF MUTATIONS INDUCED BY MUTAGENS IN THE TK GENE OF MOUSE LYMPHOMA CELLS

EPA Science Inventory

MOLECULAR ANALYSIS OF MUTATIONS INDUCED BY BROMATE AND N- ETHYL-N-NITROSOUREA IN THE TK GENE OF MOUSE L YMPHOMA CELLS

The mouse lymphoma assay is widely used to identify chemical mutagens The Tk +1- gene located on an autosome in mouse lymphoma cells may recover a wide ra...

Activated Microglia Desialylate and Phagocytose Cells via Neuraminidase, Galectin-3, and Mer Tyrosine Kinase

PubMed Central

Nomura, Koji; Vilalta, Anna; Allendorf, David H.; Hornik, Tamara C.

2017-01-01

Activated microglia can phagocytose dying, stressed, or excess neurons and synapses via the phagocytic receptor Mer tyrosine kinase (MerTK). Galectin-3 (Gal-3) can cross-link surface glycoproteins by binding galactose residues that are normally hidden below terminal sialic acid residues. Gal-3 was recently reported to opsonize cells via activating MerTK. We found that LPS-activated BV-2 microglia rapidly released Gal-3, which was blocked by calcineurin inhibitors. Gal-3 bound to MerTK on microglia and to stressed PC12 (neuron-like) cells, and it increased microglial phagocytosis of PC12 cells or primary neurons, which was blocked by inhibition of MerTK. LPS-activated microglia exhibited a sialidase activity that desialylated PC12 cells and could be inhibited by Tamiflu, a neuraminidase (sialidase) inhibitor. Sialidase treatment of PC12 cells enabled Gal-3 to bind and opsonize the live cells for phagocytosis by microglia. LPS-induced microglial phagocytosis of PC12 was prevented by small interfering RNA knockdown of Gal-3 in microglia, lactose inhibition of Gal-3 binding, inhibition of neuraminidase with Tamiflu, or inhibition of MerTK by UNC569. LPS-induced phagocytosis of primary neurons by primary microglia was also blocked by inhibition of MerTK. We conclude that activated microglia release Gal-3 and a neuraminidase that desialylates microglial and PC12 surfaces, enabling Gal-3 binding to PC12 cells and their phagocytosis via MerTK. Thus, Gal-3 acts as an opsonin of desialylated surfaces, and inflammatory loss of neurons or synapses may potentially be blocked by inhibiting neuraminidases, Gal-3, or MerTK. PMID:28500071

Synergistic and Antagonistic Mutation Responses of Human MCL-5 Cells to Mixtures of Benzo[a]pyrene and 2-Amino-1-Methyl-6-Phenylimidazo[4,5-b]pyridine: Dose-Related Variation in the Joint Effects of Common Dietary Carcinogens.

PubMed

David, Rhiannon; Ebbels, Timothy; Gooderham, Nigel

2016-01-01

Chemical carcinogens such as benzo[a]pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) may contribute to the etiology of human diet-associated cancer. Individually, these compounds are genotoxic, but the consequences of exposure to mixtures of these chemicals have not been systematically examined. We determined the mutagenic response to mixtures of BaP and PhIP at concentrations relevant to human exposure (micromolar to subnanomolar). Human MCL-5 cells (metabolically competent) were exposed to BaP or PhIP individually or in mixtures. Mutagenicity was assessed at the thymidine kinase (TK) locus, CYP1A activity was determined by ethoxyresorufin-O-deethylase (EROD) activity and qRT-PCR, and cell cycle was measured by flow cytometry. Mixtures of BaP and PhIP produced dose responses different from those of the individual chemicals; we observed remarkably increased mutant frequency (MF) at lower concentrations of the mixtures (not mutagenic individually), and decreased MF at higher concentrations of the mixtures, than the calculated predicted additive MF of the individual chemicals. EROD activity and CYP1A1 mRNA levels were correlated with TK MF, supporting involvement of the CYP1A family in mutation. Moreover, a cell cycle G2/M phase block was observed at high-dose combinations, consistent with DNA damage sensing and repair. Mixtures of these genotoxic chemicals produced mutation responses that differed from those expected for the additive effects of the individual chemicals. The increase in MF for certain combinations of chemicals at low concentrations that were not genotoxic for the individual chemicals, as well as the nonmonotonic dose response, may be important for understanding the mutagenic potential of food and the etiology of diet-associated cancers. David R, Ebbels T, Gooderham N. 2016. Synergistic and antagonistic mutation responses of human MCL-5 cells to mixtures of benzo[a]pyrene and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine: dose-related variation in the joint effects of common dietary carcinogens. Environ Health Perspect 124:88-96; http://dx.doi.org/10.1289/ehp.1409557.

Synergistic and Antagonistic Mutation Responses of Human MCL-5 Cells to Mixtures of Benzo[a]pyrene and 2-Amino-1-Methyl-6-Phenylimidazo[4,5-b]pyridine: Dose-Related Variation in the Joint Effects of Common Dietary Carcinogens

PubMed Central

David, Rhiannon; Ebbels, Timothy; Gooderham, Nigel

2015-01-01

Background Chemical carcinogens such as benzo[a]pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) may contribute to the etiology of human diet-associated cancer. Individually, these compounds are genotoxic, but the consequences of exposure to mixtures of these chemicals have not been systematically examined. Objectives We determined the mutagenic response to mixtures of BaP and PhIP at concentrations relevant to human exposure (micromolar to subnanomolar). Methods Human MCL-5 cells (metabolically competent) were exposed to BaP or PhIP individually or in mixtures. Mutagenicity was assessed at the thymidine kinase (TK) locus, CYP1A activity was determined by ethoxyresorufin-O-deethylase (EROD) activity and qRT-PCR, and cell cycle was measured by flow cytometry. Results Mixtures of BaP and PhIP produced dose responses different from those of the individual chemicals; we observed remarkably increased mutant frequency (MF) at lower concentrations of the mixtures (not mutagenic individually), and decreased MF at higher concentrations of the mixtures, than the calculated predicted additive MF of the individual chemicals. EROD activity and CYP1A1 mRNA levels were correlated with TK MF, supporting involvement of the CYP1A family in mutation. Moreover, a cell cycle G2/M phase block was observed at high-dose combinations, consistent with DNA damage sensing and repair. Conclusions Mixtures of these genotoxic chemicals produced mutation responses that differed from those expected for the additive effects of the individual chemicals. The increase in MF for certain combinations of chemicals at low concentrations that were not genotoxic for the individual chemicals, as well as the nonmonotonic dose response, may be important for understanding the mutagenic potential of food and the etiology of diet-associated cancers. Citation David R, Ebbels T, Gooderham N. 2016. Synergistic and antagonistic mutation responses of human MCL-5 cells to mixtures of benzo[a]pyrene and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine: dose-related variation in the joint effects of common dietary carcinogens. Environ Health Perspect 124:88–96; http://dx.doi.org/10.1289/ehp.1409557 PMID:26091049

Sensitivity of human cells expressing low-fidelity or weak-catalytic-activity variants of DNA polymerase ζ to genotoxic stresses.

PubMed

Suzuki, Tetsuya; Grúz, Petr; Honma, Masamitsu; Adachi, Noritaka; Nohmi, Takehiko

2016-09-01

Translesion DNA polymerases (TLS pols) play critical roles in defense mechanisms against genotoxic agents. The defects or mutations of TLS pols are predicted to result in hypersensitivity of cells to environmental mutagens. In this study, human cells expressing DNA polymerase ζ (Pol ζ) variants with low fidelity or weak catalytic activity have been established with Nalm-6-MSH+ cells and their sensitivity to mutagenicity and cytotoxicity of benzo[a]pyrene diol epoxide (BPDE) and ultraviolet-C light (UV-C) was examined. The low-fidelity mutants were engineered by knocking-in DNA sequences that direct changes of leucine 2618 to either phenylalanine (L2618F) or methionine (L2618M) of Pol ζ. The weak-catalytic-activity mutants were generated by knocking-in DNA sequences that direct changes of either tyrosine 2779 to phenylalanine (Y2779F) or aspartate 2781 to asparagine (D2781N). In addition, a +1 frameshift mutation, i.e., CCC to CCCC, was introduced in the coding region of the TK1 gene to measure the mutant frequencies. Doubling time and spontaneous TK mutant frequencies of the established cell lines were similar to those of the wild-type cells. The low-fidelity mutants displayed, however, higher sensitivity to the mutagenicity of BPDE and UV-C than the wild-type cells although their cytotoxic sensitivity was not changed. In contrast, the weak-catalytic-activity mutants were more sensitive to the cytotoxicity of BPDE and UV-C than the wild-type cells, and displayed much higher sensitivity to the clastogenicity of BPDE than the wild-type cells in an in vitro micronucleus assay. These results indicate that human Pol ζ is involved in TLS across DNA lesions induced by BPDE and UV-C and also that the TLS plays important roles in induction of mutations, clastogenicity and in cellular survival of the damaged human cells. Similarities and differences in in vivo roles of yeast and human Pol ζ in genome integrity are discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

Targeted Adenoviral Vector Demonstrates Enhanced Efficacy for In Vivo Gene Therapy of Uterine Leiomyoma

PubMed Central

Abdelaziz, Mohamed; Sherif, Lotfy; ElKhiary, Mostafa; Nair, Sanjeeta; Shalaby, Shahinaz; Mohamed, Sara; Eziba, Noura; El-Lakany, Mohamed; Curiel, David; Ismail, Nahed; Diamond, Michael P.; Al-Hendy, Ayman

2016-01-01

Background: Gene therapy is a potentially effective non-surgical approach for the treatment of uterine leiomyoma. We demonstrated that targeted adenovirus vector, Ad-SSTR-RGD-TK/GCV, was highly effective in selectively inducing apoptosis and inhibiting proliferation of human leiomyoma cells in vitro while sparing normal myometrial cells. Study design: An in-vivo study, to compare efficacy and safety of modified adenovirus vector Ad-SSTR-RGD-TK/GCV versus untargeted vector for treatment of leiomyoma. Materials and methods: Female nude mice were implanted with rat leiomyoma cells subcutaneously. Then mice were randomized into three groups. Group 1 received Ad-LacZ (marker gene), Group 2 received untargeted Ad-TK, and Group 3 received the targeted Ad-SSTR-RGD-TK. Tumors were measured weekly for 4 weeks. Then mice were sacrificed and tissue samples were collected. Evaluation of markers of apoptosis, proliferation, extracellular matrix, and angiogenesis was performed using Western Blot & Immunohistochemistry. Statistical analysis was done using ANOVA. Dissemination of adenovirus was assessed by PCR. Results: In comparison with the untargeted vector, the targeted adenoviral vector significantly shrank leiomyoma size (P < 0.05), reduced expression of proliferation marker (PCNA) (P < 0.05), induced expression of apoptotic protein, c-PARP-1, (P < 0.05) and inhibited expression of extracellular matrix-related genes (TGF beta 3) and angiogenesis-related genes (VEGF & IGF-1) (P < 0.01). There were no detectable adenovirus in tested tissues other than leiomyoma lesions with both targeted and untargeted adenovirus. Conclusion: Targeted adenovirus, effectively reduces tumor size in leiomyoma without dissemination to other organs. Further evaluation of this localized targeted strategy for gene therapy is needed in appropriate preclinical humanoid animal models in preparation for a future pilot human trial. PMID:26884457

Targeted Adenoviral Vector Demonstrates Enhanced Efficacy for In Vivo Gene Therapy of Uterine Leiomyoma.

PubMed

Abdelaziz, Mohamed; Sherif, Lotfy; ElKhiary, Mostafa; Nair, Sanjeeta; Shalaby, Shahinaz; Mohamed, Sara; Eziba, Noura; El-Lakany, Mohamed; Curiel, David; Ismail, Nahed; Diamond, Michael P; Al-Hendy, Ayman

2016-04-01

Gene therapy is a potentially effective non-surgical approach for the treatment of uterine leiomyoma. We demonstrated that targeted adenovirus vector, Ad-SSTR-RGD-TK/GCV, was highly effective in selectively inducing apoptosis and inhibiting proliferation of human leiomyoma cells in vitro while sparing normal myometrial cells. An in-vivo study, to compare efficacy and safety of modified adenovirus vector Ad-SSTR-RGD-TK/GCV versus untargeted vector for treatment of leiomyoma. Female nude mice were implanted with rat leiomyoma cells subcutaneously. Then mice were randomized into three groups. Group 1 received Ad-LacZ (marker gene), Group 2 received untargeted Ad-TK, and Group 3 received the targeted Ad-SSTR-RGD-TK. Tumors were measured weekly for 4 weeks. Then mice were sacrificed and tissue samples were collected. Evaluation of markers of apoptosis, proliferation, extracellular matrix, and angiogenesis was performed using Western Blot & Immunohistochemistry. Statistical analysis was done using ANOVA. Dissemination of adenovirus was assessed by PCR. In comparison with the untargeted vector, the targeted adenoviral vector significantly shrank leiomyoma size (P < 0.05), reduced expression of proliferation marker (PCNA) (P < 0.05), induced expression of apoptotic protein, c-PARP-1, (P < 0.05) and inhibited expression of extracellular matrix-related genes (TGF beta 3) and angiogenesis-related genes (VEGF & IGF-1) (P < 0.01). There were no detectable adenovirus in tested tissues other than leiomyoma lesions with both targeted and untargeted adenovirus. Targeted adenovirus, effectively reduces tumor size in leiomyoma without dissemination to other organs. Further evaluation of this localized targeted strategy for gene therapy is needed in appropriate preclinical humanoid animal models in preparation for a future pilot human trial. © The Author(s) 2016.

Molecular Cloning and Characterization of Taurocyamine Kinase from Clonorchis sinensis: A Candidate Chemotherapeutic Target

PubMed Central

Tokuhiro, Shinji; Nagataki, Mitsuru; Jarilla, Blanca R.; Nomura, Haruka; Kim, Tae Im; Hong, Sung-Jong; Agatsuma, Takeshi

2013-01-01

Background Adult Clonorchis sinensis lives in the bile duct and causes endemic clonorchiasis in East Asian countries. Phosphagen kinases (PK) constitute a highly conserved family of enzymes, which play a role in ATP buffering in cells, and are potential targets for chemotherapeutic agents, since variants of PK are found only in invertebrate animals, including helminthic parasites. This work is conducted to characterize a PK from C. sinensis and to address further investigation for future drug development. Methology/Principal findings A cDNA clone encoding a putative polypeptide of 717 amino acids was retrieved from a C. sinensis transcriptome. This polypeptide was homologous to taurocyamine kinase (TK) of the invertebrate animals and consisted of two contiguous domains. C. sinensis TK (CsTK) gene was reported and found consist of 13 exons intercalated with 12 introns. This suggested an evolutionary pathway originating from an arginine kinase gene group, and distinguished annelid TK from the general CK phylogenetic group. CsTK was found not to have a homologous counterpart in sequences analysis of its mammalian hosts from public databases. Individual domains of CsTK, as well as the whole two-domain enzyme, showed enzymatic activity and specificity toward taurocyamine substrate. Of the CsTK residues, R58, I60 and Y84 of domain 1, and H60, I63 and Y87 of domain 2 were found to participate in binding taurocyamine. CsTK expression was distributed in locomotive and reproductive organs of adult C. sinensis. Developmentally, CsTK was stably expressed in both the adult and metacercariae stages. Recombinant CsTK protein was found to have low sensitivity and specificity toward C. sinensis and platyhelminth-infected human sera on ELISA. Conclusion CsTK is a promising anti-C. sinensis drug target since the enzyme is found only in the C. sinensis and has a substrate specificity for taurocyamine, which is different from its mammalian counterpart, creatine. PMID:24278491

Dioscin augments HSV-tk-mediated suicide gene therapy for melanoma by promoting connexin-based intercellular communication

PubMed Central

Li, Bin; Wu, Yingya; Liu, Xijuan; Tan, Yuhui; Du, Biaoyan

2017-01-01

Suicide gene therapy is a promising strategy against melanoma. However, the low efficiency of the gene transfer technique can limit its application. Our preliminary data showed that dioscin, a glucoside saponin, could upregulate the expression of connexins Cx26 and Cx43, major components of gap junctions, in melanoma cells. We hypothesized that dioscin may increase the bystander effect of herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) through increasing the formation of gap junctions. Further analysis showed that dioscin indeed could increase the gap junctional intercellular communication in B16 melanoma cells, resulting in more efficient GCV-induced bystander killing in B16tk cells. By contrast, overexpression of dominant negative Cx43 impaired the cell-cell communication of B16 cells and subsequently weakened the bystander effect of HSV-tk/GCV gene therapy. In vivo, combination treatment with dioscin and GCV of tumor-bearing mice with 30% positive B16tk cells and 70% wild-type B16 cells caused a significant reduction in tumor volume and weight compared to treatment with GCV or dioscin alone. Taken together, these results demonstrated that dioscin could augment the bystander effect of the HSV-tk/GCV system through increasing connexin-mediated gap junction coupling. PMID:27903977

Construction of membrane-anchoring fusion protein of Thermococcus kodakaraensis glycerol kinase and its application to repetitive batchwise reactions.

PubMed

Restiawaty, Elvi; Honda, Kohsuke; Okano, Kenji; Hirota, Ryuichi; Omasa, Takeshi; Kuroda, Akio; Ohtake, Hisao

2012-04-01

We previously demonstrated the stoichiometric conversion of glycerol to glycerol-3-phosphate (G3P) using Escherichia coli recombinants producing the ATP-dependent glycerol kinase of the hyperthermophile Thermococcus kodakaraensis (TkGK) and the polyphosphate kinase of Thermus thermophilus HB27 (TtPPK). TtPPK was associated with the membrane fraction of E. coli recombinants, whereas TkGK was released from the cells during the reaction at 70°C. In this study, TkGK was fused with either TtPPK or an E. coli membrane-intrinsic protein, YedZ, to minimize the heat-induced leakage of TkGK. When the E. coli recombinants having these fusion proteins were incubated at 70°C for 2h, more than 80% of TkGK activity was retained in the heated E. coli cells. However, the yields of G3P production by E. coli having the fusion proteins of TtPPK and TkGK were only less than 35%. Polyphosphate is a strong chelator for metal ions and has an inhibitory effect on TkGK which requires magnesium. Insufficient space between TtPPK and TkGK might enhance the inhibitory effect of polyphosphate on TkGK activity of the fusion protein. The mixture of E. coli cells having TtPPK and those having TkGK fused with YedZ converted 80% of glycerol into G3P. These recombinant cells could be easily recovered from the reaction mixture by centrifugation and repeatedly used without a significant loss of enzyme activities. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Neural differentiation promoted by truncated trkC receptors in collaboration with p75(NTR).

PubMed

Hapner, S J; Boeshore, K L; Large, T H; Lefcort, F

1998-09-01

trkC receptors, which serve critical functions during the development of the nervous system, are alternatively spliced to yield isoforms containing the catalytic tyrosine kinase domain (TK+) and truncated isoforms which lack this domain (TK-). To test for potential differences in their roles during early stages of neural development, TK+ and TK- isoforms were ectopically expressed in cultures of neural crest, the stem cell population that gives rise to the vast majority of the peripheral nervous system. NT-3 activation of ectopically expressed trkC TK+ receptors promoted both proliferation of neural crest cells and neuronal differentiation. Strikingly, the trkC TK- isoform was significantly more effective at promoting neuronal differentiation, but had no effect on proliferation. Furthermore, the trkC TK- response was dependent on a conserved receptor cytoplasmic domain and required the participation of the p75(NTR) neurotrophin receptor. Antibody-mediated receptor dimerization of TK+ receptors, but not TK- receptors, was sufficient to stimulate differentiation. These data identify a phenotypic response to activation of the trkC TK- receptor and demonstrate a functional interaction with p75(NTR), indicating there may be multiple trkC receptor-mediated systems guiding neuronal differentiation. Copyright 1998 Academic Press.

Thymidine plaque autoradiography of thymidine kinase-positive and thymidine kinase-negative herpesviruses.

PubMed Central

Tenser, R B; Jones, J C; Ressel, S J; Fralish, F A

1983-01-01

Plaques formed by herpes simplex virus (HSV), pseudorabies virus, and varicella-zoster virus were studied by plaque autoradiography after [14C]thymidine labeling. Standard thymidine kinase-positive (TK+) viruses and TK- mutants of HSV types 1 and 2 and pseudorabies virus were studied, including cell cultured viruses and viruses isolated from animals. Autoradiography was performed with X-ray film with an exposure time of 5 days. After development of films, TK+ plaques showed dark rims due to isotope incorporation, whereas TK- plaques were minimally labeled. Plaque autoradiography of stock TK- viruses showed reversion frequencies to the TK+ phenotype of less than 10(-3). Autoradiography indicated that TK- virus retained the TK- phenotype after replication in vivo. In addition, it was shown that TK- HSV could be isolated from mouse trigeminal ganglion tissue after corneal inoculation of TK- HSV together with TK+ HSV. The plaque autoradiographic procedure was very useful to evaluate proportions of TK+ and TK- virus present in TK+-TK- virus mixtures. Images PMID:6826696

Characterization of BRCA2 Transcriptional Regulation

DTIC Science & Technology

2000-08-01

Renilla luciferase vector (Promega) with 4 ll of Fugene-6 was used for each transfection. The pRL-TK Renilla luciferase activity was used to control for...experiments, cells received 0.5 jig of BRCA2 promoter construct, 0.1 jig of pRL-TK Renilla luciferase vector, and 0.5 jig of the indicated expression...Myc, and pCMV-Max. Firefly lucifer- ase and renilla luciferase assays were performed using the Dual-Luciferase Reporter Assay Sys- tem (Promega

Conditional Cytotoxic Anti-HIV Gene Therapy for Selectable Cell Modification

PubMed Central

Garg, Himanshu; Joshi, Anjali

2016-01-01

Gene therapy remains one of the potential strategies to achieve a cure for HIV infection. One of the major limitations of anti-HIV gene therapy concerns recovering an adequate number of modified cells to generate an HIV-proof immune system. Our study addresses this issue by developing a methodology that can mark conditional vector-transformed cells for selection and subsequently target HIV-infected cells for elimination by treatment with ganciclovir (GCV). We used the herpes simplex virus thymidine kinase (TK) mutant SR39, which is highly potent at killing cells at low GCV concentrations. This gene was cloned into a conditional HIV vector, pNL-GFPRRESA, which expresses the gene of interest as well as green fluorescent protein (GFP) in the presence of HIV Tat protein. We show here that TK-SR39 was more potent that wild-type TK (TK-WT) at eliminating infected cells at lower concentrations of GCV. As the vector expresses GFP in the presence of Tat, transient expression of Tat either by Tat RNA transfection or transduction by a nonintegrating lentiviral (NIL) vector marked the cells with GFP for selection. In cells selected by this strategy, TK-SR39 was more potent at limiting virus replication than TK-WT. Finally, in Jurkat cells modified and selected by this approach, infection with CXCR4-tropic Lai virus could be suppressed by treatment with GCV. GCV treatment limited the number of HIV-infected cells, virus production, as well as virus-induced cytopathic effects in this model. We provide proof of principle that TK-SR39 in a conditional HIV vector can provide a safe and effective anti-HIV strategy. PMID:26800572

Conditional Cytotoxic Anti-HIV Gene Therapy for Selectable Cell Modification.

PubMed

Garg, Himanshu; Joshi, Anjali

2016-05-01

Gene therapy remains one of the potential strategies to achieve a cure for HIV infection. One of the major limitations of anti-HIV gene therapy concerns recovering an adequate number of modified cells to generate an HIV-proof immune system. Our study addresses this issue by developing a methodology that can mark conditional vector-transformed cells for selection and subsequently target HIV-infected cells for elimination by treatment with ganciclovir (GCV). We used the herpes simplex virus thymidine kinase (TK) mutant SR39, which is highly potent at killing cells at low GCV concentrations. This gene was cloned into a conditional HIV vector, pNL-GFPRRESA, which expresses the gene of interest as well as green fluorescent protein (GFP) in the presence of HIV Tat protein. We show here that TK-SR39 was more potent that wild-type TK (TK-WT) at eliminating infected cells at lower concentrations of GCV. As the vector expresses GFP in the presence of Tat, transient expression of Tat either by Tat RNA transfection or transduction by a nonintegrating lentiviral (NIL) vector marked the cells with GFP for selection. In cells selected by this strategy, TK-SR39 was more potent at limiting virus replication than TK-WT. Finally, in Jurkat cells modified and selected by this approach, infection with CXCR4-tropic Lai virus could be suppressed by treatment with GCV. GCV treatment limited the number of HIV-infected cells, virus production, as well as virus-induced cytopathic effects in this model. We provide proof of principle that TK-SR39 in a conditional HIV vector can provide a safe and effective anti-HIV strategy.

Computer-aided active-site-directed modeling of the Herpes Simplex Virus 1 and human thymidine kinase

NASA Astrophysics Data System (ADS)

Folkers, Gerd; Trumpp-Kallmeyer, Susanne; Gutbrod, Oliver; Krickl, Sabine; Fetzer, Jürgen; Keil, Günther M.

1991-10-01

Thymidine kinase (TK), which is induced by Herpes Simplex Virus 1 (HSV1), plays a key role in the antiviral activity of guanine derivatives such as aciclovir (ACV). In contrast, ACV shows only low affinity to the corresponding host cell enzyme. In order to define the differences in substrate binding of the two enzymes on molecular level, models for the three-dimensional (3-D) structures of the active sites of HSV1-TK and human TK were developed. The reconstruction of the active sites started from primary and secondary structure analysis of various kinases. The results were validated to homologous enzymes with known 3-D structures. The models predict that both enzymes consist of a central core β-sheet structure, connected by loops and α-helices very similar to the overall structure of other nucleotide binding enzymes. The phosphate binding is made up of a highly conserved glycine-rich loop at the N-terminus of the proteins and a conserved region at the C-terminus. The thymidine recognition site was found about 100 amino acids downstream from the phosphate binding loop. The differing substrate specificity of human and HSV1-TK can be explained by amino-acid substitutions in the homologous regions. To achieve a better understanding of the structure of the active site and how the thymidine kinase proteins interact with their substrates, the corresponding complexes of thymidine and dihydroxypropoxyguanine (DHPG) with HSV1 and human TK were built. For the docking of the guanine derivative, the X-ray structure of Elongation Factor Tu (EF-Tu), co-crystallized with guanosine diphosphate, was taken as reference. Fitting of thymidine into the active sites was done with respect to similar interactions found in thymidylate kinase. To complement the analysis of the 3-D structures of the two kinases and the substrate enzyme interactions, site-directed mutagenesis of the thymidine recognition site of HSV1-TK has been undertaken, changing Asp162 in the thymidine recognition site into Asn. First investigations reveal that the enzymatic activity of the mutant protein is destroyed.

T-cell suicide gene therapy prompts thymic renewal in adults after hematopoietic stem cell transplantation.

PubMed

Vago, Luca; Oliveira, Giacomo; Bondanza, Attilio; Noviello, Maddalena; Soldati, Corrado; Ghio, Domenico; Brigida, Immacolata; Greco, Raffaella; Lupo Stanghellini, Maria Teresa; Peccatori, Jacopo; Fracchia, Sergio; Del Fiacco, Matteo; Traversari, Catia; Aiuti, Alessandro; Del Maschio, Alessandro; Bordignon, Claudio; Ciceri, Fabio; Bonini, Chiara

2012-08-30

The genetic modification of T cells with a suicide gene grants a mechanism of control of adverse reactions, allowing safe infusion after partially incompatible hematopoietic stem cell transplantation (HSCT). In the TK007 clinical trial, 22 adults with hematologic malignancies experienced a rapid and sustained immune recovery after T cell-depleted HSCT and serial infusions of purified donor T cells expressing the HSV thymidine kinase suicide gene (TK+ cells). After a first wave of circulating TK+ cells, the majority of T cells supporting long-term immune reconstitution did not carry the suicide gene and displayed high numbers of naive lymphocytes, suggesting the thymus-dependent development of T cells, occurring only upon TK+ -cell engraftment. Accordingly, after the infusions, we documented an increase in circulating TCR excision circles and CD31+ recent thymic emigrants and a substantial expansion of the active thymic tissue as shown by chest tomography scans. Interestingly, a peak in the serum level of IL-7 was observed after each infusion of TK+ cells, anticipating the appearance of newly generated T cells. The results of the present study show that the infusion of genetically modified donor T cells after HSCT can drive the recovery of thymic activity in adults, leading to immune reconstitution.

CDB-4124 and its putative monodemethylated metabolite, CDB-4453, are potent antiprogestins with reduced antiglucocorticoid activity: in vitro comparison to mifepristone and CDB-2914.

PubMed

Attardi, Barbara J; Burgenson, Janet; Hild, Sheri A; Reel, Jerry R; Blye, Richard P

2002-02-25

To obtain selective antiprogestins, we have examined the in vitro antiprogestational/antiglucocorticoid properties of two novel compounds, CDB-4124 and the putative monodemethylated metabolite, CDB-4453, in transcription and receptor binding assays and compared them to CDB-2914 and mifepristone. All four antiprogestins bound with high affinity to rabbit uterine progestin receptors (PR) and recombinant human PR-A and PR-B (rhPR-A, rhPR-B) and were potent inhibitors of R5020-induced transactivation of the PRE2-tk-luciferase (PRE2-tk-LUC) reporter plasmid and endogenous alkaline phosphatase production in T47D-CO human breast cancer cells. None of these compounds exhibited agonist activity in these cells. Induction of luciferase activity was potentiated about five-fold by 8-Br-cAMP under basal conditions and to the same extent in the presence of the PR antagonists. Mifepristone bound to rabbit thymic glucocorticoid receptors (GR) with approximately twice the avidity of the CDB antiprogestins. Inhibition of GR-mediated transcription of PRE2-tk-LUC was assessed in HepG2 human hepatoblastoma cells. Mifepristone exhibited greater antiglucocorticoid activity than CDB-2914, 4124, and 4453, about 12-, 22-, and 185-fold, respectively. Thus, while there was a good correlation between binding to PR and functional activity of these antiprogestins, GR binding was not predictive of their glucocorticoid antagonist activity. In agreement with our in vivo results, CDB-4124 and CDB-4453, as well as CDB-2914, are potent antiprogestins in vitro, but show considerably less antiglucocorticoid activity than mifepristone.

Thymidine Kinase-Negative Herpes Simplex Virus 1 Can Efficiently Establish Persistent Infection in Neural Tissues of Nude Mice.

PubMed

Huang, Chih-Yu; Yao, Hui-Wen; Wang, Li-Chiu; Shen, Fang-Hsiu; Hsu, Sheng-Min; Chen, Shun-Hua

2017-02-15

Herpes simplex virus 1 (HSV-1) establishes latency in neural tissues of immunocompetent mice but persists in both peripheral and neural tissues of lymphocyte-deficient mice. Thymidine kinase (TK) is believed to be essential for HSV-1 to persist in neural tissues of immunocompromised mice, because infectious virus of a mutant with defects in both TK and UL24 is detected only in peripheral tissues, but not in neural tissues, of severe combined immunodeficiency mice (T. Valyi-Nagy, R. M. Gesser, B. Raengsakulrach, S. L. Deshmane, B. P. Randazzo, A. J. Dillner, and N. W. Fraser, Virology 199:484-490, 1994, https://doi.org/10.1006/viro.1994.1150). Here we find infiltration of CD4 and CD8 T cells in peripheral and neural tissues of mice infected with a TK-negative mutant. We therefore investigated the significance of viral TK and host T cells for HSV-1 to persist in neural tissues using three genetically engineered mutants with defects in only TK or in both TK and UL24 and two strains of nude mice. Surprisingly, all three mutants establish persistent infection in up to 100% of brain stems and 93% of trigeminal ganglia of adult nude mice at 28 days postinfection, as measured by the recovery of infectious virus. Thus, in mouse neural tissues, host T cells block persistent HSV-1 infection, and viral TK is dispensable for the virus to establish persistent infection. Furthermore, we found 30- to 200-fold more virus in neural tissues than in the eye and detected glycoprotein C, a true late viral antigen, in brainstem neurons of nude mice persistently infected with the TK-negative mutant, suggesting that adult mouse neurons can support the replication of TK-negative HSV-1. Acyclovir is used to treat herpes simplex virus 1 (HSV-1)-infected immunocompromised patients, but treatment is hindered by the emergence of drug-resistant viruses, mostly those with mutations in viral thymidine kinase (TK), which activates acyclovir. TK mutants are detected in brains of immunocompromised patients with persistent infection. However, answers to the questions as to whether TK-negative (TK - ) HSV-1 can establish persistent infection in brains of immunocompromised hosts and whether neurons in vivo are permissive for TK - HSV-1 remain elusive. Using three genetically engineered HSV-1 TK - mutants and two strains of nude mice deficient in T cells, we found that all three HSV-1 TK - mutants can efficiently establish persistent infection in the brain stem and trigeminal ganglion and detected glycoprotein C, a true late viral antigen, in brainstem neurons. Our study provides evidence that TK - HSV-1 can persist in neural tissues and replicate in brain neurons of immunocompromised hosts. Copyright © 2017 American Society for Microbiology.

Tumor Immunotherapy by Gene-circuit Recruited Immunomodulatory Systems (TIGRIS) for Prostate Cancer

DTIC Science & Technology

2017-09-01

Fu, X., Huang, W., and Cai, Z. (2014). Syn- thesizing AND gate genetic circuits based on CRISPR -Cas9 for identification of bladder cancer cells. Nat...and Lu, T.K. (2014). Multi- plexed and programmable regulation of gene networks with an integrated RNA and CRISPR /Cas toolkit in human cells. Mol

Cloning and characterization of full-length mouse thymidine kinase 2: the N-terminal sequence directs import of the precursor protein into mitochondria.

PubMed Central

Wang, L; Eriksson, S

2000-01-01

The subcellular localization of mitochondrial thymidine kinase (TK2) has been questioned, since no mitochondrial targeting sequences have been found in cloned human TK2 cDNAs. Here we report the cloning of mouse TK2 cDNA from a mouse full-length enriched cDNA library. The mouse TK2 cDNA codes for a protein of 270 amino acids, with a 40-amino-acid presumed N-terminal mitochondrial targeting signal. In vitro translation and translocation experiments with purified rat mitochondria confirmed that the N-terminal sequence directed import of the precursor TK2 into the mitochondrial matrix. A single 2.4 kb mRNA transcript was detected in most tissues examined, except in liver, where an additional shorter (1.0 kb) transcript was also observed. There was no correlation between the tissue distribution of TK2 activity and the expression of TK2 mRNA. Full-length mouse TK2 protein and two N-terminally truncated forms, one of which corresponds to the mitochondrial form of TK2 and a shorter form corresponding to the previously characterized recombinant human TK2, were expressed in Escherichia coli and affinity purified. All three forms of TK2 phosphorylated thymidine, deoxycytidine and 2'-deoxyuridine, but with different kinetic efficiencies. A number of cytostatic pyrimidine nucleoside analogues were also tested and shown to be good substrates for the various forms of TK2. The active form of full-length mouse TK2 was a dimer, as judged by Superdex 200 chromatography. These results enhance our understanding of the structure and function of TK2, and may help to explain the mitochondrial disorder, mitochondrial neurogastrointestinal encephalomyopathy. PMID:11023833

Interleukin-15-transferred cytokine-induced killer cells elevated anti-tumor activity in a gastric tumor-bearing nude mice model.

PubMed

Peng, Zheng; Liang, Wentao; Li, Zexue; Xu, Yingxin; Chen, Lin

2016-02-01

Gastric cancer is the second leading cause of cancer-related mortality worldwide. Adoptive cell therapy (ACT) for gastric cancer is a novel therapy modality. However, the therapeutic effectiveness in vivo is still limited. The objective of this study was to assess the value of interleukin-15 (IL-15)-transferred cytokine-induced killer (CIK) cells in ACT for gastric cancer. IL-15-IRES-TK retroviral vector was constructed and transferred into the CIK cells. A gastric tumor-bearing nude mice model was constructed by subcutaneously injecting gastric cancer cells, BGC-823. Gastric tumor-bearing nude mice were randomly divided into three groups (five mice each group) and injected with physiological saline, CIK cells, and IL-15-IRES-TK-transfected CIK cells for 2 weeks, respectively. IL-15-IRES-TK-transferred CIK cells were prepared successfully and flow cytometry (FCM) analysis indicated that the transfection rate reached 85.7% after 5 days culture. In vivo experiment, we found that CIK cells retarded tumor growth by reducing tumor volume and tumor weight, as well as increasing tumor inhibition rate. Furthermore, IL-15-IRES-TK-transferred CIK cells showed a much stronger inhibition on tumor growth than CIK cells alone. Tumor morphology observation and growth indexes also showed that IL-15-transfected CIK cells had stronger cytotoxicity to tumor tissue than CIK cells. IL-15-IRES-TK transfection could elevate the effects of CIK cells to gastric carcinoma. The engineered CIK cells carrying IL-15-IRES-TK may be used in the ACT for gastric carcinoma, but prudent clinical trial is still indispensable. © 2015 International Federation for Cell Biology.

Combined antitumor gene therapy with herpes simplex virus-thymidine kinase and short hairpin RNA specific for mammalian target of rapamycin.

PubMed

Woo, Ha-Na; Lee, Won Il; Kim, Ji Hyun; Ahn, Jeonghyun; Han, Jeong Hee; Lim, Sue Yeon; Lee, Won Woo; Lee, Heuiran

2015-12-01

A proof-of-concept study is presented using dual gene therapy that employed a small hairpin RNA (shRNA) specific for mammalian target of rapamycin (mTOR) and a herpes simplex virus-thymidine kinase (HSV-TK) gene to inhibit the growth of tumors. Recombinant adeno-associated virus (rAAV) vectors containing a mutant TK gene (sc39TK) were transduced into HeLa cells, and the prodrug ganciclovir (GCV) was administered to establish a suicide gene-therapy strategy. Additionally, rAAV vectors expressing an mTOR-targeted shRNA were employed to suppress mTOR-dependent tumor growth. GCV selectively induced death in tumor cells expressing TK, and the mTOR-targeted shRNA altered the cell cycle to impair tumor growth. Combining the TK-GCV system with mTOR inhibition suppressed tumor growth to a greater extent than that achieved with either treatment alone. Furthermore, HSV-TK expression and mTOR inhibition did not mutually interfere with each other. In conclusion, gene therapy that combines the TK-GCV system and mTOR inhibition shows promise as a novel strategy for cancer therapy.

Genistein and tyrphostin AG556 decrease ultra-rapidly activating delayed rectifier K+ current of human atria by inhibiting EGF receptor tyrosine kinase.

PubMed

Xiao, Guo-Sheng; Zhang, Yan-Hui; Wu, Wei; Sun, Hai-Ying; Wang, Yan; Li, Gui-Rong

2017-03-01

The ultra-rapidly activating delayed rectifier K + current I Kur (encoded by K v 1.5 or KCNA5) plays an important role in human atrial repolarization. The present study investigates the regulation of this current by protein tyrosine kinases (PTKs). Whole-cell patch voltage clamp technique and immunoprecipitation and Western blotting analysis were used to investigate whether the PTK inhibitors genistein, tyrphostin AG556 (AG556) and PP2 regulate human atrial I Kur and hKv1.5 channels stably expressed in HEK 293 cells. Human atrial I Kur was decreased by genistein (a broad-spectrum PTK inhibitor) and AG556 (a highly selective EGFR TK inhibitor) in a concentration-dependent manner. Inhibition of I Kur induced by 30 μM genistein or 10 μM AG556 was significantly reversed by 1 mM orthovanadate (a protein tyrosine phosphatase inhibitor). Similar results were observed in HEK 293 cells stably expressing hK v 1.5 channels. On the other hand, the Src family kinase inhibitor PP2 (1 μM) slightly enhanced I Kur and hK v 1.5 current, and the current increase was also reversed by orthovanadate. Immunoprecipitation and Western blotting analysis showed that genistein, AG556, and PP2 decreased tyrosine phosphorylation of hK v 1.5 channels and that the decrease was countered by orthovanadate. The PTK inhibitors genistein and AG556 decrease human atrial I Kur and cloned hK v 1.5 channels by inhibiting EGFR TK, whereas the Src kinase inhibitor PP2 increases I Kur and hK v 1.5 current. These results imply that EGFR TK and the soluble Src kinases may have opposite effects on human atrial I Kur . © 2017 The British Pharmacological Society.

Plasma thymidine kinase-1 activity predicts outcome in patients with hormone receptor positive and HER2 negative metastatic breast cancer treated with endocrine therapy

PubMed Central

Bonechi, Martina; Galardi, Francesca; Biagioni, Chiara; De Luca, Francesca; Bergqvist, Mattias; Neumüller, Magnus; Guarducci, Cristina; Boccalini, Giulia; Gabellini, Stefano; Migliaccio, Ilenia; Di Leo, Angelo; Pestrin, Marta; Malorni, Luca

2018-01-01

The aim of this study was to investigate if thymidine kinase-1 (TK1), a well-known proliferation marker, could represent a valid circulating biomarker to identify hormone receptor positive (HR+)/HER2 negative (HER2neg) metastatic breast cancer (MBC) patients most likely to benefit from endocrine therapy (ET). We used the DiviTum™ assay to analyze TK1 activity in cell lysates of three HR+/HER2neg BC cell lines and in plasma of 31 HR+/HER2neg MBC patients receiving ET. Blood samples were collected at treatment initiation, after one month and at disease progression. CTCs count and ESR1/PIK3CA mutations in circulating tumor DNA were performed and correlated with TK1 activity. TK1 activity was reduced in the two endocrine-sensitive cell lines after 2 days of treatment. In patients, high baseline TK1 activity correlated with CTCs positivity (p-value=0.014). Patients with low baseline levels of TK1 activity had a significantly better PFS compared to those with high baseline TK1 activity (p-value=0.012). Patients with an early drop of TK1 activity after one month of treatment had a significantly better PFS compared to those who experienced an increase (p-value=0.0026). Our study suggests that TK1 could be a potential prognostic, predictive and monitoring marker of early ET response in HR+/HER2neg MBC patients. PMID:29662653

Plasma thymidine kinase-1 activity predicts outcome in patients with hormone receptor positive and HER2 negative metastatic breast cancer treated with endocrine therapy.

PubMed

Bonechi, Martina; Galardi, Francesca; Biagioni, Chiara; De Luca, Francesca; Bergqvist, Mattias; Neumüller, Magnus; Guarducci, Cristina; Boccalini, Giulia; Gabellini, Stefano; Migliaccio, Ilenia; Di Leo, Angelo; Pestrin, Marta; Malorni, Luca

2018-03-27

The aim of this study was to investigate if thymidine kinase-1 (TK1), a well-known proliferation marker, could represent a valid circulating biomarker to identify hormone receptor positive (HR+)/HER2 negative (HER2neg) metastatic breast cancer (MBC) patients most likely to benefit from endocrine therapy (ET). We used the DiviTum™ assay to analyze TK1 activity in cell lysates of three HR+/HER2neg BC cell lines and in plasma of 31 HR+/HER2neg MBC patients receiving ET. Blood samples were collected at treatment initiation, after one month and at disease progression. CTCs count and ESR1 / PIK3CA mutations in circulating tumor DNA were performed and correlated with TK1 activity. TK1 activity was reduced in the two endocrine-sensitive cell lines after 2 days of treatment. In patients, high baseline TK1 activity correlated with CTCs positivity (p-value=0.014). Patients with low baseline levels of TK1 activity had a significantly better PFS compared to those with high baseline TK1 activity (p-value=0.012). Patients with an early drop of TK1 activity after one month of treatment had a significantly better PFS compared to those who experienced an increase (p-value=0.0026). Our study suggests that TK1 could be a potential prognostic, predictive and monitoring marker of early ET response in HR+/HER2neg MBC patients.

Determination of Free Radical Scavenging, Antioxidative DNA Damage Activities and Phytochemical Components of Active Fractions from Lansium domesticum Corr. Fruit

PubMed Central

Klungsupya, Prapaipat; Suthepakul, Nava; Muangman, Thanchanok; Rerk-Am, Ubon; Thongdon-A, Jeerayu

2015-01-01

Lansium domesticum Corr. or “long-kong” is one of the most popular fruits in Thailand. Its peel (skin, SK) and seeds (SD) become waste unless recycled or applied for use. This study was undertaken to determine the bioactivity and phytochemical components of L. domesticum (LD) skin and seed extracts. Following various extraction and fractionation procedures, 12 fractions were obtained. All fractions were tested for antioxidant capacity against O2−• and OH•. It was found that the peel of L. domesticum fruits exhibited higher O2−• and OH• scavenging activity than seeds. High potential antioxidant activity was found in two fractions of 50% ethanol extract of peel followed by ethyl acetate (EA) fractionation (LDSK50-EA) and its aqueous phase (LDSK50-H2O). Therefore, these two active fractions were selected for further studies on their antioxidative activity against DNA damage by hydrogen peroxide (H2O2) in human TK6 cells using comet assay. The comet results revealed DNA-protective activity of both LDSK50-EA and LDSK50-H2O fractions when TK6 human lymphoblast cells were pre-treated at 25, 50, 100, and 200 μg/mL for 24 h prior to H2O2 exposure. The phytochemical analysis illustrated the presence of phenolic substances, mainly scopoletin, rutin, and chlorogenic acid, in these two active fractions. This study generates new information on the biological activity of L. domesticum. It will promote and strengthen the utilization of L. domesticum by-products. PMID:26287238

Novel Targeting Approach for Breast Cancer Gene Therapy

DTIC Science & Technology

2010-09-01

haloperidol and ibogaine)- conjugated polyamidoamine (PAMAM) dendrimers Poly(amidoamine) (PAMAM) dendrimers of 3.5 generation with carboxylate surface...Mukherjee A, Prasad TK, Rao NM, Banerjee R. Haloperidol associated stealth liposomes. A potent carrier for delivering genes to human breast cancer cells

In vivo marking of spontaneous or vaccine-induced fibrosarcomas in the domestic house cat, using an adenoviral vector containing a bifunctional fusion protein, GAL-TEK.

PubMed

Marini, F C; Cannon, J P; Belmont, J W; Shillitoe, E J; Lapeyre, J N

1995-09-01

We evaluated the ability of a replication-deficient, recombinant adenoviral vector to transfer the bifunctional gene GAL-TEK, which expresses a marking/therapeutic gene product, to naturally occurring cat fibrosarcomas in situ. GAL-TEK contains an in-frame fusion of the bacterial LacZ gene for histochemical marking of tumors with beta-galactosidase (beta-Gal) and the HSV tk gene for enzyme-prodrug activation of the prodrug ganciclovir (GCV) to induce selective tumor cell killing. GAL-TEK bifunctional marking and cell killing activities were tested in vitro after adenoviral vector infection of HT1080 human fibrosarcoma cells. The tk activity of GAL-TEK is shown to be almost as potent as HSV tk to catalyze conversion of GCV to GCV nucleotides and promote selective cell killing. Using 8 cats with recurring 2.5-cm2 fibrosarcomas that either arose spontaneously or were induced by vaccine, we determined experimentally the administration routes and times required for optimum GAL-TEK gene transfer by beta-Gal histological staining and reverse transcriptase polymerase chain reaction to the multiple compartments of the growing fibrosarcomas consonant with minimizing collateral infection of neighboring tissues and other unwanted side effects.

Vascular endothelial growth factor receptor 1 (VEGFR1) tyrosine kinase signaling facilitates granulation tissue formation with recruitment of VEGFR1+ cells from bone marrow.

PubMed

Park, Keiichi; Amano, Hideki; Ito, Yoshiya; Mastui, Yoshio; Kamata, Mariko; Yamazaki, Yasuharu; Takeda, Akira; Shibuya, Masabumi; Majima, Masataka

2018-06-01

Vascular endothelial growth factor (VEGF)-A facilitates wound healing. VEGF-A binds to VEGF receptor 1 (VEGFR1) and VEGFR2 and induces wound healing through the receptor's tyrosine kinase (TK) domain. During blood flow recovery and lung regeneration, expression of VEGFR1 is elevated. However, the precise mechanism of wound healing, especially granulation formation on VEGFR1, is not well understood. We hypothesized that VEGFR1-TK signaling induces wound healing by promoting granulation tissue formation. A surgical sponge implantation model was made by implanting a sponge disk into dorsal subcutaneous tissue of mice. Granulation formation was estimated from the weight of the sponge and the granulation area from the immunohistochemical analysis of collagen I. The expression of fibroblast markers was estimated from the expression of transforming growth factor-beta (TGF-β) and cellular fibroblast growth factor-2 (FGF-2) using real-time PCR (polymerase chain reaction) and from the immunohistochemical analysis of S100A4. VEGFR1 TK knockout (TK -/- ) mice exhibited suppressed granulation tissue formation compared to that in wild-type (WT) mice. Expression of FGF-2, TGF-β, and VEGF-A was significantly suppressed in VEGFR1 TK -/- mice, and the accumulation of VEGFR1 + cells in granulation tissue was reduced in VEGFR1 TK -/- mice compared to that in WT mice. The numbers of VEGFR1 + cells and S100A4 + cells derived from bone marrow (BM) were higher in WT mice transplanted with green fluorescent protein (GFP) transgenic WT BM than in VEGFR1 TK -/- mice transplanted with GFP transgenic VEGFR1 TK -/- BM. These results indicated that VEGFR1-TK signaling induced the accumulation of BM-derived VEGFR1 + cells expressing F4/80 and S100A4 and contributed to granulation formation around the surgically implanted sponge area in a mouse model.

A combinatorial approach of inclusion complexation and dendrimer synthesization for effective targeting EGFR-TK.

PubMed

Shende, Pravin; Patil, Sampada; Gaud, R S

2017-07-01

The aim of the present study was to use a combinatorial approach of inclusion complexation and dendrimer synthesization of gefitinib using solvent-free technique for targeting EGFR-TK to treat Non-Small-Cell Lung Cancer (NSCLC). The inclusion complex of gefitinib with β-cyclodextrin was prepared by trituration method. This complex encapsulated G4 PAMAM dendrimers were synthesized by Michael addition and amidation reactions using green chemistry and then PEGylated by conjugation reaction. FTIR and DSC confirmed the formation of inclusion complex of gefitinib and β-cyclodextrin and PEGylation of G4 PAMAM dendrimers. Gefitinib showed higher solubility, encapsulation efficiency and controlled release profile from PEGylated dendrimers compared to inclusion complex. The PEGylated dendrimers of inclusion complex of gefitinib were found to reduce hemolytic toxicity and lesser GI 50 value on Human lung cancer cell line A-549 by effective targeting EGFR-TK. A combinatorial approach of inclusion complexation and dendrimer synthesization is one of the alternative advanced approaches to treat NSCLC. Copyright © 2017 Elsevier B.V. All rights reserved.

Tumor-specific expression of shVEGF and suicide gene as a novel strategy for esophageal cancer therapy.

PubMed

Liu, Ting; Wu, Hai-Jun; Liang, Yu; Liang, Xu-Jun; Huang, Hui-Chao; Zhao, Yan-Zhong; Liao, Qing-Chuan; Chen, Ya-Qi; Leng, Ai-Min; Yuan, Wei-Jian; Zhang, Gui-Ying; Peng, Jie; Chen, Yong-Heng

2016-06-21

To develop a potent and safe gene therapy for esophageal cancer. An expression vector carrying fusion suicide gene (yCDglyTK) and shRNA against vascular endothelial growth factor (VEGF) was constructed and delivered into EC9706 esophageal cancer cells by calcium phosphate nanoparticles (CPNP). To achieve tumor selectivity, expression of the fusion suicide gene was driven by a tumor-specific human telomerase reverse transcriptase (hTERT) promoter. The biologic properties and therapeutic efficiency of the vector, in the presence of prodrug 5-fluorocytosine (5-FC), were evaluated in vitro and in vivo. Both in vitro and in vivo testing showed that the expression vector was efficiently introduced by CPNP into tumor cells, leading to cellular expression of yCDglyTK and decreased VEGF level. With exposure to 5-FC, it exhibited strong anti-tumor effects against esophageal cancer. Combination of VEGF shRNA with the fusion suicide gene demonstrated strong anti-tumor activity. The shVEGF-hTERT-yCDglyTK/5-FC system provided a novel approach for esophageal cancer-targeted gene therapy.

Oxidative Stress Induced S-glutathionylation and Proteolytic Degradation of Mitochondrial Thymidine Kinase 2*

PubMed Central

Sun, Ren; Eriksson, Staffan; Wang, Liya

2012-01-01

Protein glutathionylation in response to oxidative stress can affect both the stability and activity of target proteins. Mitochondrial thymidine kinase 2 (TK2) is a key enzyme in mitochondrial DNA precursor synthesis. Using an antibody specific for glutathione (GSH), S-glutathionylated TK2 was detected after the addition of glutathione disulfide (GSSG) but not GSH. This was reversed by the addition of dithiothreitol, suggesting that S-glutathionylation of TK2 is reversible. Site-directed mutagenesis of the cysteine residues and subsequent analysis of mutant enzymes demonstrated that Cys-189 and Cys-264 were specifically glutathionylated by GSSG. These cysteine residues do not appear to be part of the active site, as demonstrated by kinetic studies of the mutant enzymes. Treatment of isolated rat mitochondria with hydrogen peroxide resulted in S-glutathionylation of added recombinant TK2. Treatment of intact cells with hydrogen peroxide led to reduction of mitochondrial TK2 activity and protein levels, as well as S-glutathionylation of TK2. Furthermore, the addition of S-glutathionylated recombinant TK2 to mitochondria isolated from hydrogen peroxide-treated cells led to degradation of the S-glutathionylated TK2, which was not observed with unmodified TK2. S-Glutathionylation on Cys-189 was responsible for the observed selective degradation of TK2 in mitochondria. These results strongly suggest that oxidative damage-induced S-glutathionylation and degradation of TK2 have significant impact on mitochondrial DNA precursor synthesis. PMID:22661713

Cloning of the active thymidine kinase gene of herpes simplex virus type 1 in Escherichia coli K-12.

PubMed

Colbere-Garapin, F; Chousterman, S; Horodniceanu, F; Kourilsky, P; Garapin, A C

1979-08-01

A herpes simplex virus DNA fragment that is produced by digestion with BamHI endonuclease and carries the thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene has been cloned in Escherichia coli. A recombinat plasmid, pFG5, has been analyzed extensively and a detailed restriction map is presented. pFG5 DNA efficiently transforms TK- mouse L cells. The TK coding sequence in the cloned fragment has been localized and a smaller recombinant plasmid, pAG0, also carrying an active TK gene, has been constructed to serve as a more convenient vector for transfer, into TK- cells, of genes previously cloned in E. coli.

The low molecular weight Dextran 40 inhibits the adhesion of T lymphocytes to endothelial cells

PubMed Central

TERMEER, C C; WEISS, J M; SCHÖPF, E; VANSCHEIDT, W; SIMON, J C

1998-01-01

Dextrans are complex colloidal macromolecules widely used as haemorrheologic substances and anti-thrombotic agents. Here we describe a novel function of Dextran 40 by demonstrating an inhibition of T lymphocyte adhesion to endothelial cells (EC). We applied an established microassay in which constitutive and tumour necrosis factor-alpha (TNF-α)-induced binding of mouse T lymphoma cells (TK-1) to mouse endothelioma (eEND.2) cells is mediated by the interaction of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on EC with their counter-receptors the LFA-1 heterodimer (CD11a/CD18) and VLA-4 on T cells. Dextran 40 in therapeutically achievable levels (2–32 mg/ml) reduced both constitutive and TNF-α-stimulated TK-1 adhesion to eEND.2. Selective preincubation of eEND.2 or TK-1 revealed that Dextran 40 acted exclusively on the T cells. To explore further the mechanisms by which Dextran 40 interfered with TK-1 adhesion, their LFA-1 and VLA-4 expression was analysed by FACS. The surface expression levels of neither receptor were affected by Dextran 40. However, confocal microscopy revealed that Dextran 40 interfered with the activation-dependent capping and clustering of LFA-1 and VLA-4 on the surface of TK-1. We conclude that Dextran 40 inhibits the capacity of TK-1 T cells to adhere to eEND.2 endothelial cells and thus may be useful for therapeutic intervention in diseases associated with enhanced T lymphocyte binding to microvascular endothelium. PMID:9844053

Fentanyl Buccal Tablet: A New Breakthrough Pain Medication in Early Management of Severe Vaso-Occlusive Crisis in Sickle Cell Disease.

PubMed

De Franceschi, Lucia; Mura, Paolo; Schweiger, Vittorio; Vencato, Elisa; Quaglia, Francesca Maria; Delmonte, Letizia; Evangelista, Maurizio; Polati, Enrico; Olivieri, Oliviero; Finco, Gabriele

2016-07-01

Sickle cell disease (SCD) is a worldwide distributed hereditary red cell disorder. The principal clinical manifestations of SCD are the chronic hemolytic anemia and the acute vaso-occlusive crisis (VOCs), which are mainly characterized by ischemic/reperfusion tissue injury. Pain is the main symptom of VOCs, and its management is still a challenge for hematologists, requiring a multidisciplinary approach. We carried out a crossover study on adult SCD patients, who received two different types of multimodal analgesia during two separate severe VOCs with time interval between VOCs of at least 6 months. The first VOC episode was treated with ketorolac (0.86 mg/kg/day) and tramadol (7.2 mg/kg/day) (TK treatment). In the second VOC episode, fentanyl buccal tablet (FBT; 100 μg) was introduced in a single dose after three hours from the beginning of TK analgesia (TKF treatment). We focused on the first 24 hours of acute pain management. The primary efficacy measure was the time-weighted-sum of pain intensity differences (SPID24). The secondary efficacy measures included the pain intensity difference (PID), the total pain relief (TOTPAR), and the time-wighted sum of anxiety (SAID24). SPID24 was significantly higher in TKF than in TK treatment. All the secondary measures were significantly ameliorated in TKF compared to TK treatment, without major opioid side effects. Patients satisfaction was higher with TKF treatment than with TK one. We propose that VOCs might require breakthrough pain drug strategy as vaso-occlusive phenomena and enhanced vasoconstriction promoting acute ischemic pain component exacerbate the continuous pain of VOCs. FBT might be a powerful and feasible tool in early management of acute pain during VOCs in emergency departments. © 2015 World Institute of Pain.

Assessing the concentration of phthalate esters (PAEs) and bisphenol A (BPA) and the genotoxic potential of treated wastewater (final effluent) in Saudi Arabia.

PubMed

Al-Saleh, Iman; Elkhatib, Rola; Al-Rajoudi, Tahreer; Al-Qudaihi, Ghofran

2017-02-01

Plasticizers such as phthalate esters (PAEs) and bisphenol A (BPA) are highly persistent organic pollutants that tend to bio-accumulate in humans through the soil-plant-animal food chain. Some studies have reported the potential carcinogenic and teratogenic effects in addition to their estrogenic activities. Water resources are scarce in Saudi Arabia, and several wastewater treatment plants (WTPs) have been constructed for agricultural and industrial use. This study was designed to: (1) measure the concentrations of BPA and six PAEs, dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), butyl benzyl phthalate (BBP), bis (2-ethylhexyl) phthalate (DEHP) and dioctyl phthalate (DOP), in secondary- and tertiary-treated wastewater collected from five WTPs in three Saudi cities for four to five weeks and (2) test their potential genotoxicity. Three genotoxicological parameters were used: % tail DNA (%T), tail moment (TM) and percentage micronuclei (%MN). Both DBP and DEHP were detected in all treated wastewater samples. DMP, DEP, BBP, DOP, and BPA were found in 83.3, 84.2, 79, 73.7 and 97.4% of the samples, respectively. The levels of DMP (p<0.001), DOP (p<0.001) and BPA (p=0.001) were higher in tertiary- treated wastewater than secondary-treated wastewater, perhaps due to the influence of the molecular weight and polarity of the chemicals. Both weekly sampling frequency and WTP locations significantly affected the variability in our data. Treated wastewater from Wadi Al-Araj was able to induce DNA damage (%T and TM) in human lymphoblastoid TK6 cells that was statistically higher than wastewater from all other WTPs and in untreated TK6 cells (negative control). %MN in samples from both Wadi Al-Araj and Manfouah did not differ statistically but was significantly higher than in the untreated TK6 cells. This study also showed that the samples of tertiary-treated wastewater had a higher genotoxicological potential to induce DNA damage than the samples of secondary-treated wastewater. BPA and some PAEs in the treated wastewater might have the potential to induce genetic damage, despite their low levels. Genotoxicity, however, may also have been due to the presence of other contaminants. Our preliminary findings should be of concern to Saudi agriculture because long-term irrigation with treated wastewater could lead to the accumulation of PAEs and BPA in the soil and ultimately reach the human and animal food chain. WTPs need to remove pollutants more efficiently. Until then, a cautious use of treated wastewater for irrigation is recommended to avoid serious health impacts on local populations. Copyright © 2016 Elsevier B.V. All rights reserved.

Zidovudine Induces Downregulation of Mitochondrial Deoxynucleoside Kinases: Implications for Mitochondrial Toxicity of Antiviral Nucleoside Analogs

PubMed Central

Sun, Ren; Eriksson, Staffan

2014-01-01

Mitochondrial thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) catalyze the initial phosphorylation of deoxynucleosides in the synthesis of the DNA precursors required for mitochondrial DNA (mtDNA) replication and are essential for mitochondrial function. Antiviral nucleosides are known to cause toxic mitochondrial side effects. Here, we examined the effects of 3′-azido-2′,3′-dideoxythymidine (AZT) (zidovudine) on mitochondrial TK2 and dGK levels and found that AZT treatment led to downregulation of mitochondrial TK2 and dGK in U2OS cells, whereas cytosolic deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1) levels were not affected. The AZT effects on mitochondrial TK2 and dGK were similar to those of oxidants (e.g., hydrogen peroxide); therefore, we examined the oxidative effects of AZT. We found a modest increase in cellular reactive oxygen species (ROS) levels in the AZT-treated cells. The addition of uridine to AZT-treated cells reduced ROS levels and protein oxidation and prevented the degradation of mitochondrial TK2 and dGK. In organello studies indicated that the degradation of mitochondrial TK2 and dGK is a mitochondrial event. These results suggest that downregulation of mitochondrial TK2 and dGK may lead to decreased mitochondrial DNA precursor pools and eventually mtDNA depletion, which has significant implications for the regulation of mitochondrial nucleotide biosynthesis and for antiviral therapy using nucleoside analogs. PMID:25182642

Responses of the L51781Y tk/sup +//tk/sup -/ mouse lymphoma cell forward mutation assay: III. 72 coded chemicals

DOE Office of Scientific and Technical Information (OSTI.GOV)

McGregor, D.B.; Brown, A.; Cattanach, P.

Seventy-two chemicals were tested for their mutagenic potential in the L51781Y tk/sup +///sup -/ mouse lymphoma cell forward mutation assay, using procedures based upon those described previously. Cultures were exposed to the chemicals for 4 hr, then cultured for 2 days before planting in soft agar with or without trifluorothymidine (TFT), 3 ..mu..g/ml. The chemicals were tested at least twice. Significant responses were obtained with allyl isothiocyanate, p-benzoquinone dioxime, benzyl acetate, 2-biphenylamine HCl, bis(2-chloro-1-methylethyl)ether, cadmium chloride, chlordane, chlorobenzene, chlorobenzilate, 2-chloroethanol, chlorothalonil, cytarabine x HCl, p,p'-DDE, diazinon, 2,6-dichloro-p-phenylenediamine, N,N-diethylthiourea, diglycidylresorcinol ether, 2,4-dimethoxy aniline x HCl, disperse yellow 3, endosulfan, 1,2-epoxyhexadecane, ethylmore » acrylate, ethyl benzene, ethylene thiourea, F D and C yellow Number 6, furan, heptachlor, isophorone, mercuric chloride, 4,4'-methylenedianiline x 2 HCl, methyl viologen, nickel sulfate x 6H/sub 2/O, 4,4'-oxydianiline, pentachloroethane, piperonyl butoxide, propyl gallate, quinoline, rotenone, 2,4,5,6-tetrachloro-4-nitro-anisole, 1,1,1,2-tetrachloroethane, trichlorfon, 2,4,6-trichlorophenol, 2,4,5-trimethoxybenzaldehyde, 1,1,3-trimethyl-2-thiourea, 1-vinyl-3-cyclopetene dioxide, vinyl toluene, and ziram. The assay was incapable of providing a clear indication of whether some chemicals were mutagens; these benzyl alcohol, 1,4-dichlorobenzene, phenol, succinic acid-2,2-dimethyl hydrazide, and toluene.« less

Targeted expression of suicide gene by tissue-specific promoter and microRNA regulation for cancer gene therapy.

PubMed

Danda, Ravikanth; Krishnan, Gopinath; Ganapathy, Kalaivani; Krishnan, Uma Maheswari; Vikas, Khetan; Elchuri, Sailaja; Chatterjee, Nivedita; Krishnakumar, Subramanian

2013-01-01

In order to realise the full potential of cancer suicide gene therapy that allows the precise expression of suicide gene in cancer cells, we used a tissue specific Epithelial cell adhesion molecule (EpCAM) promoter (EGP-2) that directs transgene Herpes simplex virus-thymidine kinase (HSV-TK) expression preferentially in EpCAM over expressing cancer cells. EpCAM levels are considerably higher in retinoblastoma (RB), a childhood eye cancer with limited expression in normal cells. Use of miRNA regulation, adjacent to the use of the tissue-specific promoter, would provide the second layer of control to the transgene expression only in the tumor cells while sparing the normal cells. To test this hypothesis we cloned let-7b miRNA targets in the 3'UTR region of HSV-TK suicide gene driven by EpCAM promoter because let-7 family miRNAs, including let-7b, were found to be down regulated in the RB tumors and cell lines. We used EpCAM over expressing and let-7 down regulated RB cell lines Y79, WERI-Rb1 (EpCAM (+ve)/let-7b(down-regulated)), EpCAM down regulated, let-7 over expressing normal retinal Müller glial cell line MIO-M1(EpCAM (-ve)/let-7b(up-regulated)), and EpCAM up regulated, let-7b up-regulated normal thyroid cell line N-Thy-Ori-3.1(EpCAM (+ve)/let-7b(up-regulated)) in the study. The cell proliferation was measured by MTT assay, apoptosis was measured by probing cleaved Caspase3, EpCAM and TK expression were quantified by Western blot. Our results showed that the EGP2-promoter HSV-TK (EGP2-TK) construct with 2 or 4 copies of let-7b miRNA targets expressed TK gene only in Y79, WERI-Rb-1, while the TK gene did not express in MIO-M1. In summary, we have developed a tissue-specific, miRNA-regulated dual control vector, which selectively expresses the suicide gene in EpCAM over expressing cells.

Chromatin Structure and Breast Cancer Radiosensitivity

DTIC Science & Technology

2006-10-01

for growth arrest induced by human polynucleotide phosphorylase (hPNPaseold-35) in human melanoma cells. J Biol Chem. 278:24542-24551 (2003). 5...regulatory proteins on human telomeres. Methods in Molecular Biology. 241: 329-339. 9. Hunt CR, Dix DJ, Sharma GG, Pandita RK, Gupta A, Funk M, and...break response network in meiosis . DNA Repair 3:1149-1164. 12. Shahrabani-Gargir L, Pandita TK and Werner H (2004) Ataxia-telangiectasia

Targeted transgenic overexpression of mitochondrial thymidine kinase (TK2) alters mitochondrial DNA (mtDNA) and mitochondrial polypeptide abundance: transgenic TK2, mtDNA, and antiretrovirals.

PubMed

Hosseini, Seyed H; Kohler, James J; Haase, Chad P; Tioleco, Nina; Stuart, Tami; Keebaugh, Erin; Ludaway, Tomika; Russ, Rodney; Green, Elgin; Long, Robert; Wang, Liya; Eriksson, Staffan; Lewis, William

2007-03-01

Mitochondrial toxicity limits nucleoside reverse transcriptase inhibitors (NRTIs) for acquired immune deficiency syndrome. NRTI triphosphates, the active moieties, inhibit human immunodeficiency virus reverse transcriptase and eukaryotic mitochondrial DNA polymerase pol-gamma. NRTI phosphorylation seems to correlate with mitochondrial toxicity, but experimental evidence is lacking. Transgenic mice (TGs) with cardiac overexpression of thymidine kinase isoforms (mitochondrial TK2 and cytoplasmic TK1) were used to study NRTI mitochondrial toxicity. Echocardiography and nuclear magnetic resonance imaging defined cardiac performance and structure. TK gene copy and enzyme activity, mitochondrial (mt) DNA and polypeptide abundance, succinate dehydrogenase and cytochrome oxidase histochemistry, and electron microscopy correlated with transgenesis, mitochondrial structure, and biogenesis. Antiretroviral combinations simulated therapy. Untreated hTK1 or TK2 TGs exhibited normal left ventricle mass. In TK2 TGs, cardiac TK2 gene copy doubled, activity increased 300-fold, and mtDNA abundance doubled. Abundance of the 17-kd subunit of complex I, succinate dehydrogenase histochemical activity, and cristae density increased. NRTIs increased left ventricle mass 20% in TK2 TGs. TK activity increased 3 logs in hTK1 TGs, but no cardiac phenotype resulted. NRTIs abrogated functional effects of transgenically increased TK2 activity but had no effect on TK2 mtDNA abundance. Thus, NRTI mitochondrial phosphorylation by TK2 is integral to clinical NRTI mitochondrial toxicity.

The therapeutic effect of PEI-Mn0.5Zn0.5Fe2O4 nanoparticles/pEgr1-HSV-TK/GCV associated with radiation and magnet-induced heating on hepatoma

NASA Astrophysics Data System (ADS)

Lin, Mei; Huang, Junxing; Zhang, Jia; Wang, Li; Xiao, Wei; Yu, Hong; Li, Yuntao; Li, Hongbo; Yuan, Chenyan; Hou, Xinxin; Zhang, Hao; Zhang, Dongsheng

2013-01-01

Comprehensive therapy based on the integration of hyperthermia, radiation, gene therapy and chemotherapy is a promising area of study in cancer treatment. Using PEI-Mn0.5Zn0.5Fe2O4 nanoparticles (PEI-MZF-NPs) as a gene transfer vector, the authors transfected self-prepared pEgr1-HSV-TK into HepG2 cells and measured the expression of the exogenous gene HSV-TK by RT-PCR. The results showed that HSV-TK was successfully transfected into HepG2 cells and the expression levels of HSV-TK remained stable. Besides, PEI-MZF-NPs were used as magnetic media for thermotherapy to treat hepatoma by magnet-induced heating, combined with radiation-gene therapy. Both in vitro and in vivo results suggest that this combined treatment with gene, radiation and heating has a better therapeutic effect than any of them alone. The apoptotic rate and necrotic rate of the combined treatment group was 51.84% and 15.45%, respectively. In contrast, it was only 20.55% and 6.80% in the radiation-gene group, 7.49% and 3.62% in the radiation-alone group, 15.23% and 7.90% in the heating-alone group, and only 3.52% and 2.16% in the blank control group. The inhibition rate of cell proliferation (88.5%) of the combined treatment group was significantly higher than that of the radiation-gene group (59.5%), radiation-alone group (37.6%) and heating-alone group (60.6%). The tumor volume and mass inhibition rate of the combined treatment group was 94.45% and 93.38%, respectively, significantly higher than 41.28% and 33.58% of the radiation-alone group, 60.76% and 52.18% of the radiation-gene group, 79.91% and 77.40% of the heating-alone group. It is therefore concluded that this combined application of heating, radiation and gene therapy has a good synergistic and complementary effect and PEI-MZF-NPs can act as a novel non-viral gene vector and magnetic induction medium, which offers a viable approach for the treatment of cancer.

Post-translational regulation of gene expression using the ATF4 oxygen-dependent degradation domain for hypoxia-specific gene therapy.

PubMed

Cho, Su Hee; Oh, Binna; Kim, Hyun Ah; Park, Jeong Hyun; Lee, Minhyung

2013-11-01

Solid tumors have hypoxic regions in their cores, due to low blood supply levels. Therefore, hypoxia-specific gene regulation systems have been developed for tumor-specific gene therapy. In this study, the oxygen-dependent degradation (ODD) domain on activating transcription factor-4 (ATF4) was evaluated for post-translational regulation of proteins. The ATF4 ODD cDNA was amplified by RT-PCR, and a luciferase plasmid containing the ATF4 ODD domain, pSV-Luc-ATF4-ODD, was constructed. Luciferase expression was induced under hypoxia by the ATF4 ODD domain in transfection assays into N2A neuroblastoma cells, C6 glioblastoma cells, and U87 glioblastoma cells. In the transfection assay with pSV-Luc-ATF4-ODD, RT-PCR results showed that the mRNA level did not change under hypoxia. This suggests that the induction of luciferase under hypoxia was mediated by post-translational regulation. A plasmid expressing thymidine kinase from herpes simplex virus (HSV-tk), pSV-HSVtk-ATF4-ODD, was constructed with the ATF4 ODD cDNA. The transfection assay with pSV-TK-ATF4-ODD showed that the ATF4 ODD domain induced HSV-tk expression under hypoxia and facilitated the death of C6 cells in the presence of ganciclovir (GCV). Furthermore, pSV-HSVtk-ATF4-ODD induced caspase-3 activity in the hypoxic cells. In conclusion, the ATF4 ODD may be useful for hypoxia-specific gene therapy by post-translational regulation of gene expression.

Targeted Transgenic Overexpression of Mitochondrial Thymidine Kinase (TK2) Alters Mitochondrial DNA (mtDNA) and Mitochondrial Polypeptide Abundance

PubMed Central

Hosseini, Seyed H.; Kohler, James J.; Haase, Chad P.; Tioleco, Nina; Stuart, Tami; Keebaugh, Erin; Ludaway, Tomika; Russ, Rodney; Green, Elgin; Long, Robert; Wang, Liya; Eriksson, Staffan; Lewis, William

2007-01-01

Mitochondrial toxicity limits nucleoside reverse transcriptase inhibitors (NRTIs) for acquired immune deficiency syndrome. NRTI triphosphates, the active moieties, inhibit human immunodeficiency virus reverse transcriptase and eukaryotic mitochondrial DNA polymerase pol-γ. NRTI phosphorylation seems to correlate with mitochondrial toxicity, but experimental evidence is lacking. Transgenic mice (TGs) with cardiac overexpression of thymidine kinase isoforms (mitochondrial TK2 and cytoplasmic TK1) were used to study NRTI mitochondrial toxicity. Echocardiography and nuclear magnetic resonance imaging defined cardiac performance and structure. TK gene copy and enzyme activity, mitochondrial (mt) DNA and polypeptide abundance, succinate dehydrogenase and cytochrome oxidase histochemistry, and electron microscopy correlated with transgenesis, mitochondrial structure, and biogenesis. Antiretroviral combinations simulated therapy. Untreated hTK1 or TK2 TGs exhibited normal left ventricle mass. In TK2 TGs, cardiac TK2 gene copy doubled, activity increased 300-fold, and mtDNA abundance doubled. Abundance of the 17-kd subunit of complex I, succinate dehydrogenase histochemical activity, and cristae density increased. NRTIs increased left ventricle mass 20% in TK2 TGs. TK activity increased 3 logs in hTK1 TGs, but no cardiac phenotype resulted. NRTIs abrogated functional effects of transgenically increased TK2 activity but had no effect on TK2 mtDNA abundance. Thus, NRTI mitochondrial phosphorylation by TK2 is integral to clinical NRTI mitochondrial toxicity. PMID:17322372

Hypomethylation mediated by decreased DNMTs involves in the activation of proto-oncogene MPL in TK6 cells treated with hydroquinone.

PubMed

Liu, Linhua; Ling, Xiaoxuan; Liang, Hairong; Gao, Yuting; Yang, Hui; Shao, Junli; Tang, Huanwen

2012-03-25

Hydroquinone (HQ), one of the most important metabolites derived from benzene, is known to be associated with acute myelogenous leukemia (AML) risk, however, its carcinogenic mechanism remains unclear. In this study, the epigenetic mechanism of HQ exposure was investigated. We characterized the epigenomic response of TK6 cells to HQ exposure, and examined the mRNA expression of DNA methyltransferases (DNMTs) including DNMT1, DNMT3a and DNMT3b, methyl-CpG-binding domain protein 2 (MBD2) and six proto-oncogenes (MPL, RAF1, MYB, MYC, ERBB2 and BRAF). Compared to the control cells, HQ exposure (2.5, 5.0, 10.0 and 20.0 μM for 48 h) resulted in the decrease of DNMTs and MBD2 expression, the global hypomethylation and increase of MPL at mRNA level. Meanwhile, most of these changes were in dose-dependent manner. Moreover, inhibition of DNMTs induced by 5-aza-2'-deoxycytidine (5-AZA), an identified DNMT inhibitor, caused more induction of MPL expression at mRNA level compared to the HQ (10.0 μM) pre-treated group. Furthermore, treatment of HQ potentially led to MPL itself hypomethylation (10.0 and 20.0 μM reduced by 47% and 44%, respectively), further revealing that the activation of proto-oncogene MPL was related to hypomethylation in its DNA sequences. In conclusion, hypomethylation, including global and specific hypomethylation, might be involved in the activation of MPL, and the hypomethylation could be induced by decreased DNMTs in TK6 cells exposed to HQ. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Effects of Cationic Microbubble Carrying CD/TK Double Suicide Gene and αVβ3 Integrin Antibody in Human Hepatocellular Carcinoma HepG2 Cells.

PubMed

Li, Jiale; Zhou, Ping; Li, Lan; Zhang, Yan; Shao, Yang; Tang, Li; Tian, Shuangming

2016-01-01

Hepatocellular carcinoma (HCC), mostly derived from hepatitis or cirrhosisis, is one of the most common types of liver cancer. T-cell mediated immune response elicited by CD/TK double suicide gene has shown a substantial antitumor effect in HCC. Integrin αVβ3 over expresssion has been suggested to regulate the biology behavior of HCC. In this study, we investigated the strategy of incorporating CD/TK double suicide gene and anti-αVβ3 integrin monoclonal antibodies into cationic microbubbles (CMBsαvβ3), and evaluated its killing effect in HCC cells. To improve the transfection efficiency of targeted CD/TK double suicide gene, we adopted cationic microbubbles (CMBs), a cationic delivery agent with enhanced DNA-carrying capacity. The ultrasound and high speed shearing method was used to prepare the non-targeting cationic microbubbles (CMBs). Using the biotin-avidin bridge method, αVβ3 integrin antibody was conjugated to CMBs, and CMBsαvβ3 was generated to specifically target to HepG2 cells. The morphology and physicochemical properties of the CMBsαvβ3 was detected by optical microscope and zeta detector. The conjugation of plasmid and the antibody in CMBsαvβ3 were examined by immunofluorescent microscopy and flow cytometry. The binding capacities of CMBsαvβ3 and CMBs to HCC HepG2 and normal L-02 cells were compared using rosette formation assay. To detect EGFP fluorescence and examine the transfection efficiencies of CMBsαvβ3 and CMBs in HCC cells, fluorescence microscope and contrast-enhanced sonography were adopted. mRNA and protein level of CD/TK gene were detected by RT-PCR and Western blot, respectively. To evaluate the anti-tumor effect of CMBsαvβ3, HCC cells with CMBsαvβ3 were exposed to 5-flurocytosine / ganciclovir (5-FC/GCV). Then, cell cycle distribution after treatment were detected by PI staining and flow cytometry. Apoptotic cells death were detected by optical microscope and assessed by MTT assay and TUNEL-staining assay. CMBsαvβ3 had a regular shape and good dispersion. Compared to CMBs, CMBsαvβ3 had more stable concentrations of αVβ3 ligand and pEGFP-KDRP-CD/TK, and CMBsαvβ3 was much sticker to HepG2 HCC cells than normal liver L-02cells. Moreover, after exposed to anti-αVβ3 monoclonal antibody, the adhesion of CMBsαvβ3 to HepG2 cells and L-02 cells were significantly reduced. Also, CMBsαvβ3 demonstrated a substantially higher efficiency in pEGFP-KDRP-CD/TK plasmid transfection in HepG2 cells than CMBs. In addition, CMBsαvβ3 could significantly facilitate 5-FC/GCV-induced cell cycle arrest in S phase. Moreover, treatment of 5-FC/GCV combined with CMBsαvβ3 resulted in a marked apoptotic cell death in HepG2 and SK-Herp-1 HCC cells. In vitro, treatment of 5-FC/GCV combined with CMBsαvβ3 suppresed cell proliferation. In nude mice model, 5-FU + GCV combined with plasmid + CMBsαvβ3were able to significantly suppress tumor volumes. Through biotin-avidin mediation system, CMBsαvβ3 were successfully generated to specifically target HCC HepG2 cells. More importantly, CMBsαvβ3 could significantly facilitate 5-FC/GCV-induced cell cycle arrest and apoptotic cell death in HepG2 cells. Our study demonstrated a potential strategy that could be translated clinically to improve liver tumor gene delivery.

Implementing a framework for integrating toxicokinetics into human health risk assessment for agrochemicals.

PubMed

Terry, Claire; Hays, Sean; McCoy, Alene T; McFadden, Lisa G; Aggarwal, Manoj; Rasoulpour, Reza J; Juberg, Daland R

2016-03-01

A strategic and comprehensive program in which toxicokinetic (TK) measurements are made for all agrochemicals undergoing toxicity testing (both new compounds and compounds already registered for use) is described. This approach provides the data to more accurately assess the toxicokinetics of agrochemicals and their metabolites in laboratory animals and humans. Having this knowledge provides the ability to conduct more insightful toxicity studies, refine and interpret exposure assessments and reduce uncertainty in risk assessments. By developing a better understanding of TK across species, including humans via in vitro metabolism studies, any differences across species in TK can be identified early and the most relevant species can be selected for toxicity tests. It also provides the ability to identify any non-linearities in TK as a function of dose, which in turn can be used to identify a kinetically derived maximum dose (KMD) and avoid dosing inappropriately outside of the kinetic linear range. Measuring TK in key life stages also helps to identify changes in ADME parameters from in utero to adults. A robust TK database can also be used to set internal concentration based "Reference Concentrations" and Biomonitoring Equivalents (BE), and support selection of Chemical Specific Adjustment Factors (CSAF). All of these factors support the reduction of uncertainty throughout the entire risk assessment process. This paper outlines how a TK research strategy can be integrated into new agrochemical toxicity testing programs, together with a proposed Framework for future use. Copyright © 2015 Elsevier Inc. All rights reserved.

Computational modeling and functional analysis of Herpes simplex virus type-1 thymidine kinase and Escherichia coli cytosine deaminase fusion protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Jufeng; Wang, Zhanli; Wei, Fang

2007-08-17

Herpes simplex virus type-1 thymidine kinase (HSV-1TK) and Escherichia coli cytosine deaminase (CD) fusion protein was designed using InsightII software. The structural rationality of the fusion proteins incorporating a series of flexible linker peptide was analyzed, and a suitable linker peptide was chosen for further investigated. The recombinant plasmid containing the coding regions of HSV-1TK and CD cDNA connected by this linker peptide coding sequence was generated and subsequently transfected into the human embryonic kidney 293 cells (HEK293). The Western blotting indicated that the recombinant fusion protein existed as a dimer with a molecular weight of approximately 90 kDa. Themore » toxicity of the prodrug on the recombinant plasmid-transfected human lung cancer cell line NCIH460 was evaluated, which showed that TKglyCD-expressing cells conferred upon cells prodrug sensitivities equivalent to that observed for each enzyme independently. Most noteworthy, cytotoxicity could be enhanced by concurrently treating TKglyCD-expressing cells with prodrugs GCV and 5-FC. The results indicate that we have successfully constructed a HSV-1TKglyCD fusion gene which might have a potential application for cancer gene therapy.« less

High-throughput screening platform for engineered nanoparticle-mediated genotoxicity using CometChip technology.

PubMed

Watson, Christa; Ge, Jing; Cohen, Joel; Pyrgiotakis, Georgios; Engelward, Bevin P; Demokritou, Philip

2014-03-25

The likelihood of intentional and unintentional engineered nanoparticle (ENP) exposure has dramatically increased due to the use of nanoenabled products. Indeed, ENPs have been incorporated in many useful products and have enhanced our way of life. However, there are many unanswered questions about the consequences of nanoparticle exposures, in particular, with regard to their potential to damage the genome and thus potentially promote cancer. In this study, we present a high-throughput screening assay based upon the recently developed CometChip technology, which enables evaluation of single-stranded DNA breaks, abasic sites, and alkali-sensitive sites in cells exposed to ENPs. The strategic microfabricated, 96-well design and automated processing improves efficiency, reduces processing time, and suppresses user bias in comparison to the standard comet assay. We evaluated the versatility of this assay by screening five industrially relevant ENP exposures (SiO2, ZnO, Fe2O3, Ag, and CeO2) on both suspension human lymphoblastoid (TK6) and adherent Chinese hamster ovary (H9T3) cell lines. MTT and CyQuant NF assays were employed to assess cellular viability and proliferation after ENP exposure. Exposure to ENPs at a dose range of 5, 10, and 20 μg/mL induced dose-dependent increases in DNA damage and cytotoxicity. Genotoxicity profiles of ZnO>Ag>Fe2O3>CeO2>SiO2 in TK6 cells at 4 h and Ag>Fe2O3>ZnO>CeO2>SiO2 in H9T3 cells at 24 h were observed. The presented CometChip platform enabled efficient and reliable measurement of ENP-mediated DNA damage, therefore demonstrating the efficacy of this powerful tool in nanogenotoxicity studies.

Cooperative therapeutic effects of herpes simplex virus thymidine kinase gene/ganciclovir system and chemotherapeutic agents on prostate cancer in vitro.

PubMed

Xing, Yifei; Xiao, Yajun; Lu, Gongcheng; Zeng, Fuqing; Zhao, Jun; Xiong, Ping; Feng, Wei

2006-01-01

The killing effects of herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) approach by the addition of several commonly clinical chemotherapeutic agents on hormone refractory prostate cancer (HRPC) cells PC-3m were investigated. After transferring of the HSV-tk gene into PC-3m cells, mRNA and protein expression of HSV-tk was detected by reverse-transcript polymerase chain reaction (RT-PCR) and strept avidin-biotin complex (SABC) immunohistochemical method. The killing effect of GCV, cisplatin (CDDP), etoposide (VP-16), vincristine (VCR), methotrexate (MTX), 5-fluorouracil (5-Fu), and suramin on PC-3m cells was evaluated by morphological assessment analysis, trypan blue exclusion assay and MTT assay respectively. Additionally, the cooperative effect of HSV-tk/GCV system combined with the above agents on the target cancer cells was determined by MTT. Furthermore, apoptosis and necrosis induced by GCV plus 5-Fu or suramin was analyzed by flow cytometry (FCM). The results showed that that there was HSV-tk mRNA and protein expression in pDR2-tk plasmid transduced PC-3m cell. Combination of GCV with VP-16, VCR, 5-Fu or suramin led to an enhanced cellular killing effect, but with CDDP resulted in a reduced one and with MTX in an approximate one. FCM revealed that synergistic use of GCV and 5-fu or suramin resulted in a rather large proportion of apoptosis and necrosis with the apoptosis index being 36.38% and 35.51%, and the proportion of necrosis being 33.05% and 28.87%, respectively. In conclusion, HSV-tk/CGV approach by addition of certain clinical available chemotherapeutic drugs brings on statistically significant enhanced cell killing over single-agent treatment. Our results highlight the potential for such new combination therapies for future treatments of HRPC.

ALC1/CHD1L, a chromatin-remodeling enzyme, is required for efficient base excision repair.

PubMed

Tsuda, Masataka; Cho, Kosai; Ooka, Masato; Shimizu, Naoto; Watanabe, Reiko; Yasui, Akira; Nakazawa, Yuka; Ogi, Tomoo; Harada, Hiroshi; Agama, Keli; Nakamura, Jun; Asada, Ryuta; Fujiike, Haruna; Sakuma, Tetsushi; Yamamoto, Takashi; Murai, Junko; Hiraoka, Masahiro; Koike, Kaoru; Pommier, Yves; Takeda, Shunichi; Hirota, Kouji

2017-01-01

ALC1/CHD1L is a member of the SNF2 superfamily of ATPases carrying a macrodomain that binds poly(ADP-ribose). Poly(ADP-ribose) polymerase (PARP) 1 and 2 synthesize poly(ADP-ribose) at DNA-strand cleavage sites, promoting base excision repair (BER). Although depletion of ALC1 causes increased sensitivity to various DNA-damaging agents (H2O2, UV, and phleomycin), the role played by ALC1 in BER has not yet been established. To explore this role, as well as the role of ALC1's ATPase activity in BER, we disrupted the ALC1 gene and inserted the ATPase-dead (E165Q) mutation into the ALC1 gene in chicken DT40 cells, which do not express PARP2. The resulting ALC1-/- and ALC1-/E165Q cells displayed an indistinguishable hypersensitivity to methylmethane sulfonate (MMS), an alkylating agent, and to H2O2, indicating that ATPase plays an essential role in the DNA-damage response. PARP1-/- and ALC1-/-/PARP1-/- cells exhibited a very similar sensitivity to MMS, suggesting that ALC1 and PARP1 collaborate in BER. Following pulse-exposure to H2O2, PARP1-/- and ALC1-/-/PARP1-/- cells showed similarly delayed kinetics in the repair of single-strand breaks, which arise as BER intermediates. To ascertain ALC1's role in BER in mammalian cells, we disrupted the ALC1 gene in human TK6 cells. Following exposure to MMS and to H2O2, the ALC1-/- TK6 cell line showed a delay in single-strand-break repair. We therefore conclude that ALC1 plays a role in BER. Following exposure to H2O2, ALC1-/- cells showed compromised chromatin relaxation. We thus propose that ALC1 is a unique BER factor that functions in a chromatin context, most likely as a chromatin-remodeling enzyme.

Tanshinone IIA Increases the Bystander Effect of Herpes Simplex Virus Thymidine Kinase/Ganciclovir Gene Therapy via Enhanced Gap Junctional Intercellular Communication

PubMed Central

Liu, Xijuan; Wu, Yingya; Du, Biaoyan; Li, Jiefen; Zhou, Jing; Li, Jingjing; Tan, Yuhui

2013-01-01

The bystander effect is an intriguing phenomenon by which adjacent cells become sensitized to drug treatment during gene therapy with herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV). This effect is reported to be mediated by gap junctional intercellular communication (GJIC), and therefore, we postulated that upregulation of genes that facilitate GJIC may enhance the HSV-tk/GCV bystander effect. Previous findings have shown Tanshinone IIA (Tan IIA), a chemical substance derived from a Chinese medicine herb, promotes the upregulation of the connexins Cx26 and Cx43 in B16 cells. Because gap junctions are formed by connexins, we hypothesized that Tan IIA might increase GJIC. Our results show that Tan IIA increased GJIC in B16 melanoma cells, leading to more efficient GCV-induced bystander killing in cells stably expressing HSV-tk. Additionally, in vivo experiments demonstrated that tumors in mice with 10% HSV-tk positive B16 cells and 90% wild-type B16 cells became smaller following treatment with the combination of GCV and Tan IIA as compared to GCV or Tan IIA alone. These data demonstrate that Tan IIA can augment the bystander effect of HSV-tk/GCV system through increased gap junction coupling, which adds strength to the promising strategy that develops connexins inducer to potentiate the effects of suicide gene therapy. PMID:23861780

CHROMOSOME 11 ABERRATIONS IN SMALL COLONY L5178Y TK-/-MUTANTS EARLY IN THEIR CLONAL HISTORY

EPA Science Inventory

The authors have developed a cytogenetic technique that allows observation of chromosome rearrangements associated with TK-/- mutagenesis of the L5178Y/TK+/-3.7.2C cell line early in mutant clonal history. For a series of mutagenic treatments they show that the major proportion (...

Microchip Immunoaffinity Electrophoresis of Antibody-Thymidine Kinase 1 Complex

PubMed Central

Pagaduan, Jayson V.; Ramsden, Madison; O’Neill, Kim; Woolley, Adam T.

2015-01-01

Thymidine kinase-1 (TK1) is an important cancer biomarker whose serum levels are elevated in early cancer development. We developed a microchip electrophoresis immunoaffinity assay to measure recombinant purified TK1 (pTK1) using an antibody that binds to human TK1. We fabricated poly(methyl methacrylate) microfluidic devices to test the feasibility of detecting antibody (Ab)-pTK1 immune complexes as a step towards TK1 analysis in clinical serum samples. We were able to separate immune complexes from unbound antibodies using 0.5X phosphate buffer saline (pH 7.4) containing 0.01% Tween-20, with 1% w/v methylcellulose that acts as a dynamic surface coating and sieving matrix. Separation of the antibody and Ab-pTK1 complex was observed within a 5 mm effective separation length. This method of detecting pTK1 is easy to perform, requires only a 10 μL sample volume, and takes just 1 minute for separation. PMID:25486911

Thermodynamic Studies on NdFeO 3(s)

NASA Astrophysics Data System (ADS)

Parida, S. C.; Dash, Smruti; Singh, Ziley; Prasad, R.; Jacob, K. T.; Venugopal, V.

2002-02-01

The enthalpy increments and the standard molar Gibbs energy of formation of NdFeO3(s) have been measured using a high-temperature Calvet microcalorimeter and a solid oxide galvanic cell, respectively. A λ-type transition, related to magnetic order-disorder transformation (antiferromagnetic to paramagnetic), is apparent from the heat capacity data at ∼687 K. Enthalpy increments, except in the vicinity of transition, can be represented by a polynomial expression: {H°m(T)-H°m(298.15 K)}/J·mol-1 (±0.7%)=-53625.6+146.0(T/K) +1.150×10-4(T/K)2 +3.007×106(T/K)-1; (298.15≤T/K ≤1000). The heat capacity, the first differential of {H°m(T)-H°m(298.15 K)} with respect to temperature, is given by Cop, m/J·K-1·mol-1=146.0+2.30×10-4(T/K)-3.007×106(T/K)-2. The reversible emf's of the cell, (-) Pt/{NdFeO3(s) +Nd2O3(s)+Fe(s)}//YDT/CSZ//{Fe(s)'FeO'(s)}/Pt(+), were measured in the temperature range from 1004 to 1208 K. It can be represented within experimental error by a linear equation: E/V:(0.1418±0.0003)-(3.890±0.023)×10-5(T/K). The Gibbs energy of formation of solid NdFeO3 calculated by the least-squares regression analysis of the data obtained in the present study, and data for Fe0.95O and Nd2O3 from the literature, is given by ΔfG°m(NdFeO3, s)/kJ·mol-1(±2.0)=-1345.9+0.2542(T/K); (1000≤T/K ≤1650). The error in ΔfG°m(NdFeO3, s, T) includes the standard deviation in emf and the uncertainty in the data taken from the literature. Values of ΔfH°m(NdFeO3, s, 298.15 K) and S°m(NdFeO3, s, 298.15 K) calculated by the second law method are -1362.5 (±6) kJ·mol-1 and 123.9 (±2.5) J·K-1·mol-1, respectively. Based on the thermodynamic information, an oxygen potential diagram for the system Nd-Fe-O was developed at 1350 K.

Thymidine Kinase-Negative Herpes Simplex Virus 1 Can Efficiently Establish Persistent Infection in Neural Tissues of Nude Mice

PubMed Central

Huang, Chih-Yu; Yao, Hui-Wen; Wang, Li-Chiu; Shen, Fang-Hsiu

2016-01-01

ABSTRACT Herpes simplex virus 1 (HSV-1) establishes latency in neural tissues of immunocompetent mice but persists in both peripheral and neural tissues of lymphocyte-deficient mice. Thymidine kinase (TK) is believed to be essential for HSV-1 to persist in neural tissues of immunocompromised mice, because infectious virus of a mutant with defects in both TK and UL24 is detected only in peripheral tissues, but not in neural tissues, of severe combined immunodeficiency mice (T. Valyi-Nagy, R. M. Gesser, B. Raengsakulrach, S. L. Deshmane, B. P. Randazzo, A. J. Dillner, and N. W. Fraser, Virology 199:484–490, 1994, https://doi.org/10.1006/viro.1994.1150). Here we find infiltration of CD4 and CD8 T cells in peripheral and neural tissues of mice infected with a TK-negative mutant. We therefore investigated the significance of viral TK and host T cells for HSV-1 to persist in neural tissues using three genetically engineered mutants with defects in only TK or in both TK and UL24 and two strains of nude mice. Surprisingly, all three mutants establish persistent infection in up to 100% of brain stems and 93% of trigeminal ganglia of adult nude mice at 28 days postinfection, as measured by the recovery of infectious virus. Thus, in mouse neural tissues, host T cells block persistent HSV-1 infection, and viral TK is dispensable for the virus to establish persistent infection. Furthermore, we found 30- to 200-fold more virus in neural tissues than in the eye and detected glycoprotein C, a true late viral antigen, in brainstem neurons of nude mice persistently infected with the TK-negative mutant, suggesting that adult mouse neurons can support the replication of TK-negative HSV-1. IMPORTANCE Acyclovir is used to treat herpes simplex virus 1 (HSV-1)-infected immunocompromised patients, but treatment is hindered by the emergence of drug-resistant viruses, mostly those with mutations in viral thymidine kinase (TK), which activates acyclovir. TK mutants are detected in brains of immunocompromised patients with persistent infection. However, answers to the questions as to whether TK-negative (TK−) HSV-1 can establish persistent infection in brains of immunocompromised hosts and whether neurons in vivo are permissive for TK− HSV-1 remain elusive. Using three genetically engineered HSV-1 TK− mutants and two strains of nude mice deficient in T cells, we found that all three HSV-1 TK− mutants can efficiently establish persistent infection in the brain stem and trigeminal ganglion and detected glycoprotein C, a true late viral antigen, in brainstem neurons. Our study provides evidence that TK− HSV-1 can persist in neural tissues and replicate in brain neurons of immunocompromised hosts. PMID:27974554

An autologous in situ tumor vaccination approach for hepatocellular carcinoma. 2. Tumor-specific immunity and cure after radio-inducible suicide gene therapy and systemic CD40-ligand and Flt3-ligand gene therapy in an orthotopic tumor model.

PubMed

Kawashita, Yujo; Deb, Niloy J; Garg, Madhur K; Kabarriti, Rafi; Fan, Zuoheng; Alfieri, Alan A; Roy-Chowdhury, Jayanta; Guha, Chandan

2014-08-01

Diffuse hepatocellular carcinoma (HCC) is a lethal disease that radiation therapy (RT) currently has a limited role in treating because of the potential for developing fatal radiation-induced liver disease. However, recently diffuse HCC, "radio-inducible suicide gene therapy" has been shown to enhance local tumor control and residual microscopic disease within the liver for diffuse HCC, by using a combination of chemoactivation and molecular radiosensitization. We have demonstrated that the addition of recombinant adenovirus-expressing human Flt3 ligand (Adeno-Flt3L) after radio-inducible suicide gene therapy induced a Th1-biased, immune response and enhanced tumor control in an ectopic model of HCC. We hypothesized that sequential administration of recombinant adenovirus-expressing CD40L (Adeno-CD40L) could further potentiate the efficacy of our trimodal therapy with RT + HSV-TK + Adeno-Flt3L. We examined our hypothesis in an orthotopic model of diffuse HCC using BNL1ME A.7R.1 (BNL) cells in Balb/c mice. BNL murine hepatoma cells (5 × 10(4)) transfected with an expression vector of HSV-TK under the control of a radiation-inducible promoter were injected intraportally into BALB/cJ mice. Fourteen days after the HCC injection, mice were treated with a 25 Gy dose of radiation to the whole liver, followed by ganciclovir (GCV) treatment and systemic adenoviral cytokine gene therapy (Flt3L or CD40L or both). Untreated mice died in 27 ± 4 days. Radiation therapy alone had a marginal effect on survival (median = 35 ± 7 days) and the addition of HSV-TK/GCV gene therapy improved the median survival to 47 ± 6 days. However, the addition of Adeno-Flt3L to radiation therapy and HSV-TK/GCV therapy significantly (P = 0.0005) increased survival to a median of 63 ± 20 days with 44% (7/16) of the animals still alive 116 days after tumor implantation. The curative effect of Flt3L was completely abolished when using immunodeficient nude mice or mice depleted for CD4, CD8 and natural killer cells. The addition of Adeno-CD40L further improved the median survival of animals to 80 ± 15 days and this effect was abolished only when using anti-CD8 antibodies. Chromium-51 (51Cr) release assay showed cytotoxic T lymphocyte (CTL) activation, suggesting efficient dendritic cell (DC) activation with CTL activation after the treatment. Furthermore, when surviving mice were rechallenged with BNL-ETK cells on the foot pad, RT + HSV-TK/GCV + Flt3L + CD40L-treated mice developed a small tumor on day 56 but the tumor eventually disappeared after 105 days. Mice treated with RT + HSV-TK/GCV + Flt3L showed a slowed tumor growth curve compared with untreated mice. Therefore, combination therapy using Flt3L to induce DC proliferation and CD40L to enhance DC maturation holds great promise for immunomodulation of radiation therapy to enhance HCC tumor control and prevent progression of disease in patients with diffuse HCC.

High-throughput PBPK and Microdosimetry: Cell-level Exposures in a Virtual Tissue Context (WC9)

EPA Science Inventory

Toxicokinetic (TK) models can determine whether chemical exposures produce potentially hazardous tissue concentrations. Tissue microdosimetry TK models relate whole-body chemical exposures to cell-scale concentrations. As a proof of concept, we approximated the micro-anatomic arc...

Thymidine kinase and mtDNA depletion in human cardiomyopathy: epigenetic and translational evidence for energy starvation

PubMed Central

Koczor, Christopher A.; Torres, Rebecca A.; Fields, Earl J.; Boyd, Amy; He, Stanley; Patel, Nilamkumar; Lee, Eva K.; Samarel, Allen M.

2013-01-01

This study addresses how depletion of human cardiac left ventricle (LV) mitochondrial DNA (mtDNA) and epigenetic nuclear DNA methylation promote cardiac dysfunction in human dilated cardiomyopathy (DCM) through regulation of pyrimidine nucleotide kinases. Samples of DCM LV and right ventricle (n = 18) were obtained fresh at heart transplant surgery. Parallel samples from nonfailing (NF) controls (n = 12) were from donor hearts found unsuitable for clinical use. We analyzed abundance of mtDNA and nuclear DNA (nDNA) using qPCR. LV mtDNA was depleted in DCM (50%, P < 0.05 each) compared with NF. No detectable change in RV mtDNA abundance occurred. DNA methylation and gene expression were determined using microarray analysis (GEO accession number: GSE43435). Fifty-seven gene promoters exhibited DNA hypermethylation or hypomethylation in DCM LVs. Among those, cytosolic thymidine kinase 1 (TK1) was hypermethylated. Expression arrays revealed decreased abundance of the TK1 mRNA transcript with no change in transcripts for other relevant thymidine metabolism enzymes. Quantitative immunoblots confirmed decreased TK1 polypeptide steady state abundance. TK1 activity remained unchanged in DCM samples while mitochondrial thymidine kinase (TK2) activity was significantly reduced. Compensatory TK activity was found in cardiac myocytes in the DCM LV. Diminished TK2 activity is mechanistically important to reduced mtDNA abundance and identified in DCM LV samples here. Epigenetic and genetic changes result in changes in mtDNA and in nucleotide substrates for mtDNA replication and underpin energy starvation in DCM. PMID:23695887

Bacterial delivery of RNAi effectors: transkingdom RNAi.

PubMed

Lage, Hermann; Krühn, Andrea

2010-08-18

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tkRNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter beta1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tkRNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells.

Silencing expression of the catalytic subunit of DNA-dependent protein kinase by small interfering RNA sensitizes human cells for radiation-induced chromosome damage, cell killing, and mutation

NASA Technical Reports Server (NTRS)

Peng, Yuanlin; Zhang, Qinming; Nagasawa, Hatsumi; Okayasu, Ryuichi; Liber, Howard L.; Bedford, Joel S.

2002-01-01

Targeted gene silencing in mammalian cells by RNA interference (RNAi) using small interfering RNAs (siRNAs) was recently described by Elbashir et al. (S. M. Elbashir et al., Nature (Lond.), 411: 494-498, 2001). We have used this methodology in several human cell strains to reduce expression of the Prkdc (DNA-PKcs) gene coding for the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) that is involved in the nonhomologous end joining of DNA double-strand breaks. We have also demonstrated a radiosensitization for several phenotypic endpoints of radiation damage. In low-passage normal human fibroblasts, siRNA knock-down of DNA-PKcs resulted in a reduced capacity for restitution of radiation-induced interphase chromosome breaks as measured by premature chromosome condensation, an increased yield of acentric chromosome fragments at the first postirradiation mitosis, and an increased radiosensitivity for cell killing. For three strains of related human lymphoblasts, DNA-PKcs-targeted siRNA transfection resulted in little or no increase in radiosensitivity with respect to cell killing, a 1.5-fold decrease in induced mutant yield in TK6- and p53-null NH32 cells, but about a 2-fold increase in induced mutant yield in p53-mutant WTK1 cells at both the hypoxanthine quanine phosphoribosyl transferase (hprt) and the thymidine kinase loci.

The contribution of mitochondrial thymidylate synthesis in preventing the nuclear genome stress.

PubMed

Lee, Ming-Hsiang; Wang, Liya; Chang, Zee-Fen

2014-04-01

In quiescent fibroblasts, the expression levels of cytosolic enzymes for thymidine triphosphate (dTTP) synthesis are down-regulated, causing a marked reduction in the dTTP pool. In this study, we provide evidence that mitochondrial thymidylate synthesis via thymidine kinase 2 (TK2) is a limiting factor for the repair of ultraviolet (UV) damage in the nuclear compartment in quiescent fibroblasts. We found that TK2 deficiency causes secondary DNA double-strand breaks formation in the nuclear genome of quiescent cells at the late stage of recovery from UV damage. Despite slower repair of quiescent fibroblast deficient in TK2, DNA damage signals eventually disappeared, and these cells were capable of re-entering the S phase after serum stimulation. However, these cells displayed severe genome stress as revealed by the dramatic increase in 53BP1 nuclear body in the G1 phase of the successive cell cycle. Here, we conclude that mitochondrial thymidylate synthesis via TK2 plays a role in facilitating the quality repair of UV damage for the maintenance of genome integrity in the cells that are temporarily arrested in the quiescent state.

Analysis of negative historical control group data from the in vitro micronucleus assay using TK6 cells.

PubMed

Lovell, David P; Fellows, Mick; Marchetti, Francesco; Christiansen, Joan; Elhajouji, Azeddine; Hashimoto, Kiyohiro; Kasamoto, Sawako; Li, Yan; Masayasu, Ozaki; Moore, Martha M; Schuler, Maik; Smith, Robert; Stankowski, Leon F; Tanaka, Jin; Tanir, Jennifer Y; Thybaud, Veronique; Van Goethem, Freddy; Whitwell, James

2018-01-01

The recent revisions of the Organisation for Economic Co-operation and Development (OECD) genetic toxicology test guidelines emphasize the importance of historical negative controls both for data quality and interpretation. The goal of a HESI Genetic Toxicology Technical Committee (GTTC) workgroup was to collect data from participating laboratories and to conduct a statistical analysis to understand and publish the range of values that are normally seen in experienced laboratories using TK6 cells to conduct the in vitro micronucleus assay. Data from negative control samples from in vitro micronucleus assays using TK6 cells from 13 laboratories were collected using a standard collection form. Although in some cases statistically significant differences can be seen within laboratories for different test conditions, they were very small. The mean incidence of micronucleated cells/1000 cells ranged from 3.2/1000 to 13.8/1000. These almost four-fold differences in micronucleus levels cannot be explained by differences in scoring method, presence or absence of exogenous metabolic activation (S9), length of treatment, presence or absence of cytochalasin B or different solvents used as vehicles. The range of means from the four laboratories using flow cytometry methods (3.7-fold: 3.5-12.9 micronucleated cells/1000 cells) was similar to that from the nine laboratories using other scoring methods (4.3-fold: 3.2-13.8 micronucleated cells/1000 cells). No laboratory could be identified as an outlier or as showing unacceptably high variability. Quality Control (QC) methods applied to analyse the intra-laboratory variability showed that there was evidence of inter-experimental variability greater than would be expected by chance (i.e. over-dispersion). However, in general, this was low. This study demonstrates the value of QC methods in helping to analyse the reproducibility of results, building up a 'normal' range of values, and as an aid to identify variability within a laboratory in order to implement processes to maintain and improve uniformity. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Lack of complex I activity in human cells carrying a mutation in MtDNA-encoded ND4 subunit is corrected by the Saccharomyces cerevisiae NADH-quinone oxidoreductase (NDI1) gene.

PubMed

Bai, Y; Hájek, P; Chomyn, A; Chan, E; Seo, B B; Matsuno-Yagi, A; Yagi, T; Attardi, G

2001-10-19

The gene for the single subunit, rotenone-insensitive, and flavone-sensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae (NDI1) can completely restore the NADH dehydrogenase activity in mutant human cells that lack the essential mitochondrial DNA (mtDNA)-encoded subunit ND4. In particular, the NDI1 gene was introduced into the nuclear genome of the human 143B.TK(-) cell line derivative C4T, which carries a homoplasmic frameshift mutation in the ND4 gene. Two transformants with a low or high level of expression of the exogenous gene were chosen for a detailed analysis. In these cells the corresponding protein is localized in mitochondria, its NADH-binding site faces the matrix compartment as in yeast mitochondria, and in perfect correlation with its abundance restores partially or fully NADH-dependent respiration that is rotenone-insensitive, flavone-sensitive, and antimycin A-sensitive. Thus the yeast enzyme has become coupled to the downstream portion of the human respiratory chain. Furthermore, the P:O ratio with malate/glutamate-dependent respiration in the transformants is approximately two-thirds of that of the wild-type 143B.TK(-) cells, as expected from the lack of proton pumping activity in the yeast enzyme. Finally, whereas the original mutant cell line C4T fails to grow in medium containing galactose instead of glucose, the high NDI1-expressing transformant has a fully restored capacity to grow in galactose medium. The present observations substantially expand the potential of the yeast NDI1 gene for the therapy of mitochondrial diseases involving complex I deficiency.

Feasibility Analysis of Incorporating In-Vitro Toxicokinetic Data ...

EPA Pesticide Factsheets

The underlying principle of read-across is that biological activity is a function of physical and structural properties of chemicals. Analogs are typically identified on the basis of structural similarity and subsequently evaluated for their use in read-across on the basis of their bioavailability, reactivity and metabolic similarity. While the concept of similarity is the major tenet in grouping chemicals for read-across, a critical consideration is to evaluate if structural differences significantly impact toxicological activity. This is a key source of uncertainty in read-across predictions. We hypothesize that inclusion of toxicokinetic (TK) information will reduce the uncertainty in read-across predictions. TK information can help substantiate whether chemicals within a category have similar ADME properties and, hence, increase the likelihood of exhibiting similar toxicological properties. This current case study is part of a larger study aimed at performing a systematic assessment of the extent to which in-vitro TK data can obviate in-vivo TK data, while maintaining or increasing scientific confidence in read-across predictions. The analysis relied on a dataset of ~7k chemicals with predicted exposure data (chemical inventory), of which 819 chemicals had rat and/or human in-vitro TK data (analog inventory), and 33 chemicals had rat in-vivo TK data (target inventory). The set of chemicals with human in vitro TK data was investigated to determine whether str

Thymidine kinase 2 (H126N) knockin mice show the essential role of balanced deoxynucleotide pools for mitochondrial DNA maintenance.

PubMed

Akman, Hasan O; Dorado, Beatriz; López, Luis C; García-Cazorla, Angeles; Vilà, Maya R; Tanabe, Lauren M; Dauer, William T; Bonilla, Eduardo; Tanji, Kurenai; Hirano, Michio

2008-08-15

Mitochondrial DNA (mtDNA) depletion syndrome (MDS), an autosomal recessive condition, is characterized by variable organ involvement with decreased mtDNA copy number and activities of respiratory chain enzymes in affected tissues. MtDNA depletion has been associated with mutations in nine autosomal genes, including thymidine kinase (TK2), which encodes a ubiquitous mitochondrial protein. To study the pathogenesis of TK2-deficiency, we generated mice harboring an H126N Tk2 mutation. Homozygous Tk2 mutant (Tk2(-/-)) mice developed rapidly progressive weakness after age 10 days and died between ages 2 and 3 weeks. Tk2(-/-) animals showed Tk2 deficiency, unbalanced dNTP pools, mtDNA depletion and defects of respiratory chain enzymes containing mtDNA-encoded subunits that were most prominent in the central nervous system. Histopathology revealed an encephalomyelopathy with prominent vacuolar changes in the anterior horn of the spinal cord. The H126N TK2 mouse is the first knock-in animal model of human MDS and demonstrates that the severity of TK2 deficiency in tissues may determine the organ-specific phenotype.

Thymidine kinase 2 (H126N) knockin mice show the essential role of balanced deoxynucleotide pools for mitochondrial DNA maintenance

PubMed Central

Akman, Hasan O.; Dorado, Beatriz; López, Luis C.; García-Cazorla, Ángeles; Vilà, Maya R.; Tanabe, Lauren M.; Dauer, William T.; Bonilla, Eduardo; Tanji, Kurenai; Hirano, Michio

2008-01-01

Mitochondrial DNA (mtDNA) depletion syndrome (MDS), an autosomal recessive condition, is characterized by variable organ involvement with decreased mtDNA copy number and activities of respiratory chain enzymes in affected tissues. MtDNA depletion has been associated with mutations in nine autosomal genes, including thymidine kinase (TK2), which encodes a ubiquitous mitochondrial protein. To study the pathogenesis of TK2-deficiency, we generated mice harboring an H126N Tk2 mutation. Homozygous Tk2 mutant (Tk2−/−) mice developed rapidly progressive weakness after age 10 days and died between ages 2 and 3 weeks. Tk2−/− animals showed Tk2 deficiency, unbalanced dNTP pools, mtDNA depletion and defects of respiratory chain enzymes containing mtDNA-encoded subunits that were most prominent in the central nervous system. Histopathology revealed an encephalomyelopathy with prominent vacuolar changes in the anterior horn of the spinal cord. The H126N TK2 mouse is the first knock-in animal model of human MDS and demonstrates that the severity of TK2 deficiency in tissues may determine the organ-specific phenotype. PMID:18467430

Molecular switch of Cre/loxP for radiation modulated gene therapy on hepatoma

NASA Astrophysics Data System (ADS)

Hsieh, Ya-Ju; Chen, Fu-Du; Wang, Fu Hui; Ke, Chien Chih; Wang, Hsin-Ell; Liu, Ren-Shyan

2007-02-01

For the purpose of enhancement of AFP promoter for the use of radiation modulated gene therapy for hepatocellular carcinoma (HCC), we combined hepatitis B virus (HBV) enhancer II with AFP promoter which shows the selectivity to the target cells to control the Cre/loxP system. Different gene constructs, pE4luc, pE4Tk, EIIAPA-Cre, E4CMV-STOP-Tk and chimeric promoters combined with HBV enhancer were constructed and transfected into HepG2, HeLa and NIH-3T3 cell lines. Cell experiments revealed that E4 enhancer responses to radiation best after 60 h irradiation at a dose range of 5-7 Gy in HepG2 stable clone. The EIIAPA promoter provided high specificity to hepatoma and activated the Cre downstream and removed the stop cassette only in hepatoma cells. After removal of the stop cassette, the E4 response to radiation could encode more Tk protein and kill more tumor cells. In summary, the chimeric EIIAPA promoter can stringently control the expression of Cre recombinase only in HCC. The radiation effect of the EIIAPA-Cre and E4CMV-STOP-Tk system shows promising results in terms of cell survival of HCC.

Sulforaphane rescues amyloid-β peptide-mediated decrease in MerTK expression through its anti-inflammatory effect in human THP-1 macrophages.

PubMed

Jhang, Kyoung A; Park, Jin-Sun; Kim, Hee-Sun; Chong, Young Hae

2018-03-12

Mer tyrosine kinase (MerTK) activity necessary for amyloid-stimulated phagocytosis strongly implicates that MerTK dysregulation might contribute to chronic inflammation implicated in Alzheimer's disease (AD) pathology. However, the precise mechanism involved in the regulation of MerTK expression by amyloid-β (Aβ) in proinflammatory environment has not yet been ascertained. The objective of this study was to determine the underlying mechanism involved in Aβ-mediated decrease in MerTK expression through Aβ-mediated regulation of MerTK expression and its modulation by sulforaphane in human THP-1 macrophages challenged with Aβ1-42. We used protein preparation, Ca 2+ influx fluorescence imaging, nuclear fractionation, Western blotting techniques, and small interfering RNA (siRNA) knockdown to perform our study. Aβ1-42 elicited a marked decrease in MerTK expression along with increased intracellular Ca 2+ level and induction of proinflammatory cytokines such as IL-1β and TNF-α. Ionomycin A and thapsigargin also increased intracellular Ca 2+ levels and production of IL-1β and TNF-α, mimicking the effect of Aβ1-42. In contrast, the Aβ1-42-evoked responses were attenuated by depletion of Ca 2+ with ethylene glycol tetraacetic acid. Furthermore, recombinant IL-1β or TNF-α elicited a decrease in MerTK expression. However, immunodepletion of IL-1β or TNF-α with neutralizing antibodies significantly inhibited Aβ1-42-mediated downregulation of MerTK expression. Notably, sulforaphane treatment potently inhibited Aβ1-42-induced intracellular Ca 2+ level and rescued the decrease in MerTK expression by blocking nuclear factor-κB (NF-κB) nuclear translocation, thereby decreasing IL-1β and TNF-α production upon Aβ1-42 stimulation. Such adverse effects of sulforaphane were replicated by BAY 11-7082, a NF-κB inhibitor. Moreover, sulforaphane's anti-inflammatory effects on Aβ1-42-induced production of IL-1β and TNF-α were significantly diminished by siRNA-mediated knockdown of MerTK, confirming a critical role of MerTK in suppressing Aβ1-42-induced innate immune response. These findings implicate that targeting of MerTK with phytochemical sulforaphane as a mechanism for preventing Aβ1-42-induced neuroinflammation has potential to be applied in AD therapeutics.

Lymphocyte thymidine kinase and treatment response in acute lymphocytic leukemia.

PubMed

Russo, S A; Harris, M B; Greengard, O

1987-01-01

The activity of thymidine kinase (TK) and the proportion of its isozymes (TK1/TK2) were studied in peripheral lymphoid cells of 37 children with acute lymphocytic leukemia (ALL). The high TK in 25 untreated subjects (31.5 +/- 8.9) decreased during chemotherapy-induced remission to uniformly low (5.3 +/- 0.4) normal values, and rose again during relapse to a mean of (24.8 +/- 8.1). The proportion of isozyme 1 followed the same pattern but TK was a more sensitive indicator of disease state. The lymphocyte fractions' TK (per mg protein) correlated with the number (per ml blood) of WBCs, blasts and lymphocytes. Although the higher TK of blasts than of apparently normal lymphocytes was confirmed in cases permitting clean physical separation, the lymphocyte fraction of several untreated subjects with minimal blast counts also exhibited elevated TK. Moreover, this elevation was also seen in relapsed cases even if their blood (unlike bone marrow) was devoid of blasts. The results indicate that quantification of TK can reveal a subpopulation of maldifferentiated lymphocytes which are microscopically normal and that it may provide an objective parameter of prognostic differences between ALL subjects with similar hematological characteristics.

Photoinduced PEG deshielding from ROS-sensitive linkage-bridged block copolymer-based nanocarriers for on-demand drug delivery.

PubMed

Li, Jie; Sun, Chunyang; Tao, Wei; Cao, Ziyang; Qian, Haisheng; Yang, Xianzhu; Wang, Jun

2018-07-01

Controlling poly(ethylene glycol) (PEG) shielding/deshielding at the desired site of action exhibits great advantages for nanocarrier-based on-demand drug delivery in vivo. However, the current PEG deshielding strategies were mainly designed for anticancer drug delivery; even so, their applications are also limited by tumor heterogeneity. As a proof-of-concept, we explored a photoinduced PEG deshielding nanocarrier TK-NP Ce6&PTX to circumvent the aforementioned challenge. The TK-NP Ce6&PTX encapsulating chlorin e6 (Ce6) and paclitaxel (PTX) was self-assembled from an innovative thioketal (TK) linkage-bridged diblock copolymer of PEG with poly(d,l-lactic acid) (PEG-TK-PLA). We demonstrated that the high PEGylation of TK-NP Ce6&PTX in blood helps the nanocarrier efficiently avoid rapid clearance and consequently prolongs its circulation time. At the desired site (tumor), 660-nm red light irradiation led to ROS generation in situ, which readily cleaved the TK linkage, resulting in PEG deshielding. Such photoinduced PEG deshielding at the desired site significantly enhances the cellular uptake of the nanocarriers, achieving on-demand drug delivery and superior therapeutic efficacy. More importantly, this strategy of photoinducing PEG deshielding of nanocarriers could potentially extend to a variety of therapeutic agents beyond anticancer drugs for on-demand delivery. Copyright © 2018 Elsevier Ltd. All rights reserved.

Bovine Herpesvirus-4-Based Vector Delivering Peste des Petits Ruminants Virus Hemagglutinin ORF Induces both Neutralizing Antibodies and Cytotoxic T Cell Responses

PubMed Central

Macchi, Francesca; Rojas, José Manuel; Verna, Andrea Elizabeth; Sevilla, Noemí; Franceschi, Valentina; Tebaldi, Giulia; Cavirani, Sandro; Martín, Verónica; Donofrio, Gaetano

2018-01-01

Peste des Petits Ruminants Virus (PPRV) is an extremely infective morbillivirus that primarily affects goats and sheep. In underdeveloped countries where livestock are the main economical resource, PPRV causes considerable economic losses. Protective live attenuated vaccines are currently available but they induce antibody responses similar to those produced in PPRV naturally infected animals. Effective vaccines able to distinguish between vaccinated and naturally infected animals are required to PPRV control and eradication programs. Hemagglutinin (H) is a highly immunogenic PPRV envelope glycoprotein displaying both hemagglutinin and neuraminidase activities, playing a crucial role in virus attachment and penetration. In this study, a recombinant Bovine Herpesvirus-4 (BoHV-4)-based vector delivering an optimized PPRV-Hemagglutinin expression cassette, BoHV-4-A-PPRV-H-ΔTK, was assessed in immunocompetent C57BL/6 mice. BoHV-4-A-PPRV-H-ΔTK-immunization elicited both cellular and humoral immune responses with specific T cell, cytotoxic T lymphocyte, and sero-neutralizing antibody against PPRV. These data suggest recombinant BoHV-4-A-PPRV-H-ΔTK as an effective vaccine candidate to protect against PPRV herd infection and potentially applicable for eradication programs. PMID:29556236

Dasatinib inhibits the growth and survival of neoplastic human eosinophils (EOL-1) through targeting of FIP1L1-PDGFRalpha.

PubMed

Baumgartner, Christian; Gleixner, Karoline V; Peter, Barbara; Ferenc, Veronika; Gruze, Alexander; Remsing Rix, Lily L; Bennett, Keiryn L; Samorapoompichit, Puchit; Lee, Francis Y; Pickl, Winfried F; Esterbauer, Harald; Sillaber, Christian; Superti-Furga, Giulio; Valent, Peter

2008-10-01

Chronic eosinophilic leukemia (CEL) is a myeloproliferative disorder characterized by molecular and/or cytogenetic evidence of clonality of eosinophils, marked eosinophilia, and organ damage. In many patients, the transforming mutation FIP1L1-PDGFRalpha and the related CHIC2 deletion are found. The respective oncoprotein, FIP1L1-PDGFRalpha, is considered to play a major role in malignant cell growth in CEL. The tyrosine kinase (TK) inhibitor imatinib (STI571) has been described to counteract the TK activity of FIP1L1-PDGFRalpha in most patients. However, not all patients with CEL show a response to imatinib. Therefore, several attempts have been made to identify other TK inhibitors that counteract growth of neoplastic eosinophils. We provide evidence that dasatinib, a multi-targeted kinase inhibitor, blocks the growth and survival of EOL-1, an eosinophil leukemia cell line carrying FIP1L1-PDGFRalpha. The effects of dasatinib on proliferation of EOL-1 cells were dose-dependent, with an IC50 of 0.5 to 1 nM, which was found to be in the same range when compared to IC50 values produced with imatinib. Dasatinib was also found to induce apoptosis in EOL-1 cells in a dose-dependent manner (IC50: 1-10 nM). The apoptosis-inducing effects of dasatinib on EOL-1 cells were demonstrable by light microscopy, flow cytometry, and in a TUNEL assay. In Western blot experiments, dasatinib completely blocked the phosphorylation of FIP1L1-PDGFRalpha in EOL-1 cells. Dasatinib inhibits the growth of leukemic eosinophils through targeting of the disease-related oncoprotein FIP1L1-PDGFRalpha. Based on this observation, dasatinib may be considered as a new interesting treatment option for patients with CEL.

Surfactant protein C dampens inflammation by decreasing JAK/STAT activation during lung repair.

PubMed

Jin, Huiyan; Ciechanowicz, Andrzej K; Kaplan, Alanna R; Wang, Lin; Zhang, Ping-Xia; Lu, Yi-Chien; Tobin, Rachel E; Tobin, Brooke A; Cohn, Lauren; Zeiss, Caroline J; Lee, Patty J; Bruscia, Emanuela M; Krause, Diane S

2018-05-01

Surfactant protein C (SPC), a key component of pulmonary surfactant, also plays a role in regulating inflammation. SPC deficiency in patients and mouse models is associated with increased inflammation and delayed repair, but the key drivers of SPC-regulated inflammation in response to injury are largely unknown. This study focuses on a new mechanism of SPC as an anti-inflammatory molecule using SPC-TK/SPC-KO (surfactant protein C-thymidine kinase/surfactant protein C knockout) mice, which represent a novel sterile injury model that mimics clinical acute respiratory distress syndrome (ARDS). SPC-TK mice express the inducible suicide gene thymidine kinase from by the SPC promoter, which targets alveolar type 2 (AT2) cells for depletion in response to ganciclovir (GCV). We compared GCV-induced injury and repair in SPC-TK mice that have normal endogenous SPC expression with SPC-TK/SPC-KO mice lacking SPC expression. In contrast to SPC-TK mice, SPC-TK/SPC-KO mice treated with GCV exhibited more severe inflammation, resulting in over 90% mortality; there was only 8% mortality of SPC-TK animals. SPC-TK/SPC-KO mice had highly elevated inflammatory cytokines and granulocyte infiltration in the bronchoalveolar lavage (BAL) fluid. Consistent with a proinflammatory phenotype, immunofluorescence revealed increased phosphorylated signal transduction and activation of transcription 3 (pSTAT3), suggesting enhanced Janus kinase (JAK)/STAT activation in inflammatory and AT2 cells of SPC-TK/SPC-KO mice. The level of suppressor of cytokine signaling 3, an anti-inflammatory mediator that decreases pSTAT3 signaling, was significantly decreased in the BAL fluid of SPC-TK/SPC-KO mice. Hyperactivation of pSTAT3 and inflammation were rescued by AZD1480, a JAK1/2 inhibitor. Our findings showing a novel role for SPC in regulating inflammation via JAK/STAT may have clinical applications.

Imaging grafted cells with [18F]FHBG using an optimized HSV1-TK mammalian expression vector in a brain injury rodent model.

PubMed

Salabert, Anne-Sophie; Vaysse, Laurence; Beaurain, Marie; Alonso, Mathieu; Arribarat, Germain; Lotterie, Jean-Albert; Loubinoux, Isabelle; Tafani, Mathieu; Payoux, Pierre

2017-01-01

Cell transplantation is an innovative therapeutic approach after brain injury to compensate for tissue damage. To have real-time longitudinal monitoring of intracerebrally grafted cells, we explored the feasibility of a molecular imaging approach using thymidine kinase HSV1-TK gene encoding and [18F]FHBG as a reporter probe to image enzyme expression. A stable neuronal cell line expressing HSV1-TK was developed with an optimised mammalian expression vector to ensure long-term transgene expression. After [18F]FHBG incubation under defined parameters, calibration ranges from 1 X 104 to 3 X 106 Neuro2A-TK cells were analysed by gamma counter or by PET-camera. In parallel, grafting with different quantities of [18F]FHBG prelabelled Neuro2A-TK cells was carried out in a rat brain injury model induced by stereotaxic injection of malonate toxin. Image acquisition of the rats was then performed with PET/CT camera to study the [18F]FHBG signal of transplanted cells in vivo. Under the optimised incubation conditions, [18F]FHBG cell uptake rate was around 2.52%. In-vitro calibration range analysis shows a clear linear correlation between the number of cells and the signal intensity. The PET signal emitted into rat brain correlated well with the number of cells injected and the number of surviving grafted cells was recorded via the in-vitro calibration range. PET/CT acquisitions also allowed validation of the stereotaxic injection procedure. Technique sensitivity was evaluated under 5 X 104 grafted cells in vivo. No [18F]FHBG or [18F]metabolite release was observed showing a stable cell uptake even 2 h post-graft. The development of this kind of approach will allow grafting to be controlled and ensure longitudinal follow-up of cell viability and biodistribution after intracerebral injection.

Formation of the accumulative human metabolite and human-specific glutathione conjugate of diclofenac in TK-NOG chimeric mice with humanized livers.

PubMed

Kamimura, Hidetaka; Ito, Satoshi; Nozawa, Kohei; Nakamura, Shota; Chijiwa, Hiroyuki; Nagatsuka, Shin-ichiro; Kuronuma, Miyuki; Ohnishi, Yasuyuki; Suemizu, Hiroshi; Ninomiya, Shin-ichi

2015-03-01

3'-Hydroxy-4'-methoxydiclofenac (VI) is a human-specific metabolite known to accumulate in the plasma of patients after repeated administration of diclofenac sodium. Diclofenac also produces glutathione-conjugated metabolites, some of which are human-specific. In the present study, we investigated whether these metabolites could be generated in humanized chimeric mice produced from TK-NOG mice. After a single oral administration of diclofenac to humanized mice, the unchanged drug in plasma peaked at 0.25 hour and then declined with a half-life (t1/2) of 2.4 hours. 4'-Hydroxydiclofenac (II) and 3'-hydroxydiclofenac also peaked at 0.25 hour and were undetectable within 24 hours. However, VI peaked at 8 hours and declined with a t1/2 of 13 hours. When diclofenac was given once per day, peak and trough levels of VI reached plateau within 3 days. Studies with administration of II suggested VI was generated via II as an intermediate. Among six reported glutathione-conjugated metabolites of diclofenac, M1 (5-hydroxy-4-(glutathion-S-yl)diclofenac) to M6 (2'-(glutathion-S-yl)monoclofenac), we found three dichlorinated conjugates [M1, M2 (4'-hydroxy-3'-(glutathion-S-yl)diclofenac), and M3 (5-hydroxy-6-(glutathion-S-yl)diclofenac)], and a single monochlorinated conjugate [M4 (2'-hydroxy-3'-(glutathion-S-yl)monoclofenac) or M5 (4'-hydroxy-2'-(glutathion-S-yl)monoclofenac)], in the bile of humanized chimeric mice. M4 and M5 are positional isomers and have been previously reported as human-specific in vitro metabolites likely generated via arene oxide and quinone imine-type intermediates, respectively. The biliary monochlorinated metabolite exhibited the same mass spectrum as those of M4 and M5, and we discuss whether this conjugate corresponded to M4 or M5. Overall, humanized TK-NOG chimeric mice were considered to be a functional tool for the study of drug metabolism of diclofenac in humans. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

A gene involved in control of human cellular senescence on human chromosome 1q

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hensler, P.J.; Pereira-Smith, O.M.; Annab, L.A.

1994-04-01

Normal cells in culture exhibit limited division potential and have been used as a model for cellular senescence. In contrast, tumor-derived or carcinogen- or virus-transformed cells are capable of indefinite division. Fusion of normal human diploid fibroblasts with immortal human cells yielded hybrids having limited life spans, indicating that cellular senescence was dominant. Fusions of various immortal human cell lines with each other led to the identification of four complementation groups for indefinite division. The purpose of this study was to determine whether human chromosome 1 could complement the recessive immortal defect of human cell lines assigned to one ofmore » the four complementation groups. Using microcell fusion, the authors introduced a single normal human chromosome 1 into immortal human cell lines representing the complementation groups and determined that it caused loss of proliferative potential of an osteosarcoma-derived cell line (TE85), a cytomegalovirus-transformed lung fibroblast cell line (CMV-Mj-HEL-1), and a Ki-ras[sup +]-transformed derivative of TE85 (143B TK[sup [minus

The contribution of mitochondrial thymidylate synthesis in preventing the nuclear genome stress

PubMed Central

Lee, Ming-Hsiang; Wang, Liya; Chang, Zee-Fen

2014-01-01

In quiescent fibroblasts, the expression levels of cytosolic enzymes for thymidine triphosphate (dTTP) synthesis are down-regulated, causing a marked reduction in the dTTP pool. In this study, we provide evidence that mitochondrial thymidylate synthesis via thymidine kinase 2 (TK2) is a limiting factor for the repair of ultraviolet (UV) damage in the nuclear compartment in quiescent fibroblasts. We found that TK2 deficiency causes secondary DNA double-strand breaks formation in the nuclear genome of quiescent cells at the late stage of recovery from UV damage. Despite slower repair of quiescent fibroblast deficient in TK2, DNA damage signals eventually disappeared, and these cells were capable of re-entering the S phase after serum stimulation. However, these cells displayed severe genome stress as revealed by the dramatic increase in 53BP1 nuclear body in the G1 phase of the successive cell cycle. Here, we conclude that mitochondrial thymidylate synthesis via TK2 plays a role in facilitating the quality repair of UV damage for the maintenance of genome integrity in the cells that are temporarily arrested in the quiescent state. PMID:24561807

Tuberculosis is associated with expansion of a motile, permissive and immunomodulatory CD16+ monocyte population via the IL-10/STAT3 axis

PubMed Central

Lastrucci, Claire; Bénard, Alan; Balboa, Luciana; Pingris, Karine; Souriant, Shanti; Poincloux, Renaud; Al Saati, Talal; Rasolofo, Voahangy; González-Montaner, Pablo; Inwentarz, Sandra; Moraña, Eduardo Jose; Kondova, Ivanela; Verreck, Frank AW; Sasiain, Maria del Carmen; Neyrolles, Olivier; Maridonneau-Parini, Isabelle; Lugo-Villarino, Geanncarlo; Cougoule, Céline

2015-01-01

The human CD14+ monocyte compartment is composed by two subsets based on CD16 expression. We previously reported that this compartment is perturbed in tuberculosis (TB) patients, as reflected by the expansion of CD16+ monocytes along with disease severity. Whether this unbalance is beneficial or detrimental to host defense remains to be elucidated. Here in the context of active TB, we demonstrate that human monocytes are predisposed to differentiate towards an anti-inflammatory (M2-like) macrophage activation program characterized by the CD16+CD163+MerTK+pSTAT3+ phenotype and functional properties such as enhanced protease-dependent motility, pathogen permissivity and immunomodulation. This process is dependent on STAT3 activation, and loss-of-function experiments point towards a detrimental role in host defense against TB. Importantly, we provide a critical correlation between the abundance of the CD16+CD163+MerTK+pSTAT3+ cells and the progression of the disease either at the local level in a non-human primate tuberculous granuloma context, or at the systemic level through the detection of the soluble form of CD163 in human sera. Collectively, this study argues for the pathogenic role of the CD16+CD163+MerTK+pSTAT3+ monocyte-to-macrophage differentiation program and its potential as a target for TB therapy, and promotes the detection of circulating CD163 as a potential biomarker for disease progression and monitoring of treatment efficacy. PMID:26482950

A Genetic Approach to Promoter Recognition during Trans Induction of Viral Gene Expression

NASA Astrophysics Data System (ADS)

Coen, Donald M.; Weinheimer, Steven P.; McKnight, Steven L.

1986-10-01

Viral infection of mammalian cells entails the regulated induction of viral gene expression. The induction of many viral genes, including the herpes simplex virus gene encoding thymidine kinase (tk), depends on viral regulatory proteins that act in trans. Because recognition of the tk promoter by cellular transcription factors is well understood, its trans induction by viral regulatory proteins may serve as a useful model for the regulation of eukaryotic gene expression. A comprehensive set of mutations was therefore introduced into the chromosome of herpes simplex virus at the tk promoter to directly analyze the effects of promoter mutations on tk transcription. The promoter domains required for efficient tk expression under conditions of trans induction corresponded to those important for recognition by cellular transcription factors. Thus, trans induction of tk expression may be catalyzed initially by the interaction of viral regulatory proteins with cellular transcription factors.

Systems Ln-Fe-O ( Ln=Eu, Gd): thermodynamic properties of ternary oxides using solid-state electrochemical cells

NASA Astrophysics Data System (ADS)

Parida, S. C.; Rakshit, S. K.; Dash, S.; Singh, Ziley; Prasad, R.; Venugopal, V.

2003-05-01

The standard molar Gibbs energies of formation of LnFeO 3(s) and Ln3Fe 5O 12(s) where Ln=Eu and Gd have been determined using solid-state electrochemical technique employing different solid electrolytes. The reversible e.m.f.s of the following solid-state electrochemical cells have been measured in the temperature range from 1050 to 1255 K. Cell (I): (-)Pt / { LnFeO 3(s)+ Ln2O 3(s)+Fe(s)} // YDT/CSZ // {Fe(s)+Fe 0.95O(s)} / Pt(+); Cell (II): (-)Pt/{Fe(s)+Fe 0.95O(s)}//CSZ//{ LnFeO 3(s)+ Ln3Fe 5O 12(s)+Fe 3O 4(s)}/Pt(+); Cell (III): (-)Pt/{ LnFeO 3(s)+ Ln3Fe 5O 12(s)+Fe 3O 4(s)}//YSZ//{Ni(s)+NiO(s)}/Pt(+); and Cell(IV):(-)Pt/{Fe(s)+Fe 0.95O(s)}//YDT/CSZ//{ LnFeO 3(s)+ Ln3Fe 5O 12(s)+Fe 3O 4(s)}/Pt(+). The oxygen chemical potentials corresponding to the three-phase equilibria involving the ternary oxides have been computed from the e.m.f. data. The standard Gibbs energies of formation of solid EuFeO 3, Eu 3Fe 5O 12, GdFeO 3 and Gd 3Fe 5O 12 calculated by the least-squares regression analysis of the data obtained in the present study are given by Δ fG°m(EuFeO 3, s) /kJ mol -1 (± 3.2)=-1265.5+0.2687( T/K) (1050 ⩽ T/K ⩽ 1570), Δ fG°m(Eu 3Fe 5O 12, s)/kJ mol -1 (± 3.5)=-4626.2+1.0474( T/K) (1050 ⩽ T/K ⩽ 1255), Δ fG°m(GdFeO 3, s) /kJ mol -1 (± 3.2)=-1342.5+0.2539( T/K) (1050 ⩽ T/K ⩽ 1570), and Δ fG°m(Gd 3Fe 5O 12, s)/kJ·mol -1 (± 3.5)=-4856.0+1.0021( T/K) (1050 ⩽ T/K ⩽ 1255). The uncertainty estimates for Δ fG°m include the standard deviation in the e.m.f. and uncertainty in the data taken from the literature. Based on the thermodynamic information, oxygen potential diagrams for the systems Eu-Fe-O and Gd-Fe-O and chemical potential diagrams for the system Gd-Fe-O were computed at 1250 K.

Thin Film Photovoltaic Cells on Flexible Substrates Integrated with Energy Storage

DTIC Science & Technology

2011-11-30

mV[19]. In 2008, Katagiri et al ’ obtained an efficiency of 6.7% by Physical vapor methods[20]. In 2010, Mitzi et al reported an efficiency of 9.6...Films 517 (2009) 2455. [21] T.K. Todorov, K.B. Reuter, D.B. Mitzi , Adv Mater 22 (2010) El 56. 3. Energy Storage: Ultracapacitors - Professor Rastogi

SEQUENCE ANALYSIS OF MUTATIONS INDUCED BY N-ETHYL-N-NITROSOUREA IN THE TK AND HPRT GENES OF MOUSE LYMPHOMA CELLS.

EPA Science Inventory

The mouse lymphoma assay is widely used to identify chemicals that are capable of inducing mutational damages. The Tk+/- gene located on an autosome in mouse lymphoma cells may recover a wider range of mutational events than the X-linked Hprt locus. However, chemical-induced muta...

Differential suppression of glial nitric oxide synthase induction by structurally related tyrosine kinase inhibitors.

PubMed

Galea, E; Reddi, J; Feinstein, D L

1995-11-24

Incubation of C6 astrocytoma cells with bacterial endotoxin (lipopolysaccharide; LPS) plus interferon-gamma (IFN-gamma), or with a combination of cytokines (TNF-alpha, IL1-beta, and IFN-gamma) leads to high levels of inducible nitric oxide synthase (iNOS) expression. Previous results demonstrated a requirement for tyrosine kinase (TK) activities for iNOS induction. In the present study, a set of structurally related TK inhibitors, the tyrphostins (TYRs), were used to characterize possible differences between LPS and cytokine iNOS induction. All TYRs tested suppressed both types of induction. However, dose-response curves revealed significant differences in the IC50 values obtained for some TYRs (T25 and T56), and significant differences in the IC50 potency rank order when comparing inhibition of LPS versus cytokine-dependent iNOS induction. These results are consistent with differential TK utilization by the LPS versus cytokine pathways of iNOS induction, and establish a basis for developing further selective inhibitors of iNOS expression.

6,7-Dimorpholinoalkoxy quinazoline derivatives as potent EGFR inhibitors with enhanced antiproliferative activities against tumor cells.

PubMed

Zhang, Yaling; Chen, Li; Xu, Hongjiang; Li, Xiabing; Zhao, Lijun; Wang, Wei; Li, Baolin; Zhang, Xiquan

2018-03-10

A series of novel 6,7-dimorpholinoalkoxy quinazoline derivatives was designed, synthesized and evaluated as potent EGFR inhibitors. Most of synthesized derivatives exhibited moderate to excellent antiproliferative activities against five human tumor cell lines. Compound 8d displayed the most remarkable inhibitory activities against tumor cells expressing wild type (A431, A549 and SW480 cells) or mutant (HCC827 and NCI-H1975 cells) epidermal growth factor receptor (EGFR) (with IC 50 values in the range of 0.37-4.87 μM), as well as more potent inhibitory effects against recombinant EGFR tyrosine kinase (EGFR-TK, wt or T790M) (with the IC 50 values of 7.0 and 9.3 nM, respectively). Molecular docking showed that 8d can form four hydrogen bonds with EGFR, and two of them were located in the Asp855-Phe856-Gly857 (DFG) motif of EGFR. Meanwhile, 8d can significantly block EGF-induced EGFR activation and the phosphorylation of its downstream proteins such as Akt and Erk1/2 in human NSCLC cells. Also, 8d mediated cell apoptosis and the prolongation of cell cycle progression in G0/G1-phase in A549 cells. The work would have remarkable implications for further design and development of more potent EGFR tyrosine kinase inhibitors (TKIs). Copyright © 2018 Elsevier Masson SAS. All rights reserved.

TrkB overexpression in mice buffers against memory deficits and depression-like behavior but not all anxiety- and stress-related symptoms induced by developmental exposure to methylmercury.

PubMed

Karpova, Nina N; Lindholm, Jesse Saku Olavi; Kulesskaya, Natalia; Onishchenko, Natalia; Vahter, Marie; Popova, Dina; Ceccatelli, Sandra; Castrén, Eero

2014-01-01

Developmental exposure to low dose of methylmercury (MeHg) has a long-lasting effect on memory and attention deficits in humans, as well as cognitive performance, depression-like behavior and the hippocampal levels of the brain-derived neurotrophic factor (Bdnf)in mice. The Bdnf receptor TrkB is a key player of Bdnf signaling. Using transgenic animals, here we analyzed the effect of the full-length TrkB overexpression (TK+) on behavior impairments induced by perinatal MeHg. TK overexpression in the MeHg-exposed mice enhanced generalized anxiety and cue memory in the fear conditioning (FC) test. Early exposure to MeHg induced deficits in reversal spatial memory in the Morris water maze (MWM) test and depression-like behavior in the forced swim test (FST) in only wild-type (WT) mice but did not affect these parameters in TK+ mice. These changes were associated with TK+ effect on the increase in Bdnf 2, 3, 4 and 6 transcription in the hippocampus as well as with interaction of TK+ and MeHg factors for Bdnf 1, 9a and truncated TrkB.T1 transcripts in the prefrontal cortex. However, the MeHg-induced anxiety-like behavior in the elevated plus maze (EPM) and open field (OF) tests was ameliorated by TK+ background only in the OF test. Moreover, TK overexpression in the MeHg mice did not prevent significant stress-induced weight loss during the period of adaptation to individual housing in metabolic cages. These TK genotype-independent changes were primarily accompanied by the MeHg-induced hippocampal deficits in the activity-dependent Bdnf 1, 4 and 9a variants, TrkB.T1, and transcripts for important antioxidant enzymes glyoxalases Glo1 and Glo2 and glutathione reductase Gsr. Our data suggest a role of full-length TrkB in buffering against memory deficits and depression-like behavior in the MeHg mice but propose the involvement of additional pathways, such as the antioxidant system or TrkB.T1 signaling, in stress- or anxiety-related responses induced by developmental MeHg exposure.

TrkB overexpression in mice buffers against memory deficits and depression-like behavior but not all anxiety- and stress-related symptoms induced by developmental exposure to methylmercury

PubMed Central

Karpova, Nina N.; Lindholm, Jesse Saku Olavi; Kulesskaya, Natalia; Onishchenko, Natalia; Vahter, Marie; Popova, Dina; Ceccatelli, Sandra; Castrén, Eero

2014-01-01

Developmental exposure to low dose of methylmercury (MeHg) has a long-lasting effect on memory and attention deficits in humans, as well as cognitive performance, depression-like behavior and the hippocampal levels of the brain-derived neurotrophic factor (Bdnf)in mice. The Bdnf receptor TrkB is a key player of Bdnf signaling. Using transgenic animals, here we analyzed the effect of the full-length TrkB overexpression (TK+) on behavior impairments induced by perinatal MeHg. TK overexpression in the MeHg-exposed mice enhanced generalized anxiety and cue memory in the fear conditioning (FC) test. Early exposure to MeHg induced deficits in reversal spatial memory in the Morris water maze (MWM) test and depression-like behavior in the forced swim test (FST) in only wild-type (WT) mice but did not affect these parameters in TK+ mice. These changes were associated with TK+ effect on the increase in Bdnf 2, 3, 4 and 6 transcription in the hippocampus as well as with interaction of TK+ and MeHg factors for Bdnf 1, 9a and truncated TrkB.T1 transcripts in the prefrontal cortex. However, the MeHg-induced anxiety-like behavior in the elevated plus maze (EPM) and open field (OF) tests was ameliorated by TK+ background only in the OF test. Moreover, TK overexpression in the MeHg mice did not prevent significant stress-induced weight loss during the period of adaptation to individual housing in metabolic cages. These TK genotype-independent changes were primarily accompanied by the MeHg-induced hippocampal deficits in the activity-dependent Bdnf 1, 4 and 9a variants, TrkB.T1, and transcripts for important antioxidant enzymes glyoxalases Glo1 and Glo2 and glutathione reductase Gsr. Our data suggest a role of full-length TrkB in buffering against memory deficits and depression-like behavior in the MeHg mice but propose the involvement of additional pathways, such as the antioxidant system or TrkB.T1 signaling, in stress- or anxiety-related responses induced by developmental MeHg exposure. PMID:25309367

Heat capacities and entropies at 298.15 K of MgTiO3 (geikielite), ZnO (zincite), and ZnCO3 (smithsonite)

USGS Publications Warehouse

Robie, R.A.; Haselton, H.T.; Hemingway, B.S.

1989-01-01

Heat capacities of synthetic MgTiO3 (geikielite), ZnO (zincite), and natural crystals of smithsonite (ZnCO3) were measured between 9 and 366 K using an automatic adiabatically shielded calorimeter. At 298.15 K the standard molar entropies Smo of MgTiO3, ZnO, and ZnCO3 are (74.64 ?? 0.15), (43.16 ?? 0.09), and (81.19 ?? 0.16) J??K-1??mol-1, respectively. Debye temperatures for MgTiO3 and ZnO calculated from our Cp, mo values below 20 K are (900 ?? 20) K and (440 ?? 25) K respectively. Heat capacities for MgTiO3 and ZnO were combined with enthalpy increments from the literature to derive heat-capacity equations for these phases from 260 to about 1800 K. The heat capacities of MgTiO3 between 260 and 1720 K were fitted with an average deviation of 0.3 per cent by the equation: C??p,m/(J??K-1??mol-1) = 222.5-0.05274(T/K)-6.092x105(T/K)-1-1874.6(T/K) -1/2+1.878x10-5(T/K)2 and for ZnO the equation: C??p,m/(J??K-1??mol-1) = 53.999+7.851x10-4(T/K)-5.868x105(T/K)-2 -127.50(T/K)-:1/2+1.9376x10-6(T/K)2 fits the heat capacities in the temperature interval of 250 to 1800 K with an average deviation of 0.7 per cent. ?? 1989.

Computer Models for Two-Dimensional Transient Heat Conduction,

DTIC Science & Technology

1983-04-01

8217) 88) WRITE(6910) (DSDELTDITDEL) 89) WRITE(6923)U90 23 FORMAT(/ hlA 4x,’x𔃾x ’Y’,1X:NISO ITRT IMIAX ITPC ’) 91) WRITE( 6 1) X YoNISOITAT.IMAX.ITPC) 92... 1042 ) E8(KONT)=-(TK4.TK(IJ) )/(2.0DO*Dl)-.50ODI2*R4 *1043) C(KONT)=TK4/(2.ODO*DI) 1044) D(KONT)=-H(INDEX) *D12*T54P8(J)-(TK(IJ)I(2.ODO*O1) )*THPL(I

Allelic polymorphism in the T cell receptor and its impact on immune responses.

PubMed

Gras, Stephanie; Chen, Zhenjun; Miles, John J; Liu, Yu Chih; Bell, Melissa J; Sullivan, Lucy C; Kjer-Nielsen, Lars; Brennan, Rebekah M; Burrows, Jacqueline M; Neller, Michelle A; Khanna, Rajiv; Purcell, Anthony W; Brooks, Andrew G; McCluskey, James; Rossjohn, Jamie; Burrows, Scott R

2010-07-05

In comparison to human leukocyte antigen (HLA) polymorphism, the impact of allelic sequence variation within T cell receptor (TCR) loci is much less understood. Particular TCR loci have been associated with autoimmunity, but the molecular basis for this phenomenon is undefined. We examined the T cell response to an HLA-B*3501-restricted epitope (HPVGEADYFEY) from Epstein-Barr virus (EBV), which is frequently dominated by a TRBV9*01(+) public TCR (TK3). However, the common allelic variant TRBV9*02, which differs by a single amino acid near the CDR2beta loop (Gln55-->His55), was never used in this response. The structure of the TK3 TCR, its allelic variant, and a nonnaturally occurring mutant (Gln55-->Ala55) in complex with HLA-B*3501(HPVGEADYFEY) revealed that the Gln55-->His55 polymorphism affected the charge complementarity at the TCR-peptide-MHC interface, resulting in reduced functional recognition of the cognate and naturally occurring variants of this EBV peptide. Thus, polymorphism in the TCR loci may contribute toward variability in immune responses and the outcome of infection.

Thymidine kinases share a conserved function for nucleotide salvage and play an essential role in Arabidopsis thaliana growth and development.

PubMed

Xu, Jing; Zhang, Lin; Yang, Dong-Lei; Li, Qun; He, Zuhua

2015-12-01

Thymidine kinases (TKs) are important components in the nucleotide salvage pathway. However, knowledge about plant TKs is quite limited. In this study, the molecular function of TKs in Arabidopsis thaliana was investigated. Two TKs were identified and named AtTK1 and AtTK2. Expression of both genes was ubiquitous, but AtTK1 was strongly expressed in high-proliferation tissues. AtTK1 was localized to the cytosol, whereas AtTK2 was localized to the mitochondria. Mutant analysis indicated that the two genes function coordinately to sustain normal plant development. Enzymatic assays showed that the two TK proteins shared similar catalytic specificity for pyrimidine nucleosides. They were able to complement an Escherichia coli strain lacking TK activity. 5'-Fluorodeoxyuridine (FdU) resistance and 5-ethynyl 2'-deoxyuridine (EdU) incorporation assays confirmed their activity in vivo. Furthermore, the tk mutant phenotype could be alleviated by nucleotide feeding, establishing that the biosynthesis of pyrimidine nucleotides was disrupted by the TK deficiency. Finally, both human and rice (Oryza sativa) TKs were able to rescue the tk mutants, demonstrating the functional conservation of TKs across organisms. Taken together, our findings clarify the specialized function of two TKs in A. thaliana and establish that the salvage pathway mediated by the kinases is essential for plant growth and development. © 2015 Institute of Plant Physiology and Ecology, SIBS, CAS New Phytologist © 2015 New Phytologist Trust.

Assessment of α-fetoprotein targeted HSV1-tk expression in hepatocellular carcinoma with in vivo imaging.

PubMed

Park, Ju Hui; Kim, Kwang Il; Lee, Kyo Chul; Lee, Yong Jin; Lee, Tae Sup; Chung, Wee Sup; Lim, Sang Moo; Kang, Joo Hyun

2015-02-01

Tumor-specific enhancer/promoter is applicable for targeting gene expression in tumors and helpful for tumor-targeting imaging and therapy. We aimed to acquire α-fetoprotein (AFP)-producing hepatocellular carcinoma (HCC) specific images using adenovirus containing HSV1-tk gene controlled by AFP enhancer/promoter and evaluate in vivo ganciclovir (GCV)-medicated therapeutic effects on AFP-targeted HSV1-tk expression with (18)F-FDG positron emission tomography (PET). Recombinant adenovirus expressing HSV1-tk under AFP enhancer/promoter was produced (AdAFP-TK) and the expression levels were evaluated by RT-PCR and (125)I-IVDU uptake. GCV-mediated HSV1-tk cytotoxicity was determined by MTT assay. After the mixture of AdAFP-fLuc and AdAFP-TK was administrated, bioluminescent images (BLIs) and (18)F-FHBG PET images were obtained in tumor-bearing mice. In vivo therapeutic effects of AdAFP-TK and GCV in the HuH-7 xenograft model were monitored by (18)F-FDG PET. When infected with AdAFP-TK, cell viability in HuH-7 was reduced, but those in HT-29 and SK-Hep-1 were not significantly decreased at any GCV concentration less than 100 μM. AFP-targeted fLuc and HSV1-tk expression were clearly visualized by BLI and (18)F-FHBG PET images in AFP-producing HCC, respectively. In vivo GCV-mediated tumor growth inhibition by AFP-targeted HSV1-tk expression was monitored by (18)F-FDG PET. Recombinant AdAFP-TK could be applied for AFP-targeted HCC gene therapy and imaging in AFP-producing HCC.

Mechanisms of Cellular Membrane Effects of TCDD, Selected Perfluorinated Acids and Polyhalogenated Aromatic Hydrocarbons

DTIC Science & Technology

1985-02-01

toxic response of both cell lines after treatment with the perfluorinated acids ( perfluoro -n-octanoic acid, 9-H hexadecafluoro-n-nonanoic acid, and...throughout the experiments to eliminate the effect of serum on toxicity. The results for perfluoro -n-octanoic acid ( PFOA ) in both cell lines are presented in...the TK+/+ cells but not in the TK+/- cells. The results for the perfluoro -n-decanoic acid ( PFDA ) are presented in Table 3. A dose-response reldtionship

Novel infectivity-enhanced oncolytic adenovirus with a capsid-incorporated dual-imaging moiety for monitoring virotherapy in ovarian cancer.

PubMed

Kimball, Kristopher J; Rivera, Angel A; Zinn, Kurt R; Icyuz, Mert; Saini, Vaibhav; Li, Jing; Zhu, Zeng B; Siegal, Gene P; Douglas, Joanne T; Curiel, David T; Alvarez, Ronald D; Borovjagin, Anton V

2009-01-01

We sought to develop a cancer-targeted, infectivity-enhanced oncolytic adenovirus that embodies a capsid-labeling fusion for noninvasive dual-modality imaging of ovarian cancer virotherapy. A functional fusion protein composed of fluorescent and nuclear imaging tags was genetically incorporated into the capsid of an infectivity-enhanced conditionally replicative adenovirus. Incorporation of herpes simplex virus thymidine kinase (HSV-tk) and monomeric red fluorescent protein 1 (mRFP1) into the viral capsid and its genomic stability were verified by molecular analyses. Replication and oncolysis were evaluated in ovarian cancer cells. Fusion functionality was confirmed by in vitro gamma camera and fluorescent microscopy imaging. Comparison of tk-mRFP virus to single-modality controls revealed similar replication efficiency and oncolytic potency. Molecular fusion did not abolish enzymatic activity of HSV-tk as the virus effectively phosphorylated thymidine both ex vivo and in vitro. In vitro fluorescence imaging demonstrated a strong correlation between the intensity of fluorescent signal and cytopathic effect in infected ovarian cancer cells, suggesting that fluorescence can be used to monitor viral replication. We have in vitro validated a new infectivity-enhanced oncolytic adenovirus with a dual-imaging modality-labeled capsid, optimized for ovarian cancer virotherapy. The new agent could provide incremental gains toward climbing the barriers for achieving conditionally replicated adenovirus efficacy in human trials.

Disruption of a sugar transporter gene cluster in a hyperthermophilic archaeon using a host-marker system based on antibiotic resistance.

PubMed

Matsumi, Rie; Manabe, Kenji; Fukui, Toshiaki; Atomi, Haruyuki; Imanaka, Tadayuki

2007-04-01

We have developed a gene disruption system in the hyperthermophilic archaeon Thermococcus kodakaraensis using the antibiotic simvastatin and a fusion gene designed to overexpress the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene (hmg(Tk)) with the glutamate dehydrogenase promoter. With this system, we disrupted the T. kodakaraensis amylopullulanase gene (apu(Tk)) or a gene cluster which includes apu(Tk) and genes encoding components of a putative sugar transporter. Disruption plasmids were introduced into wild-type T. kodakaraensis KOD1 cells, and transformants exhibiting resistance to 4 microM simvastatin were isolated. The transformants exhibited growth in the presence of 20 microM simvastatin, and we observed a 30-fold increase in intracellular HMG-CoA reductase activity. The expected gene disruption via double-crossover recombination occurred at the target locus, but we also observed recombination events at the hmg(Tk) locus when the endogenous hmg(Tk) gene was used. This could be avoided by using the corresponding gene from Pyrococcus furiosus (hmg(Pf)) or by linearizing the plasmid prior to transformation. While both gene disruption strains displayed normal growth on amino acids or pyruvate, cells without the sugar transporter genes could not grow on maltooligosaccharides or polysaccharides, indicating that the gene cluster encodes the only sugar transporter involved in the uptake of these compounds. The Deltaapu(Tk) strain could not grow on pullulan and displayed only low levels of growth on amylose, suggesting that Apu(Tk) is a major polysaccharide-degrading enzyme in T. kodakaraensis.

Homologous Recombination and Translesion DNA Synthesis Play Critical Roles on Tolerating DNA Damage Caused by Trace Levels of Hexavalent Chromium

PubMed Central

Chen, Youjun; Zhou, Yi-Hui; Neo, Dayna; Clement, Jean; Takata, Minoru; Takeda, Shunichi; Sale, Julian; Wright, Fred A.; Swenberg, James A.; Nakamura, Jun

2016-01-01

Contamination of potentially carcinogenic hexavalent chromium (Cr(VI)) in the drinking water is a major public health concern worldwide. However, little information is available regarding the biological effects of a nanomoler amount of Cr(VI). Here, we investigated the genotoxic effects of Cr(VI) at nanomoler levels and their repair pathways. We found that DNA damage response analyzed based on differential toxicity of isogenic cells deficient in various DNA repair proteins is observed after a three-day incubation with K2CrO4 in REV1-deficient DT40 cells at 19.2 μg/L or higher as well as in TK6 cells deficient in polymerase delta subunit 3 (POLD3) at 9.8 μg/L or higher. The genotoxicity of Cr(VI) decreased ~3000 times when the incubation time was reduced from three days to ten minutes. TK mutation rate also significantly decreased from 6 day to 1 day exposure to Cr(VI). The DNA damage response analysis suggest that DNA repair pathways, including the homologous recombination and REV1- and POLD3-mediated error-prone translesion synthesis pathways, are critical for the cells to tolerate to DNA damage caused by trace amount of Cr(VI). PMID:27907204

DNA sequence responsible for the amplification of adjacent genes.

PubMed

Pasion, S G; Hartigan, J A; Kumar, V; Biswas, D K

1987-10-01

A 10.3-kb DNA fragment in the 5'-flanking region of the rat prolactin (rPRL) gene was isolated from F1BGH(1)2C1, a strain of rat pituitary tumor cells (GH cells) that produces prolactin in response to 5-bromodeoxyuridine (BrdU). Following transfection and integration into genomic DNA of recipient mouse L cells, this DNA induced amplification of the adjacent thymidine kinase gene from Herpes simplex virus type 1 (HSV1TK). We confirmed the ability of this "Amplicon" sequence to induce amplification of other linked or unlinked genes in DNA-mediated gene transfer studies. When transferred into the mouse L cells with the 10.3-5'rPRL gene sequence of BrdU-responsive cells, both the human growth hormone and the HSV1TK genes are amplified in response to 5-bromodeoxyuridine. This observation is substantiated by BrdU-induced amplification of the cotransferred bacterial Neo gene. Cotransfection studies reveal that the BrdU-induced amplification capability is associated with a 4-kb DNA sequence in the 5'-flanking region of the rPRL gene of BrdU-responsive cells. These results demonstrate that genes of heterologous origin, linked or unlinked, and selected or unselected, can be coamplified when located within the amplification boundary of the Amplicon sequence.

Isolation and characterization of acyclovir-resistant mutants of herpes simplex virus.

PubMed

Field, H J; Darby, G; Wildy, P

1980-07-01

Mutants of HSV which are resistant to acyclovir (acycloguanosine) have been isolated following serial passages of several herpes simplex virus (HSV) strains in the presence of the drug. The majority of the mutants isolated are defective in induction of thymidine kinase (TK) and this is consistent with the observation that independently isolated TK- viruses are naturally resistant to ACV. One mutant is described (SC16 R9C2) which is resistant in biochemically transformed cells which express HSV TK. This suggests that its resistance resides at a level other than TK. It is also resistant to phosphonoacetic acid, suggesting that the DNA polymerase locus may be involved. A further mutant is described [Cl (101) P2C5] which induces normal levels of TK, although the nature of resistance of this virus is not yet elucidated.

A Novel Branching Enzyme of the GH-57 Family in the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

PubMed Central

Murakami, Taira; Kanai, Tamotsu; Takata, Hiroki; Kuriki, Takashi; Imanaka, Tadayuki

2006-01-01

Branching enzyme (BE) catalyzes formation of the branch points in glycogen and amylopectin by cleavage of the α-1,4 linkage and its subsequent transfer to the α-1,6 position. We have identified a novel BE encoded by an uncharacterized open reading frame (TK1436) of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. TK1436 encodes a conserved protein showing similarity to members of glycoside hydrolase family 57 (GH-57 family). At the C terminus of the TK1436 protein, two copies of a helix-hairpin-helix (HhH) motif were found. TK1436 orthologs are distributed in archaea of the order Thermococcales, cyanobacteria, some actinobacteria, and a few other bacterial species. When recombinant TK1436 protein was incubated with amylose used as the substrate, a product peak was detected by high-performance anion-exchange chromatography, eluting more slowly than the substrate. Isoamylase treatment of the reaction mixture significantly increased the level of short-chain α-glucans, indicating that the reaction product contained many α-1,6 branching points. The TK1436 protein showed an optimal pH of 7.0, an optimal temperature of 70°C, and thermostability up to 90°C, as determined by the iodine-staining assay. These properties were the same when a protein devoid of HhH motifs (the TK1436ΔH protein) was used. The average molecular weight of branched glucan after reaction with the TK1436ΔH protein was over 100 times larger than that of the starting substrate. These results clearly indicate that TK1436 encodes a structurally novel BE belonging to the GH-57 family. Identification of an overlooked BE species provides new insights into glycogen biosynthesis in microorganisms. PMID:16885460

Investigation on culturable microflora in Tibetan kefir grains from different areas of China.

PubMed

Gao, Jie; Gu, Fengying; Abdella, Nesredin H; Ruan, Hui; He, Guoqing

2012-08-01

Four samples of Tibetan kefir grains (TK-ZJUJ 01-04) from Tibet and surrounding areas were investigated via phenotypic and genotypic methods to compare and analyze the diversity of culturable microflora among different origins. As a result, 4 genera of microorganisms from TK-ZJUJ01: Bacillus subtilis (2.9 × 10(7) cfu/mL), Lactococcus lactis (8.2 × 10(7) cfu/mL), Kluyveromyces marxianus (3.0 × 10(6) cfu/mL), Saccharomyces cerevisiae (9.0 × 10(6) cfu/mL); 4 genera from TK-ZJUJ02: Lactobacillus kefiri (1.0 × 10(8) cfu/mL), Pichia kudriavzevii (5.0 × 10(6) cfu/mL), K. marxianus (1.9 × 10(7) cfu/mL), Kazachstania unispora (6.2 × 10(7) cfu/mL); 6 genera from TK-ZJUJ03: Leuconostoc lactis (4.6 × 10(7) cfu/mL), L. lactis (3.0 × 10(7) cfu/mL), Lactobacillus plantarum (3.0 × 10(7) cfu/mL), K. unispora (3.0 × 10(6) cfu/mL), K. marxianus (2.0 × 10(6) cfu/mL), (1.7 × 10(7) cfu/mL); and 4 genera from TK-ZJUJ04: L. plantarum (1.8 × 10(7) cfu/mL), Acetobacter fabarum (5.0 × 10(6) cfu/mL), K. unispora (6.2 × 10(7) cfu/mL), Pichia guilliermondii (6.2 × 10(7) cfu/mL) were identified. Yeasts like P. kudriavzevii and P. guilliermondii isolated in this study were the first time reported in Tibetan kefir grains. For TK-ZJUJ 01-03, lactic acid bacteria were the major microorganisms, which accounted for more than 50% of all the microbial population, while for TK-ZJUJ04, the largest microbial group was yeasts which accounted for more than 50%. In a word, study of diversity and composition of microflora provided us theoretical foundation for further investigation and application of Tibetan kefir grains. This is the basic research in order to develop and industrialize a new kind of yogurt starter which is naturally formed microbiota with both lactic acid bacteria and yeasts in it. © 2012 Institute of Food Technologists®

Perceptions of Healthy-Looking and Sexy-Looking Brow Thickness.

PubMed

Hwang, Kun; Kim, Hun

2018-06-01

This study aims to determine which brow thickness levels were considered ideal, healthy-looking, young-looking, and sexy-looking by Koreans.The 163 women and 38 men who visited a brow bar were asked to view illustrations of 4 brow thickness levels defined in terms of the percentage of the eye width at the lateral canthus: very thin (VTn-1/6) (16% of eye width), thin (Tn-1/5) (20%), medium (Med-1/4) (25%), and thick (Tk-1/3) (33%) and to choose which they thought were ideal, young-looking, healthy-looking, and sexy-looking.Med-1/4 (61.2%) was the brow thickness most often considered to be ideal, followed by Tk-1/3 (17.4%) and Tn-1/5 (15.9%). VTn-1/6 was the least preferred (5.5%). Med-1/4 was the most preferred ideal thickness among both sexes. Tk-1/3 (40.8%) was the thickness most often considered to be young-looking, followed by Med-1/4 (35.3%) and Tn-1/5 (18.4%). VTn-1/6 was the least preferred (5.5%). Tk-1/3 was the most preferred young-looking thickness among both sexes. Tk-1/3 (48.8%) was the thickness most often considered to be healthy-looking, followed by Med-1/4 (30.8%) and Tn-1/5 (11.9%). VTn-1/6 was the least preferred (8.5%). Tk-1/3 was the most preferred healthy-looking thickness by women (52.2%), whereas Med-1/4 was the most common choice among men (39.5%). Tn-1/5 (40.8%) was the thickness most often considered to be sexy-looking, followed by VTn-1/6 (25.9%) and Med-1/4 (21.4%). Tk-1/3 was the least preferred (11.9%). Tn-1/5 was the most preferred sexy-looking thickness among both sexes.The results may be used as guidelines for patient or customer preferences in facial rejuvenation operations, brow esthetics, or tattooing.

Introduction of Exogenous HSV-TK Suicide Gene Increases Safety of Keratinocyte-Derived Induced Pluripotent Stem Cells by Providing Genetic "Emergency Exit" Switch.

PubMed

Sułkowski, Maciej; Konieczny, Paweł; Chlebanowska, Paula; Majka, Marcin

2018-01-09

Since their invention in 2006, induced Pluripotent Stem (iPS) cells remain a great promise for regenerative medicine circumventing the ethical issues linked to Embryonic Stem (ES) cell research. iPS cells can be generated in a patient-specific manner as an unlimited source of various cell types for in vitro drug screening, developmental biology studies and regenerative use. Having the capacity of differentiating into the cells of all three primary germ layers, iPS cells have high potential to form teratoma tumors. This remains their main disadvantage and hazard which, until resolved, prevents utilization of iPS cells in clinic. Here, we present an approach for increasing iPS cells safety by introducing genetic modification-exogenous suicide gene Herpes Simplex Virus Thymidine Kinase ( HSV-TK ). Its expression results in specific vulnerability of genetically modified cells to prodrug-ganciclovir (GCV). We show that HSV-TK expressing cells can be eradicated both in vitro and in vivo with high specificity and efficiency with low doses of GCV. Described strategy increases iPS cells safety for future clinical applications by generating "emergency exit" switch allowing eradication of transplanted cells in case of their malfunction.

Correlation of In Vivo Versus In Vitro Benchmark Doses (BMDs) Derived From Micronucleus Test Data: A Proof of Concept Study.

PubMed

Soeteman-Hernández, Lya G; Fellows, Mick D; Johnson, George E; Slob, Wout

2015-12-01

In this study, we explored the applicability of using in vitro micronucleus (MN) data from human lymphoblastoid TK6 cells to derive in vivo genotoxicity potency information. Nineteen chemicals covering a broad spectrum of genotoxic modes of action were tested in an in vitro MN test using TK6 cells using the same study protocol. Several of these chemicals were considered to need metabolic activation, and these were administered in the presence of S9. The Benchmark dose (BMD) approach was applied using the dose-response modeling program PROAST to estimate the genotoxic potency from the in vitro data. The resulting in vitro BMDs were compared with previously derived BMDs from in vivo MN and carcinogenicity studies. A proportional correlation was observed between the BMDs from the in vitro MN and the BMDs from the in vivo MN assays. Further, a clear correlation was found between the BMDs from in vitro MN and the associated BMDs for malignant tumors. Although these results are based on only 19 compounds, they show that genotoxicity potencies estimated from in vitro tests may result in useful information regarding in vivo genotoxic potency, as well as expected cancer potency. Extension of the number of compounds and further investigation of metabolic activation (S9) and of other toxicokinetic factors would be needed to validate our initial conclusions. However, this initial work suggests that this approach could be used for in vitro to in vivo extrapolations which would support the reduction of animals used in research (3Rs: replacement, reduction, and refinement). © The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology.

The combi-targeting concept: synthesis of stable nitrosoureas designed to inhibit the epidermal growth factor receptor (EGFR).

PubMed

Domarkas, Juozas; Dudouit, Fabienne; Williams, Christopher; Qiyu, Qiu; Banerjee, Ranjita; Brahimi, Fouad; Jean-Claude, Bertrand Jacques

2006-06-15

According to the "combi-targeting" concept, the EGFR tyrosine kinase (TK) inhibitory potency of compounds termed "combi-molecules" is critical for selective growth inhibition of tumor cells with disordered expression of EGFR or its closest family member erbB2. Here we report on the optimization of the EGFR TK inhibitory potency of the combi-molecules of the nitrosourea class by comparison with their aminoquinazoline and ureidoquinazoline precursors. This led to the discovery of a new structural parameter that influences their EGFR TK inhibitory potency, i.e., the torsion angle between the plane of the quinazoline ring and the ureido or the nitrosoureido moiety of the synthesized drugs. Compounds (3'-Cl and Br series) with small angles (0.5-3 degrees ) were generally stronger EGFR TK inhibitors than those with large angles (18-21 degrees ). This was further corroborated by ligand-receptor van der Waals interaction calculations that showed significant binding hindrance imposed by large torsion angles in the narrow ATP cleft of EGFR. Selective antiproliferative studies in a pair of mouse fibroblast NIH3T3 cells, one of which NIH3T3/neu being transfected with the erbB2 oncogene, showed that IC(50) values for inhibition of EGFR TK could be good predictors of their selective potency against the serum-stimulated growth of the erbB2-tranfected cell line (Pearson r = 0.8). On the basis of stability (t(1/2)), EGFR TK inhibitory potency (IC(50)), and selective erbB2 targeting, compound 23, a stable nitrosourea, was considered to have the structural requirements for further development.

A Numerical Simulation of a Carbon Black Suspension Cell Via a Time-Reversed, Double Layer Compute Algorithm

DTIC Science & Technology

1999-12-01

be accounted for by conventional descriptions of the system response. To remedy this deficiency , researchers developed a theory or model of the...timex,tO, tev, tps REAL*8 uO, width, x, xx, yy, zz, zr REAL*8 FRACi, FRAC2,F0_XX,F0_YY,F0_ZZ REAL*8 TKl, TK2 ,TQl,Tq2 INTEGER I, J, JJ, K, KK, L, NUM...UU2(J+1) !KK = Layer J+i’s time counter. TK1 = TAU(J+1) TK2 = TK1 + DELTAT(KK) j LOOP MCM C: DO KQ = UU2(J+1), KSUM PLTTIME = TIME * 1E+09 DO

Fibroblast growth factor receptor signaling affects development and function of dopamine neurons - inhibition results in a schizophrenia-like syndrome in transgenic mice.

PubMed

Klejbor, Ilona; Myers, Jason M; Hausknecht, Kathy; Corso, Thomas D; Gambino, Angelo S; Morys, Janusz; Maher, Pamela A; Hard, Robert; Richards, Jerry; Stachowiak, Ewa K; Stachowiak, Michal K

2006-06-01

Developing and mature midbrain dopamine (DA) neurons express fibroblast growth factor (FGF) receptor-1 (FGFR1). To determine the role of FGFR1 signaling in the development of DA neurons, we generated transgenic mice expressing a dominant negative mutant [FGFR1(TK-)] from the catecholaminergic, neuron-specific tyrosine hydroxylase (TH) gene promoter. In homozygous th(tk-)/th(tk-) mice, significant reductions in the size of TH-immunoreactive neurons were found in the substantia nigra compacta (SNc) and the ventral tegmental area (VTA) at postnatal days 0 and 360. Newborn th(tk-)/th(tk-) mice had a reduced density of DA neurons in both SNc and VTA, and the changes in SNc were maintained into adulthood. The reduced density of DA transporter in the striatum further demonstrated an impaired development of the nigro-striatal DA system. Paradoxically, the th(tk-)/th(tk-) mice had increased levels of DA, homovanilic acid and 3-methoxytyramine in the striatum, indicative of excessive DA transmission. These structural and biochemical changes in DA neurons are similar to those reported in human patients with schizophrenia and, furthermore, these th(tk-)/th(tk-) mice displayed an impaired prepulse inhibition that was reversed by a DA receptor antagonist. Thus, this study establishes a new developmental model for a schizophrenia-like disorder in which the inhibition of FGF signaling leads to alterations in DA neurons and DA-mediated behavior.

Enhanced combined tumor-specific oncolysis and suicide gene therapy for prostate cancer using M6 promoter.

PubMed

Ahn, M; Lee, S-J; Li, X; Jiménez, J A; Zhang, Y-P; Bae, K-H; Mohammadi, Y; Kao, C; Gardner, T A

2009-01-01

Enzyme pro-drug suicide gene therapy has been hindered by inefficient viral delivery and gene transduction. To further explore the potential of this approach, we have developed AdIU1, a prostate-restricted replicative adenovirus (PRRA) armed with the herpes simplex virus thymidine kinase (HSV-TK). In our previous Ad-OC-TK/ACV phase I clinical trial, we demonstrated safety and proof of principle with a tissue-specific promoter-based TK/pro-drug therapy using a replication-defective adenovirus for the treatment of prostate cancer metastases. In this study, we aimed to inhibit the growth of androgen-independent (AI), PSA/PSMA-positive prostate cancer cells by AdIU1. In vitro the viability of an AI- PSA/PSMA-expressing prostate cancer cell line, CWR22rv, was significantly inhibited by treatment with AdIU1 plus GCV (10 microg ml(-1)), compared with AdIU1 treatment alone and also cytotoxicity was observed following treatment with AdIU1 plus GCV only in PSA/PSMA-positive CWR22rv and C4-2 cells, but not in the PSA/PSMA-negative cell line, DU-145. In vivo assessment of AdIU1 plus GCV treatment revealed a stronger therapeutic effect against CWR22rv tumors in nude mice than treatment with AdIU1 alone, AdE4PSESE1a alone or in combination with GCV. Our results demonstrate the therapeutic potential of specific-oncolysis and suicide gene therapy for AI-PSA/PSMA-positive prostate cancer gene therapy.

Xeroderma Pigmentosum Group A Suppresses Mutagenesis Caused by Clustered Oxidative DNA Adducts in the Human Genome.

PubMed

Sassa, Akira; Kamoshita, Nagisa; Kanemaru, Yuki; Honma, Masamitsu; Yasui, Manabu

2015-01-01

Clustered DNA damage is defined as multiple sites of DNA damage within one or two helical turns of the duplex DNA. This complex damage is often formed by exposure of the genome to ionizing radiation and is difficult to repair. The mutagenic potential and repair mechanisms of clustered DNA damage in human cells remain to be elucidated. In this study, we investigated the involvement of nucleotide excision repair (NER) in clustered oxidative DNA adducts. To identify the in vivo protective roles of NER, we established a human cell line lacking the NER gene xeroderma pigmentosum group A (XPA). XPA knockout (KO) cells were generated from TSCER122 cells derived from the human lymphoblastoid TK6 cell line. To analyze the mutagenic events in DNA adducts in vivo, we previously employed a system of tracing DNA adducts in the targeted mutagenesis (TATAM), in which DNA adducts were site-specifically introduced into intron 4 of thymidine kinase genes. Using the TATAM system, one or two tandem 7,8-dihydro-8-oxoguanine (8-oxoG) adducts were introduced into the genomes of TSCER122 or XPA KO cells. In XPA KO cells, the proportion of mutants induced by a single 8-oxoG (7.6%) was comparable with that in TSCER122 cells (8.1%). In contrast, the lack of XPA significantly enhanced the mutant proportion of tandem 8-oxoG in the transcribed strand (12%) compared with that in TSCER122 cells (7.4%) but not in the non-transcribed strand (12% and 11% in XPA KO and TSCER122 cells, respectively). By sequencing the tandem 8-oxoG-integrated loci in the transcribed strand, we found that the proportion of tandem mutations was markedly increased in XPA KO cells. These results indicate that NER is involved in repairing clustered DNA adducts in the transcribed strand in vivo.

Xeroderma Pigmentosum Group A Suppresses Mutagenesis Caused by Clustered Oxidative DNA Adducts in the Human Genome

PubMed Central

Sassa, Akira; Kamoshita, Nagisa; Kanemaru, Yuki; Honma, Masamitsu; Yasui, Manabu

2015-01-01

Clustered DNA damage is defined as multiple sites of DNA damage within one or two helical turns of the duplex DNA. This complex damage is often formed by exposure of the genome to ionizing radiation and is difficult to repair. The mutagenic potential and repair mechanisms of clustered DNA damage in human cells remain to be elucidated. In this study, we investigated the involvement of nucleotide excision repair (NER) in clustered oxidative DNA adducts. To identify the in vivo protective roles of NER, we established a human cell line lacking the NER gene xeroderma pigmentosum group A (XPA). XPA knockout (KO) cells were generated from TSCER122 cells derived from the human lymphoblastoid TK6 cell line. To analyze the mutagenic events in DNA adducts in vivo, we previously employed a system of tracing DNA adducts in the targeted mutagenesis (TATAM), in which DNA adducts were site-specifically introduced into intron 4 of thymidine kinase genes. Using the TATAM system, one or two tandem 7,8-dihydro-8-oxoguanine (8-oxoG) adducts were introduced into the genomes of TSCER122 or XPA KO cells. In XPA KO cells, the proportion of mutants induced by a single 8-oxoG (7.6%) was comparable with that in TSCER122 cells (8.1%). In contrast, the lack of XPA significantly enhanced the mutant proportion of tandem 8-oxoG in the transcribed strand (12%) compared with that in TSCER122 cells (7.4%) but not in the non-transcribed strand (12% and 11% in XPA KO and TSCER122 cells, respectively). By sequencing the tandem 8-oxoG-integrated loci in the transcribed strand, we found that the proportion of tandem mutations was markedly increased in XPA KO cells. These results indicate that NER is involved in repairing clustered DNA adducts in the transcribed strand in vivo. PMID:26559182

Genetic Dissection of PTEN Signaling Mechanisms in Prostate Cancer

DTIC Science & Technology

2005-03-01

Renilla luciferase reporter to be used as a control for cell number and transfection efficiency. Luciferase assays were performed using the Dual...Luciferase Assay Kit from Promega. Reporter activities were normalized with the TK- Renilla luciferase control obtained from Promega. All assays were...Invitrogen). In addition to these components all transfections included 100 nanograms on the TK- Renilla luciferase reporter to be used as a control for cell

Synthesis of novel amides based on acridone scaffold with interesting antineoplastic activity.

PubMed

Mahajan, Anand A; Rane, Rajesh A; Amritkar, Anish A; Naphade, Shital S; Miniyar, Pankaj B; Bangalore, Pavan Kumar; Karpoormath, Rajshekhar

2015-01-01

In search of novel cytotoxic agents based on acridone scaffold, twenty five derivatives of acridone-2- carboxamide were synthesized and evaluated against a panel of eleven cancer cell lines by using MTT assay. Amides, A5 and A8 (IC50 = 0.3 µM) exhibited good cytotoxicity against MCF7. Compound A22 (IC50 = 4.3 µM) was found to be selectively cytotoxic against cancer cell line MCF7 and KB403. Particularly, promising cytotoxic activities were shown by amides A6 (IC50 = 0.7 µM), A16 (IC50 = 6.3 µM), A8 (IC50 = 0.9 µM ), A21 (IC50 = 1.3 µM), A5 (IC50 = 2.9 µM), A8 (IC50 = 2.8 µM), A14 (IC50 = 0.8 µM), A9 (IC50 = 0.8 µM) and A8 (IC50 = 0.4 µM) against cell lines; PA1, WRL68, CaCO2, TK-10, K-562, PC-3, HOP-92, ECV-304 and UACC-257, respectively. The favorable cytotoxic profile and non-toxicity towards normal human cells displayed by the derivative revealed their potential for further anticancer drug developments.

The Substrate-free and -bound Crystal Structures of the Duplicated Taurocyamine Kinase from the Human Parasite Schistosoma mansoni*

PubMed Central

Merceron, Romain; Awama, Ayman M.; Montserret, Roland; Marcillat, Olivier; Gouet, Patrice

2015-01-01

The taurocyamine kinase from the blood fluke Schistosoma mansoni (SmTK) belongs to the phosphagen kinase (PK) family and catalyzes the reversible Mg2+-dependent transfer of a phosphoryl group between ATP and taurocyamine. SmTK is derived from gene duplication, as are all known trematode TKs. Our crystallographic study of SmTK reveals the first atomic structure of both a TK and a PK with a bilobal structure. The two unliganded lobes present a canonical open conformation and interact via their respective C- and N-terminal domains at a helix-mediated interface. This spatial arrangement differs from that observed in true dimeric PKs, in which both N-terminal domains make contact. Our structures of SmTK complexed with taurocyamine or l-arginine compounds explain the mechanism by which an arginine residue of the phosphagen specificity loop is crucial for substrate specificity. An SmTK crystal was soaked with the dead end transition state analog (TSA) components taurocyamine-NO32−-MgADP. One SmTK monomer was observed with two bound TSAs and an asymmetric conformation, with the first lobe semiclosed and the second closed. However, isothermal titration calorimetry and enzyme kinetics experiments showed that the two lobes function independently. A small angle x-ray scattering model of SmTK-TSA in solution with two closed active sites was generated. PMID:25837252

The mouse lymphoma assay detects recombination, deletion, and aneuploidy.

PubMed

Wang, Jianyong; Sawyer, Jeffrey R; Chen, Ling; Chen, Tao; Honma, Masamitsu; Mei, Nan; Moore, Martha M

2009-05-01

The mouse lymphoma assay (MLA) uses the thymidine kinase (Tk) gene of the L5178Y/Tk(+/-)-3.7.2C mouse lymphoma cell line as a reporter gene to evaluate the mutagenicity of chemical and physical agents. The MLA is recommended by both the United States Food and Drug Administration and the United States Environmental Protection Agency as the preferred in vitro mammalian cell mutation assay for genetic toxicology screening because it detects a wide range of genetic alterations, including both point mutations and chromosomal mutations. However, the specific types of chromosomal mutations that can be detected by the MLA need further clarification. For this purpose, three chemicals, including two clastogens and an aneugen (3'-azido-3'-deoxythymidine, mitomycin C, and taxol), were used to induce Tk mutants. Loss of heterozygosity (LOH) analysis was used to select mutants that could be informative as to whether they resulted from deletion, mitotic recombination, or aneuploidy. A combination of additional methods, G-banding analysis, chromosome painting, and a real-time PCR method to detect the copy number (CN) of the Tk gene was then used to provide a detailed analysis. LOH involving at least 25% of chromosome 11, a normal karyotype, and a Tk CN of 2 would indicate that the mutant resulted from recombination, whereas LOH combined with a karyotypically visible deletion of chromosome 11 and a Tk CN of 1 would indicate a deletion. Aneuploidy was confirmed using G-banding combined with chromosome painting analysis for mutants showing LOH at every microsatellite marker on chromosome 11. From this analysis, it is clear that mouse lymphoma Tk mutants can result from recombination, deletion, and aneuploidy.

The combi-targeting concept: a novel 3,3-disubstituted nitrosourea with EGFR tyrosine kinase inhibitory properties.

PubMed

Qiu, Qiyu; Dudouit, Fabienne; Matheson, Stephanie L; Brahimi, Fouad; Banerjee, Ranjita; McNamee, James P; Jean-Claude, Bertrand J

2003-01-01

To study the dual mechanism of action of FD137, a 3,3-disubstituted nitrosourea designed to block signaling mediated by the epidermal growth factor receptor (EGFR) on its own and to be hydrolyzed to an inhibitor of EGFR plus a DNA-damaging species. HPLC was used to determine the half-life (t(1/2)) of FD137 and to characterize its derived metabolite FD110. The dual mechanisms of DNA damaging and EGFR tyrosine kinase (TK) targeting were ascertained by the comet assay for DNA damage and by inmunodetection of phosphotyrosine in an ELISA and a whole-cell assay for EGFR-mediated signaling. The antiproliferative effects of the different drugs and their combinations were determined by the sulforhodamine B (SRB) assay. In contrast to BCNU, FD137 significantly blocked EGF-induced EGFR autophosphorylation (IC(50) 4 micro M) in the human solid tumor cell line A431. DNA damage induced by FD137 could only be observed after 24 h exposure, but the level of DNA damage remained 3.6-fold lower than that induced by BCNU. This difference was rationalized by the 160-fold greater stability of FD137 when compared with BCNU in serum-containing medium. Further, degradation of FD137 was accompanied by the slow release of FD110, an extremely potent inhibitor of EGFR TK [IC(50) (EGFR autophosphorylation) <0.3 micro M]. The complex properties of FD137 translated into a 55-fold greater antiproliferative activity than BCNU against the EGFR-overexpressing A431 cells that coexpresses the O(6)-alkylguanine transferase (AGT). Depletion of AGT in these cells by the use of O(6)-benzylguanine (O(6)-BG) enhanced their sensitivity to BCNU by 8-fold, but only by 3-fold to FD137. The results overall suggest that the superior antiproliferative activity of FD137 when compared with BCNU may be associated with its ability to behave as a combination of many species with different mechanisms of action. However, the enhancement of its potency by O(6)-BG suggests that its antiproliferative effect was at least partially mitigated by AGT and perhaps it may be largely dominated by its signal transduction inhibitory component.

Remediation of Cd-contaminated soil around metal sulfide mines

NASA Astrophysics Data System (ADS)

Lu, Xinzhe; Hu, Xuefeng; Kang, Zhanjun; Luo, Fan

2017-04-01

The mines of metal sulfides are widely distributed in the southwestern part of Zhejiang Province, Southeast China. The activities of mining, however, often lead to the severe pollution of heavy metals in soils, especially Cd contamination. According to our field investigations, the spatial distribution of Cd-contaminated soils is highly consistent with the presence of metal sulfide mines in the areas, further proving that the mining activities are responsible for Cd accumulation in the soils. To study the remediation of Cd-contaminated soils, a paddy field nearby large sulfide mines, with soil pH 6 and Cd more than 1.56 mg kg-1, five times higher than the national recommended threshold, was selected. Plastic boards were deeply inserted into soil to separate the field and make experimental plots, with each plot being 4 m×4 m. Six treatments, TK01˜TK06, were designed to study the effects of different experimental materials on remediating Cd-contaminated soils. The treatment of TK01 was the addition of 100 kg zeolites to the plot; TK02, 100 kg apatites; TK03, 100 kg humid manure; TK04, 50 kg zeolites + 50 kg apatites; TK05, 50 kg zeolites + 50 kg humid manure; TK06 was blank control (CK). One month after the treatments, soil samples at the plots were collected to study the possible change of chemical forms of Cd in the soils. The results indicated that these treatments reduced the content of available Cd in the soils effectively, by a decreasing sequence of TK04 (33%) > TK02 (25%) > TK01 (23%) > TK05 (22%) > TK03 (15%), on the basis of CK. Correspondingly, the treatments also reduced the content of Cd in rice grains significantly, by a similar decreasing sequence of TK04 (83%) > TK02 (77%) > TK05 (63%) > TK01 (47%) > TK03 (27%). The content of Cd in the rice grains was 0.071 mg kg-1, 0.094 mg kg-1, 0.159 mg kg-1, 0.22 mg kg-1 and 0.306 mg kg-1, respectively, compared with CK, 0.418 mg kg-1. This experiment suggested that the reduction of available Cd in the soils is the key to the remediation of Cd-contaminated soils, and apply the composite material of zeolite combining apatite is the best choice for the remediation of Cd-contaminated soils.

Cellular and molecular insight into the inhibition of primary root growth of Arabidopsis induced by peptaibols, a class of linear peptide antibiotics mainly produced by Trichoderma spp.

PubMed

Shi, Wei-Ling; Chen, Xiu-Lan; Wang, Li-Xia; Gong, Zhi-Ting; Li, Shuyu; Li, Chun-Long; Xie, Bin-Bin; Zhang, Wei; Shi, Mei; Li, Chuanyou; Zhang, Yu-Zhong; Song, Xiao-Yan

2016-04-01

Trichoderma spp. are well known biocontrol agents that produce a variety of antibiotics. Peptaibols are a class of linear peptide antibiotics mainly produced by Trichoderma Alamethicin, the most studied peptaibol, is reported as toxic to plants at certain concentrations, while the mechanisms involved are unclear. We illustrated the toxic mechanisms of peptaibols by studying the growth-inhibitory effect of Trichokonin VI (TK VI), a peptaibol from Trichoderma longibrachiatum SMF2, on Arabidopsis primary roots. TK VI inhibited root growth by suppressing cell division and cell elongation, and disrupting root stem cell niche maintenance. TK VI increased auxin content and disrupted auxin response gradients in root tips. Further, we screened the Arabidopsis TK VI-resistant mutant tkr1. tkr1 harbors a point mutation in GORK, which encodes gated outwardly rectifying K(+)channel proteins. This mutation alleviated TK VI-induced suppression of K(+)efflux in roots, thereby stabilizing the auxin gradient. The tkr1 mutant also resisted the phytotoxicity of alamethicin. Our results indicate that GORK channels play a key role in peptaibol-plant interaction and that there is an inter-relationship between GORK channels and maintenance of auxin homeostasis. The cellular and molecular insight into the peptaibol-induced inhibition of plant root growth advances our understanding of Trichoderma-plant interactions. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

INDUCTION OF CELL CYCLE ARREST AND APOPTOSIS BY ORMENIS ERIOLEPIS A MORROCAN ENDEMIC PLANT IN VARIOUS HUMAN CANCER CELL LINES.

PubMed

Belayachi, Lamiae; Aceves-Luquero, Clara; Merghoub, Nawel; de Mattos, Silvia Fernández; Amzazi, Saaîd; Villalonga, Priam; Bakri, Youssef

2017-01-01

Ormenis eriolepis Coss (Asteraceae) is an endemic Moroccan subspecies, traditionally named "Hellala" or "Fergoga". It's usually used for its hypoglycemic effect as well as for the treatment of stomacal pain. As far as we know, there is no scientific exploration of anti tumoral activity of Ormenis eriolepis extracts. In this regard, we performed a screening of organic extracts and fractions in a panel of both hematological and solid cancer cell lines, to evaluate the potential in vitro anti tumoral activity and to elucidate the respective mechanisms that may be responsible for growth arrest and cell death induction. The plant was extracted using organic solvents, and four different extracts were screened on Jurkat, Jeko-1, TK-6, LN229, SW620, U2OS, PC-3 and NIH3T3 cells. Cell viability assays revealed that, the IC50 values were (11,63±5,37μg/ml) for Jurkat, (13,33±1,67μg/ml) for Jeko-1, (41,67±1,98μg/ml) for LN229 and (19,31±4,88μg/ml) for PC-3 cells upon treatment with Oe-DF and Oe-HE respectively. Both the fraction and extract exhibited no effects on TK6 and NIH3T3. Cytometry analysis accompanied by DNA damage signaling protein levels monitoring (p-H2A.X), showed that both the Dichloromethane Fraction and Hexanic extract induce DNA double stranded breaks (DSBs) accompanied by cell cycle arrest in G1 (Jurkat, Jeko -1 and LN22) and G2/M (PC-3) phases which is agreed with the caspase activity observed. Additional experiments with selective inhibitors of stress and survival pathways (JNK, MAPK, Rho, p53, and JAK3) indicated that none of these pathways was significantly involved in apoptosis induction. The bioactive compound analysis by CG/MS indicated that the major compounds in Oe-DF were: Linoleic Acid (15,89%), Podophyllotoxin (17,89%) and Quercetin (22,95%). For Oe-HE the major molecules were: Linoleic Acid (9,76%), α-curcumene (7,07%), α-bisabolol (5,49%), Campesterol (4,41%), Stigmasterol (14,08%) and β-sitosterol (7,49%). Our data suggest that bioactive compounds present in Ormenis eriolepis show significant anti proliferative activity inducing cell cycle arrest and cell death operating through apoptosis pathway.

Lack of dependence on p53 for DNA double strand break repair of episomal vectors in human lymphoblasts

NASA Technical Reports Server (NTRS)

Kohli, M.; Jorgensen, T. J.

1999-01-01

The p53 tumor suppressor gene has been shown to be involved in a variety of repair processes, and recent findings have suggested that p53 may be involved in DNA double strand break repair in irradiated cells. The role of p53 in DNA double strand break repair, however, has not been fully investigated. In this study, we have constructed a novel Epstein-Barr virus (EBV)-based shuttle vector, designated as pZEBNA, to explore the influence of p53 on DNA strand break repair in human lymphoblasts, since EBV-based vectors do not inactivate the p53 pathway. We have compared plasmid survival of irradiated, restriction enzyme linearized, and calf intestinal alkaline phosphatase (CIP)-treated pZEBNA with a Simian virus 40 (SV40)-based shuttle vector, pZ189, in TK6 (wild-type p53) and WTK1 (mutant p53) lymphoblasts and determined that p53 does not modulate DNA double strand break repair in these cell lines. Copyright 1999 Academic Press.

The Kinetic Effects on Thymidine Kinase 2 by Enzyme-Bound dTTP May Explain the Mitochondrial Side Effects of Antiviral Thymidine Analogs▿†

PubMed Central

Wang, Liya; Sun, Ren; Eriksson, Staffan

2011-01-01

Mitochondrial thymidine kinase 2 (TK2) is a key enzyme in the salvage of pyrimidine deoxynucleosides needed for mitochondrial DNA synthesis. TK2 phosphorylates thymidine (dThd), deoxycytidine (dCyd), and many other antiviral pyrimidine nucleoside analogs. Zidovudine (AZT) is the first nucleoside analog approved for anti-HIV therapy, and it is still used in combination with other drugs. One of the side effects of long-term treatment with nucleoside analogs is mitochondrial DNA depletion, which has been ascribed to competition by AZT for the endogenous dThd phosphorylation carried out by TK2. Here we studied the kinetics of AZT and 3′-fluorothymidine phosphorylation by recombinant human TK2 and the effects of these and other pyrimidine nucleoside analogs on the phosphorylation of dThd and dCyd. Thymidine analogs strongly inhibited dThd phosphorylation but not dCyd phosphorylation, which instead was stimulated ∼30%. We found that recombinant human TK2 contained the feedback inhibitor dTTP in a 1:1 molar ratio and that incubation with dThd and AZT could completely remove the enzyme-bound dTTP, but dCyd was less efficient in this regard. The release of feedback inhibitor by dThd and dThd analogs most likely accounts for the observed kinetics. Similar effects were also observed with native rat liver mitochondrial TK2, strongly indicating a physiologic role for this process, which most likely is an important factor in the mitochondrial toxicity observed with antiviral nucleoside analogs. PMID:21444706

Viral drug sensitivity testing using quantitative PCR: effect of tyrosine kinase inhibitors on polyomavirus BK replication.

PubMed

Randhawa, Parmjeet S; Farasati, Noush A; Huang, Yuchen; Mapara, Markus Y; Shapiro, Ron

2010-12-01

Our objective was to determine whether quantitative polymerase chain reaction (PCR) can be used to measure the effect of tyrosine kinase (TK) inhibition on polyomavirus BK (BKV) replication. The BKV was grown in a cell culture system. The rate of viral replication in the presence or absence of the drug being tested was assessed by amplifying the viral genome using primers directed against the viral capsid 1 protein. Dasatinib, erlotinib, gefitinib, imatinib, sunitinib, and sorafenib all showed antiviral activity at micromolar concentrations. The 50% effective concentration for erlotinib and sorafenib was within blood concentrations readily achieved in human subjects. Quantitative PCR is a convenient method for viral drug sensitivity testing for slow-growing viruses that do not readily produce cytopathic effect. TK inhibitors deserve further consideration as a potential therapeutic option for BKV-associated nephropathy and hemorrhagic cystitis.

Characterization of a thymidine kinase-deficient mutant of equine herpesvirus 4 and in vitro susceptibility of the virus to antiviral agents.

PubMed

Azab, Walid; Tsujimura, Koji; Kato, Kentaro; Arii, Jun; Morimoto, Tomomi; Kawaguchi, Yasushi; Tohya, Yukinobu; Matsumura, Tomio; Akashi, Hiroomi

2010-02-01

Equine herpesvirus 4 (EHV-4) is an important equine pathogen that causes respiratory tract disease among horses worldwide. A thymidine kinase (TK)-deletion mutant has been generated by using bacterial artificial chromosome (BAC) technology to investigate the role of TK in pathogenesis. Deletion of TK had virtually no effect on the growth characteristics of WA79DeltaTK in cell culture when compared to the parent virus. Also, virus titers and plaque formation were unaffected in the absence of the TK gene. The sensitivity of EHV-4 to inhibition by acyclovir (ACV) and ganciclovir (GCV) was studied by means of a plaque reduction assay. GCV proved to be more potent and showed a superior anti-EHV-4 activity. On the other hand, ACV showed very poor ability to inhibit EHV-4 replication. As predicted, WA79DeltaTK was insensitive to GCV. Although EHV-4 is normally insensitive to ACV, it showed >20-fold increase in sensitivity when the equine herpesvirus-1 (EHV-1) TK was supplied in trans. Furthermore, both ACV and GCV resulted in a significant reduction of plaque size induced by EHV-4 and 1. Taken together, these data provided direct evidence that GCV is a potent selective inhibitor of EHV-4 and that the virus-encoded TK is an important determinant of the virus susceptibility to nucleoside analogues. Copyright 2009 Elsevier B.V. All rights reserved.

Size- and coating-dependent cytotoxicity and genotoxicity of silver nanoparticles evaluated using in vitro standard assays.

PubMed

Guo, Xiaoqing; Li, Yan; Yan, Jian; Ingle, Taylor; Jones, Margie Yvonne; Mei, Nan; Boudreau, Mary D; Cunningham, Candice K; Abbas, Mazhar; Paredes, Angel M; Zhou, Tong; Moore, Martha M; Howard, Paul C; Chen, Tao

2016-11-01

The physicochemical characteristics of silver nanoparticles (AgNPs) may greatly alter their toxicological potential. To explore the effects of size and coating on the cytotoxicity and genotoxicity of AgNPs, six different types of AgNPs, having three different sizes and two different coatings, were investigated using the Ames test, mouse lymphoma assay (MLA) and in vitro micronucleus assay. The genotoxicities of silver acetate and silver nitrate were evaluated to compare the genotoxicity of nanosilver to that of ionic silver. The Ames test produced inconclusive results for all types of the silver materials due to the high toxicity of silver to the test bacteria and the lack of entry of the nanoparticles into the cells. Treatment of L5718Y cells with AgNPs and ionic silver resulted in concentration-dependent cytotoxicity, mutagenicity in the Tk gene and the induction of micronuclei from exposure to nearly every type of the silver materials. Treatment of TK6 cells with these silver materials also resulted in concentration-dependent cytotoxicity and significantly increased micronucleus frequency. With both the MLA and micronucleus assays, the smaller the AgNPs, the greater the cytotoxicity and genotoxicity. The coatings had less effect on the relative genotoxicity of AgNPs than the particle size. Loss of heterozygosity analysis of the induced Tk mutants indicated that the types of mutations induced by AgNPs were different from those of ionic silver. These results suggest that AgNPs induce cytotoxicity and genotoxicity in a size- and coating-dependent manner. Furthermore, while the MLA and in vitro micronucleus assay (in both types of cells) are useful to quantitatively measure the genotoxic potencies of AgNPs, the Ames test cannot.

[Cytosine deaminase and thymidine kinase double suicide gene system driven by carcinoembryonic antigen promoter for the treatment of colorectal carcinoma xenograft in nude mice].

PubMed

Huang, Zheng-jie; Feng, Qing-zhao; You, Jun; Xu, Lin; Luo, Wei-yuan; Yi, Wen-cheng; Zeng, Yue-yue; Luo, Qi

2013-07-23

To explore the therapeutic efficacy of double suicide gene system driven by carcinoembryonic antigen (CEA) promoter (Cp-CDglyTK) on colorectal carcinoma xenograft in nude mice. The plasmid pcDNA3.1(-)Cp-CDglyTK was transfected into the CEA-positive SW480 and CEA-negative HeLa cells respectively. The expression of suicide gene was detected by RT-PCR. And the transfected cells were treated with 5-fluorocytosine (5-FC) and ganciclovir (GCV) at different concentrations and the cell-killing and bystander effects assayed by methyl thiazolyl tetrazolium (MTT). By a transplantation of cultivated cells, SW480 or HeLa cell lines were injected subcutaneously into right axillary of nude mice to establish 96 SW480 and 72 HeLa tumor animal models. Nude mice were completely randomized with statistical software according to tumor volume. For prodrug therapy, 48 SW480-bearing mice were divided equally into 4 groups of I-IV. At the same time, 48 HeLa-bearing mice were divided equally into 4 groups of V-VIII. Groups I & V received an intratumoral injection of PBS, groups II & VIGCV and 5-FC intratumorally, groups III & VII PBS intraperitoneally and groups IV & VIII GCV and 5-FC intraperitoneally. Forty-eight SW480-bearing mice were divided equally into 4 groups of IX∼XII and 24 Hela-bearing ones into groups of & in therapy experiment by suicide gene plus prodrug. Six groups received an intratumoral injection of liposome Lipofectamine and plasmid CP-CDglyTK and then an intraperitoneal injection of drug. The groups of IX and received an injection of PBS, group X GCV, group XI 5-FC and groups XII & GCV and 5-FC. The observation parameters included tumor bulk, tumor weight, survival time and treatment effect in each group. SW480 cells transfected by plasmid pcDNA3.1(-)Cp- CDglyTK expressed CDglyTK gene. The inhibition rates of GCV and 5-FC were significantly higher than those of HeLa cells (59.87% ± 0.21% vs 9.90% ± 0.09%, P < 0.01). And higher inhibition rates and stronger bystander effect existed in double versus single produg (all P < 0.05). Tumor size, final tumor weight and survival time of nude mice in groups ofII, IV, VI & VIII had no significant difference with groups ofI, III, V & VII (all P < 0.05). Final tumor size and weight of group XII was significantly smaller than those of groups of IX, X and XI ((150.0 ± 3.2) vs (522.5 ± 1.9) and (256.8 ± 10.4) and (260.7 ± 2.2) mm(3), (54.1 ± 10.4) vs (682.0 ± 12.0) and (251.8 ± 15.1) and (271.6 ± 17.7) mg, all P < 0.05). Meanwhile, the tumor inhibition rate and survival time of group XII(92.1% and (25.7 ± 0.8)d) were significant higher and longer than group X (63.1% and (21.8 ± 0.5) d) and group XI (60.2% and (18.0 ± 0.9) d) (all P < 0.05). However, no significant difference existed in tumor size, final tumor weight and survival time between groups and (all P > 0.05). The inhibition rate of group was merely 0.9%. CDglyTK double suicide gene system driven by CEA promoter may inhibit CEA positive colorectal cancer xenograft in prodrug-treated nude mice.

A defect in the thymidine kinase 2 gene causing isolated mitochondrial myopathy without mtDNA depletion.

PubMed

Leshinsky-Silver, E; Michelson, M; Cohen, S; Ginsberg, M; Sadeh, M; Barash, V; Lerman-Sagie, T; Lev, D

2008-07-01

Isolated mitochondrial myopathies (IMM) are either due to primary defects in mtDNA, in nuclear genes that control mtDNA abundance and structure such as thymidine kinase 2 (TK2), or due to CoQ deficiency. Defects in the TK2 gene have been found to be associated with mtDNA depletion attributed to a depleted mitochondrial dNTP pool in non-dividing cells. We report an unusual case of IMM, homozygous for the H90N mutation in the TK2 gene but unlike other cases with the same mutation, does not demonstrate mtDNA depletion. The patient's clinical course is relatively mild and a muscle biopsy showed ragged red muscle fibers with a mild decrease in complexes I and an increase in complexes IV and II activities. This report extends the phenotypic expression of TK2 defects and suggests that all patients who present with an IMM even with normal quantities of mtDNA should be screened for TK2 mutations.

Epstein-Barr virus thymidine kinase is a centrosomal resident precisely localized to the periphery of centrioles.

PubMed

Gill, Michael B; Kutok, Jeffery L; Fingeroth, Joyce D

2007-06-01

The thymidine kinase (TK) encoded by Epstein-Barr virus (EBV) differs not only from that of the alphaherpesviruses but also from that of the gamma-2 herpesvirus subfamily. Because cellular location is frequently a determinant of regulatory function, to gain insight into additional role(s) of EBV TK and to uncover how the lymphocryptovirus and rhadinovirus enzymes differ, the subcellular localizations of EBV TK and the related cercopithecine herpesvirus-15 TK were investigated. We show that in contrast to those of the other family members, the gamma-1 herpesvirus TKs localize to the centrosome and even more precisely to the periphery of the centriole, tightly encircling the tubulin-rich centrioles in a microtubule-independent fashion. Centrosomal localization is observed in diverse cell types and occurs whether the protein is expressed independently or in the context of lytic EBV infection. Surprisingly, analysis of mutants revealed that the unique N-terminal domain was not critical for targeting to the centrosome, but rather, peptide sequences located C terminal to this domain were key. This is the first herpesvirus protein documented to reside in the centrosome, or microtubule-organizing center, an amembranous organelle that regulates the structural biology of the cell cycle through control of chromosome separation and cytokinesis. More recently, proteasome-mediated degradation of cell cycle regulatory proteins, production and loading of antigenic peptides onto HLA molecules, and transient homing of diverse virion proteins required for entry and/or egress have been shown to be coordinated at the centrosome. Potential implications of centrosomal localization for EBV TK function are discussed.

Tissue kallikrein deficiency, insulin resistance, and diabetes in mouse and man.

PubMed

Potier, Louis; Waeckel, Ludovic; Fumeron, Fréderic; Bodin, Sophie; Fysekidis, Marinos; Chollet, Catherine; Bellili, Naima; Bonnet, Fabrice; Gusto, Gaëlle; Velho, Gilberto; Marre, Michel; Alhenc-Gelas, François; Roussel, Ronan; Bouby, Nadine

2014-05-01

The kallikrein-kinin system has been suggested to participate in the control of glucose metabolism. Its role and the role of angiotensin-I-converting enzyme, a major kinin-inactivating enzyme, are however the subject of debate. We have evaluated the consequence of deficiency in tissue kallikrein (TK), the main kinin-forming enzyme, on the development of insulin resistance and diabetes in mice and man. Mice with inactivation of the TK gene were fed a high-fat diet (HFD) for 3 months, or crossed with obese, leptin-deficient (ob/ob) mice to generate double ob/ob-TK-deficient mutants. In man, a loss-of-function polymorphism of the TK gene (R53H) was studied in a large general population cohort tested for insulin resistance, the DESIR study (4843 participants, 9 year follow-up). Mice deficient in TK gained less weight on the HFD than their WT littermates. Fasting glucose level was increased and responses to glucose (GTT) and insulin (ITT) tolerance tests were altered at 10 and 16 weeks on the HFD compared with standard on the diet, but TK deficiency had no influence on these parameters. Likewise, ob-TK⁻/⁻ mice had similar GTT and ITT responses to those of ob-TK⁺/⁺ mice. TK deficiency had no effect on blood pressure in either model. In humans, changes over time in BMI, fasting plasma glucose, insulinemia, and blood pressure were not influenced by the defective 53H-coding TK allele. The incidence of diabetes was not influenced by this allele. These data do not support a role for the TK-kinin system, protective or deleterious, in the development of insulin resistance and diabetes.

Remnant living cells that escape cell loss in late-stage tumors exhibit cancer stem cell-like characteristics

PubMed Central

Chen, Y-L; Wang, S-Y; Liu, R-S; Wang, H-E; Chen, J-C; Chiou, S-H; Chang, C A; Lin, L-T; Tan, D T W; Lee, Y-J

2012-01-01

A balance between cell proliferation and cell loss is essential for tumor progression. Although up to 90% of cells are lost in late-stage carcinomas, the progression and characteristics of remnant living cells in tumor mass are unclear. Here we used molecular imaging to track the progression of living cells in a syngeneic tumor model, and ex vivo investigated the properties of this population at late-stage tumor. The piggyBac transposon system was used to stably introduce the dual reporter genes, including monomeric red fluorescent protein (mRFP) and herpes simplex virus type-1 thymidine kinase (HSV1-tk) genes for fluorescence-based and radionuclide-based imaging of tumor growth in small animals, respectively. Iodine-123-labeled 5-iodo-2′-fluoro-1-beta-𝒟-arabinofuranosyluracil was used as a radiotracer for HSV1-tk gene expression in tumors. The fluorescence- and radionuclide-based imaging using the single-photon emission computed tomography/computed tomography revealed that the number of living cells reached the maximum at 1 week after implantation of 4T1 tumors, and gradually decreased and clustered near the side of the body until 4 weeks accompanied by enlargement of tumor mass. The remnant living cells at late-stage tumor were isolated and investigated ex vivo. The results showed that these living cells could form mammospheres and express cancer stem cell (CSC)-related biomarkers, including octamer-binding transcription factor 4, SRY (sex-determining region Y)-box 2, and CD133 genes compared with those cultured in vitro. Furthermore, this HSV1-tk-expressing CSC-like population was sensitive to ganciclovir applied for the suicide therapy. Taken together, the current data suggested that cells escaping from cell loss in late-stage tumors exhibit CSC-like characteristics, and HSV1-tk may be considered a theranostic agent for targeting this population in vivo. PMID:23034334

Is the renal kallikrein-kinin system a factor that modulates calciuria?

PubMed

Negri, Armando Luis

Renal tubular calcium reabsorption is one of the principal factors that determine serum calcium concentration and calcium excretion. Calcium excretion is regulated by the distal convoluted tubule and connecting tubule, where the epithelial calcium channel TRPV5 can be found, which limits the rate of transcellular calcium transport. The dynamic presence of the TRPV5 channel on the surface of the tubular cell is mediated by an endosomal recycling process. Different intrarenal factors are involved in calcium channel fixation in the apical membrane, including the anti-ageing hormone klotho and tissue kallikrein (TK). Both proteins are synthesised in the distal tubule and secreted in the tubular fluid. TK stimulates active calcium reabsorption through the bradykinin receptor B2 that compromises TRPV5 activation through the protein kinase C pathway. TK-deficient mice show hypercalciuria of renal origin comparable to that seen in TRPV5 knockout mice. There is a polymorphism with loss of function of the human TK gene R53H (allele H) that causes a marked decrease in enzymatic activity. The presence of the allele H seems to be common at least in the Japanese population (24%). These individuals have a tendency to greater calcium and sodium excretion in urine that is more evident during furosemide infusion. Future studies should analyse if manipulating the renal kallikrein-kinin system can correct idiopathic hypercalciuria with drugs other than thiazide diuretics. Copyright © 2016 Sociedad Española de Nefrología. Published by Elsevier España, S.L.U. All rights reserved.

Plasmacytoid Dendritic Cells in the Tumor Microenvironment: Immune Targets for Glioma Therapeutics12

PubMed Central

Candolfi, Marianela; King, Gwendalyn D; Yagiz, Kader; Curtin, James F; Mineharu, Yohei; Muhammad, AKM Ghulam; Foulad, David; Kroeger, Kurt M; Barnett, Nick; Josien, Regis; Lowenstein, Pedro R; Castro, Maria G

2012-01-01

Adenovirus-mediated delivery of the immune-stimulatory cytokine Flt3L and the conditionally cytotoxic thymidine kinase (TK) induces tumor regression and long-term survival in preclinical glioma (glioblastoma multiforme [GBM]) models. Flt3L induces expansion and recruitment of plasmacytoid dendritic cells (pDCs) into the brain. Although pDCs can present antigen and produce powerful inflammatory cytokines, that is, interferon α (IFN-α), their role in tumor immunology remains debated. Thus, we studied the role of pDCs and IFN-α in Ad.TK/GCV+ Ad.Flt3L-mediated anti-GBM therapeutic efficacy. Our data indicate that the combined gene therapy induced recruitment of plasmacytoid DCs (pDCs) into the tumor mass; which were capable of in vivo phagocytosis, IFN-α release, and T-cell priming. Thus, we next used either pDCs or an Ad vector encoding IFN-α delivered within the tumor microenvironment. When rats were treated with Ad.TK/GCV in combination with pDCs or Ad-IFN-α, they exhibited 35% and 50% survival, respectively. However, whereas intracranial administration of Ad.TK/GCV + Ad.Flt3L exhibited a high safety profile, Ad-IFN-α led to severe local inflammation, with neurologic and systemic adverse effects. To elucidate whether the efficacy of the immunotherapy was dependent on IFN-α-secreting pDCs, we administered an Ad vector encoding B18R, an IFN-α antagonist, which abrogated the antitumoral effect of Ad.TK/GCV + Ad.Flt3L. Our data suggest that IFN-α release by activated pDCs plays a critical role in the antitumor effect mediated by Ad.TK/GCV + Ad.Flt3L. In summary, taken together, our results demonstrate that pDCs mediate anti-GBM therapeutic efficacy through the production of IFN-α, thus manipulation of pDCs constitutes an attractive new therapeutic target for the treatment of GBM. PMID:22952428

Poor recognition of O6-isopropyl dG by MGMT triggers double strand break-mediated cell death and micronucleus induction in FANC-deficient cells

PubMed Central

Hashimoto, Kiyohiro; Sharma, Vyom; Sasanuma, Hiroyuki; Tian, Xu; Takata, Minoru; Takeda, Shunichi; Swenberg, James A.; Nakamura, Jun

2016-01-01

Isopropyl methanesulfonate (IPMS) is the most potent genotoxic compound among methanesulfonic acid esters. The genotoxic potential of alkyl sulfonate esters is believed to be due to their alkylating ability of the O6 position of guanine. Understanding the primary repair pathway activated in response to IPMS-induced DNA damage is important to profile the genotoxic potential of IPMS. In the present study, both chicken DT40 and human TK6 cell-based DNA damage response (DDR) assays revealed that dysfunction of the FANC pathway resulted in higher sensitivity to IPMS compared to EMS or MMS. O6-alkyl dG is primarily repaired by methyl guanine methyltransferase (MGMT), while isopropyl dG is less likely to be a substrate for MGMT. Comparison of the cytotoxic potential of IPMS and its isomer n-propyl methanesulfonate (nPMS) revealed that the isopropyl moiety avoids recognition by MGMT and leads to higher cytotoxicity. Next, the micronucleus (MN) assay showed that FANC deficiency increases the sensitivity of DT40 cells to MN induction by IPMS. Pretreatment with O6-benzyl guanine (OBG), an inhibitor of MGMT, increased the MN frequency in DT40 cells treated with nPMS, but not IPMS. Lastly, IPMS induced more double strand breaks in FANC-deficient cells compared to wild-type cells in a time-dependent manner. All together, these results suggest that IPMS-derived O6-isopropyl dG escapes recognition by MGMT, and the unrepaired DNA damage leads to double strand breaks, resulting in MN induction. FANC, therefore, plays a pivotal role in preventing MN induction and cell death caused by IPMS. PMID:27486975

Low p16INK4a Expression in Early Passage Human Prostate Basal Epithelial Cells Enables Immortalization by Telomerase Expression Alone.

PubMed

Graham, Mindy Kim; Principessa, Lorenzo; Antony, Lizamma; Meeker, Alan K; Isaacs, John T

2017-03-01

There are two principal senescence barriers that must be overcome to successfully immortalize primary human epithelial cells in culture, stress-induced senescence, and replicative senescence. The p16 INK4a /retinoblastoma protein (p16/Rb) pathway mediates stress-induced senescence, and is generally upregulated by primary epithelial cells in response to the artificial conditions from tissue culture. Replicative senescence is associated with telomere loss. Following each round of cell division, telomeres progressively shorten. Once telomeres shorten to a critical length, the DNA damage response pathway is activated, and the tumor suppressor p53 pathway triggers replicative senescence. Exogenous expression of telomerase in normal human epithelial cells extends the replicative capacity of cells, and in some cases, immortalizes cells. However reliable immortalization of epithelial cells usually requires telomerase activity coupled with inactivation of the p16/Rb pathway. A lentiviral vector, pLOX-TERT-iresTK (Addgene #12245), containing a CMV promoter upstream of a bicistronic coding cassette that includes loxP sites flanking the catalytic subunit of human telomerase gene (TERT) and herpes simplex virus type-1 thymidine kinase gene (HSV1-tk) was used to transduce normal prostate basal epithelial cells (PrECs) initiated in cell culture from prostate cancer patients undergoing radical prostatectomies. Transduction of early (i.e., <7) passage PrECs with TERT led to successful immortalization. However, attempts to immortalize late (i.e., >7) passage PrECs were unsuccessful. Late passage PrECs, which acquired elevated p16, were unable to overcome the senescence barrier. Immortalized PrECs (TERT-PrECs) retained a normal male karyotype and low p16 expression. Additionally, TERT-PrECs were non-tumorigenic when inoculated into intact male immunodeficient NSG mice. The present studies document that early passage human PrECs have sufficiently low p16 to permit immortalization by TERT expression alone. TERT-PrECs developed using this transduction approach provides an appropriate and experimentally facile model for clarifying the molecular mechanism(s) involved in both immortalization of human PrECs, as well as identifying genetic/epigenetic "drivers" for conversion of these immortalized non-tumorigenic cells into fully lethal prostate cancers. Notably, loxP sites flank the exogenous TERT gene in the TERT-PrECs. Cre recombinase can be used to excise TERT, and resolve whether TERT expression is required for these cells to be fully transformed into lethal cancer. Prostate 77: 374-384, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

The Ron Receptor Regulates Kupffer Cell-Dependent Cytokine Production and Hepatocyte Survival Following Endotoxin Exposure in Mice

PubMed Central

Stuart, William D.; Kulkarni, Rishikesh M.; Gray, Jerilyn K.; Vasiliauskas, Juozas; Leonis, Mike A.; Waltz, Susan E.

2011-01-01

Previous studies demonstrated that targeted deletion of the Ron receptor tyrosine kinase (TK) domain in mice leads to marked hepatocyte protection in a well-characterized model of lipopolysaccharide (LPS)-induced acute liver failure in D-galactosamine (GalN)-sensitized mice. Hepatocyte protection in TK−/− mice was observed despite paradoxically elevated serum levels of tumor necrosis factor alpha (TNFα). To understand the role of Ron in the liver, purified populations of Kupffer cells and hepatocytes from wild-type (TK+/+) and TK−/− mice were studied. Utilizing quantitative RT-PCR, we demonstrated that Ron is expressed in these cell-types. Moreover, we also recapitulated the protected hepatocyte phenotype and exaggerated cytokine production observed in the TK−/− mice in vivo through the use of purified cultured cells ex vivo. We show that isolated TK−/− Kupffer cells produce increased levels of TNFα and select cytokines compared to TK+/+ cells following LPS stimulation. We also show that conditioned media from LPS-treated TK−/− Kupffer cells was more toxic to hepatocytes than control media, suggesting the exaggerated levels of cytokines produced from the TK−/− Kupffer cells are detrimental to wild type hepatocytes. In addition, we observed that TK−/− hepatocytes were more resistant to cell death compared to TK+/+ hepatocytes, suggesting that Ron functions in both the epithelial and inflammatory cell compartments to regulate acute liver injury. These findings were confirmed in vivo in mice with hepatocyte and macrophage cell-type-specific conditional Ron deletions. Mice with Ron loss selectively in hepatocytes exhibited less liver damage and increased survival compared to mice with Ron loss in macrophages. In conclusion, we have dissected cell-type-specific roles for Ron such that this receptor modulates cytokine production from Kupffer cells and inhibits hepatocyte survival in response to injury. PMID:21520175

Synthesis of a probe for monitoring HSV1-tk reporter gene expression using chemical exchange saturation transfer MRI

PubMed Central

Bar-Shir, Amnon; Liu, Guanshu; Greenberg, Marc M; Bulte, Jeff W M; Gilad, Assaf A

2013-01-01

In experiments involving transgenic animals or animals treated with transgenic cells, it is important to have a method to monitor the expression of the relevant genes longitudinally and noninvasively. An MRI-based reporter gene enables monitoring of gene expression in the deep tissues of living subjects. This information can be co-registered with detailed high-resolution anatomical and functional information. We describe here the synthesis of the reporter probe, 5-methyl-5,6-dihydrothymidine (5-MDHT), which can be used for imaging of the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene expression in rodents by MRI. The protocol also includes data acquisition and data processing routines customized for chemical exchange saturation transfer (CEST) contrast mechanisms. The dihydropyrimidine 5-MDHT is synthesized through a catalytic hydrogenation of the 5,6-double bond of thymidine to yield 5,6-dihydrothymidine, which is methylated on the C-5 position of the resulting saturated pyrimidine ring. The synthesis of 5-MDHT can be completed within 5 d, and the compound is stable for more than 1 year. PMID:24177294

MUTANT FREQUENCY AND MUTATIONAL SPECTRA IN THETK AND HPRT GENES OF N-ETHYL-N-NITROSOUREA TREATED MOUSE LYMPHOMA CELLS

EPA Science Inventory

Abstract

The mouse lymphoma assay (MLA) utilizing the Tk locus is widely used to identify chemical mutagens. The autosomal location of the Tk locus allows for the detection of a wide range of mutational events, from point mutations to chromosome alterations. However, the ...

Functional characterization of the turkey macrophage migration inhibitory factor.

PubMed

Park, Myeongseon; Kim, Sungwon; Fetterer, Raymond H; Dalloul, Rami A

2016-08-01

Macrophage migration inhibitory factor (MIF) is a soluble protein that inhibits the random migration of macrophages and plays a pivotal immunoregulatory function in innate and adaptive immunity. The aim of this study was to clone the turkey MIF (TkMIF) gene, express the active protein, and characterize its basic function. The full-length TkMIF gene was amplified from total RNA extracted from turkey spleen, followed by cloning into a prokaryotic (pET11a) expression vector. Sequence analysis revealed that TkMIF consists of 115 amino acids with 12.5 kDa molecular weight. Multiple sequence alignment revealed 100%, 65%, 95% and 92% identity with chicken, duck, eagle and zebra finch MIFs, respectively. Recombinant TkMIF (rTkMIF) was expressed in Escherichia coli and purified through HPLC and endotoxin removal. SDS-PAGE analysis revealed an approximately 13.5 kDa of rTkMIF monomer containing T7 tag in soluble form. Western blot analysis showed that anti-chicken MIF (ChMIF) polyclonal antisera detected a monomer form of TkMIF at approximately 13.5 kDa size. Further functional analysis revealed that rTkMIF inhibits migration of both mononuclear cells and splenocytes in a dose-dependent manner, but was abolished by the addition of anti-ChMIF polyclonal antisera. qRT-PCR analysis revealed elevated transcripts of pro-inflammatory cytokines by rTkMIF in LPS-stimulated monocytes. rTkMIF also led to increased levels of IFN-γ and IL-17F transcripts in Con A-activated splenocytes, while IL-10 and IL-13 transcripts were decreased. Overall, the sequences of both the turkey and chicken MIF have high similarity and comparable biological functions with respect to migration inhibitory activities of macrophages and enhancement of pro-inflammatory cytokine expression, suggesting that turkey and chicken MIFs would be biologically cross-reactive. Copyright © 2016 Elsevier Ltd. All rights reserved.

Application of oligonucleotide array CGH to the simultaneous detection of a deletion in the nuclear TK2 gene and mtDNA depletion.

PubMed

Zhang, Shulin; Li, Fang-Yuan; Bass, Harold N; Pursley, Amber; Schmitt, Eric S; Brown, Blaire L; Brundage, Ellen K; Mardach, Rebecca; Wong, Lee-Jun

2010-01-01

Thymidine kinase 2 (TK2), encoded by the TK2 gene on chromosome 16q22, is one of the deoxyribonucleoside kinases responsible for the maintenance of mitochondrial deoxyribonucleotide pools. Defects in TK2 mainly cause a myopathic form of the mitochondrial DNA depletion syndrome (MDDS). Currently, only point mutations and small insertions and deletions have been reported in TK2 gene; gross rearrangements of TK2 gene and possible hepatic involvement in patients with TK2 mutations have not been described. We report a non-consanguineous Jordanian family with three deceased siblings due to mtDNA depletion. Sequence analysis of the father detected a heterozygous c.761T>A (p.I254N) mutation in his TK2 gene; however, point mutations in the mother were not detected. Subsequent gene dosage analysis using oligonucleotide array CGH identified an intragenic approximately 5.8-kb deletion encompassing the 5'UTR to intron 2 of her TK2 gene. Sequence analysis confirmed that the deletion spans c.1-495 to c.283-2899 of the TK2 gene (nucleotide 65,136,256-65,142,086 of chromosome 16). Analysis of liver and muscle specimens from one of the deceased infants in this family revealed compound heterozygosity for the paternal point mutation and maternal intragenic deletion. In addition, a significant reduction of the mtDNA content in liver and muscle was detected (10% and 20% of age- and tissue-matched controls, respectively). Prenatal diagnosis was performed in the third pregnancy. The fetus was found to carry both the point mutation and the deletion. This child died 6months after birth due to myopathy. A serum specimen demonstrated elevated liver transaminases in two of the infants from whom results were available. This report expands the mutation spectrum associated with TK2 deficiency. While the myopathic form of MDDS appears to be the main phenotype of TK2 mutations, liver dysfunction may also be a part of the mitochondrial depletion syndrome caused by TK2 gene defects.

Zinc oxide tetrapods inhibit herpes simplex virus infection of cultured corneas

PubMed Central

Duggal, Neil; Jaishankar, Dinesh; Yadavalli, Tejabhiram; Hadigal, Satvik; Mishra, Yogendra Kumar; Adelung, Rainer

2017-01-01

Purpose Infection of the human cornea by herpes simplex virus type-1 (HSV-1) can cause significant vision loss. The purpose of this study was to develop an ex vivo model to visualize viral growth and spread in the cornea. The model was also used to analyze cytokine production and study the antiviral effects of zinc oxide tetrapods. Methods A β-galactosidase-expressing recombinant virus, HSV-1(KOS)tk12, was used to demonstrate the ability of the virus to enter and develop blue plaques on human corneal epithelial (HCE) cells and corneal tissues. Freshly obtained porcine corneas were cultured and then scratched before infection with HSV-1(KOS)tk12. The blue plaques on the corneas were imaged using a stereomicroscope. Western blot analysis for HSV-1 proteins was performed to verify HSV-1 infection of the cornea. Using the ex vivo model, zinc oxide tetrapods were tested for their anti-HSV-1 potential, and a cytokine profile was developed to assess the effects of the treatment. Results Cultured corneas and the use of β-galactosidase-expressing HSV-1(KOS)tk12 virus can provide an attractive ex vivo model to visualize and study HSV-1 entry and spread of the infection in tissues. We found that unlike cultured HCE cells, which demonstrated nearly 100% infectivity, HSV-1 infection of the cultured cornea was more restrictive and took longer to develop. We also found that the zinc oxide tetrapod–shaped nano- and microstructures inhibited HSV infection of the cultured cells, as well as the cultured corneas. The cytokine profile of the infected samples was consistent with previous studies of HSV-1 corneal infection. Conclusions The ability to visualize HSV-1 growth and spread in corneal tissues can provide new details about HSV-1 infection of the cornea and the efficacy of new cornea-specific antiviral drug candidates. The ex vivo model also demonstrates antiviral effects of zinc oxide tetrapods and adequately portrays the drug delivery issues that cornea-specific treatments face. PMID:28275313

Long Term Expression of Drosophila melanogaster Nucleoside Kinase in Thymidine Kinase 2-deficient Mice with No Lethal Effects Caused by Nucleotide Pool Imbalances*

PubMed Central

Krishnan, Shuba; Paredes, João A.; Zhou, Xiaoshan; Kuiper, Raoul V.; Hultenby, Kjell; Curbo, Sophie; Karlsson, Anna

2014-01-01

Mitochondrial DNA depletion caused by thymidine kinase 2 (TK2) deficiency can be compensated by a nucleoside kinase from Drosophila melanogaster (Dm-dNK) in mice. We show that transgene expression of Dm-dNK in Tk2 knock-out (Tk2−/−) mice extended the life span of Tk2−/− mice from 3 weeks to at least 20 months. The Dm-dNK+/−Tk2−/− mice maintained normal mitochondrial DNA levels throughout the observation time. A significant difference in total body weight due to the reduction of subcutaneous and visceral fat in the Dm-dNK+/−Tk2−/− mice was the only visible difference compared with control mice. This indicates an effect on fat metabolism mediated through residual Tk2 deficiency because Dm-dNK expression was low in both liver and fat tissues. Dm-dNK expression led to increased dNTP pools and an increase in the catabolism of purine and pyrimidine nucleotides but these alterations did not apparently affect the mice during the 20 months of observation. In conclusion, Dm-dNK expression in the cell nucleus expanded the total dNTP pools to levels required for efficient mitochondrial DNA synthesis, thereby compensated the Tk2 deficiency, during a normal life span of the mice. The Dm-dNK+/− mouse serves as a model for nucleoside gene or enzyme substitutions, nucleotide imbalances, and dNTP alterations in different tissues. PMID:25296759

Long term expression of Drosophila melanogaster nucleoside kinase in thymidine kinase 2-deficient mice with no lethal effects caused by nucleotide pool imbalances.

PubMed

Krishnan, Shuba; Paredes, João A; Zhou, Xiaoshan; Kuiper, Raoul V; Hultenby, Kjell; Curbo, Sophie; Karlsson, Anna

2014-11-21

Mitochondrial DNA depletion caused by thymidine kinase 2 (TK2) deficiency can be compensated by a nucleoside kinase from Drosophila melanogaster (Dm-dNK) in mice. We show that transgene expression of Dm-dNK in Tk2 knock-out (Tk2(-/-)) mice extended the life span of Tk2(-/-) mice from 3 weeks to at least 20 months. The Dm-dNK(+/-)Tk2(-/-) mice maintained normal mitochondrial DNA levels throughout the observation time. A significant difference in total body weight due to the reduction of subcutaneous and visceral fat in the Dm-dNK(+/-)Tk2(-/-) mice was the only visible difference compared with control mice. This indicates an effect on fat metabolism mediated through residual Tk2 deficiency because Dm-dNK expression was low in both liver and fat tissues. Dm-dNK expression led to increased dNTP pools and an increase in the catabolism of purine and pyrimidine nucleotides but these alterations did not apparently affect the mice during the 20 months of observation. In conclusion, Dm-dNK expression in the cell nucleus expanded the total dNTP pools to levels required for efficient mitochondrial DNA synthesis, thereby compensated the Tk2 deficiency, during a normal life span of the mice. The Dm-dNK(+/-) mouse serves as a model for nucleoside gene or enzyme substitutions, nucleotide imbalances, and dNTP alterations in different tissues. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Deletion of the Ron receptor tyrosine kinase domain in mice provides protection from endotoxin-induced acute liver failure.

PubMed

Leonis, Mike A; Toney-Earley, Kenya; Degen, Sandra J F; Waltz, Susan E

2002-11-01

The targeted deletion of the cytoplasmic domain of the Ron receptor tyrosine kinase (TK) in mice leads to exaggerated responses to injury in several murine models of inflammation as well as increased lethality in response to endotoxin (lipopolysaccharide [LPS]). Using a well-characterized model of LPS-induced acute liver failure (ALF) in galactosamine (GalN)-sensitized mice, we show that Ron TK(-/-) mice display marked protection compared with control Ron TK(+/+) mice. Whereas control mice have profound elevation of serum aminotransferase levels (a marker of hepatocyte injury) and hemorrhagic necrosis of the liver, in dramatic contrast, Ron TK(-/-) mice have mild elevation of aminotransferase levels and relatively normal liver histology. These findings are associated with a reduction in the number of liver cells undergoing apoptosis in Ron TK(-/-) mice. Paradoxically, treatment of Ron TK(-/-) mice with LPS/GalN leads to markedly elevated (3.5-fold) serum levels of tumor necrosis factor (TNF) alpha, a key inflammatory mediator in this liver injury model, as well as reduced amounts of interleukin (IL) 10 (a suppressor of TNF-alpha production) and interferon (IFN)-gamma (a TNF-alpha sensitizer). These results show that ablation of the TK activity of the Ron receptor leads to protection from the development of hepatocellular apoptosis in response to treatment with LPS/GalN, even in the presence of excessive levels of serum TNF-alpha. In conclusion, our studies show that the Ron receptor TK plays a critical role in modulating the response of the liver to endotoxin.

Nuclear localisation of calreticulin in vivo is enhanced by its interaction with glucocorticoid receptors.

PubMed

Roderick, H L; Campbell, A K; Llewellyn, D H

1997-03-24

The multi-functional protein calreticulin (CRT) is normally found within the lumen of the endoplasmic reticulum (ER). However, some of its proposed functions require it to be located within the nucleus, where its presence is contentious. We have investigated this in live COS7, HeLa and LM(TK-) cells using green fluorescent protein (GFP)-fusion proteins. GFP-CRT, and GFP, with an ER signal peptide and a KDEL sequence (ER-GFP), were localised to the ER. In addition, GFP-CRT was located in the nucleus of all the cell types at low levels. The higher levels of nuclear fluorescence in LM(TK-) and HeLa cells suggested that glucocorticoid receptors might enhance nuclear localisation of calreticulin. Dexamethasone treatment of LM(TK-) cells doubled the amount of nuclear GFP-CRT, but did not affect the localisation of a GFP-CRT fusion in which the glucocorticoid receptor-binding N-domain of calreticulin had been deleted. Thus, despite ER targeting and retention signals, calreticulin is also located within the nucleus where its presence increases due to its interaction with glucocorticoid receptors.

A combination hepatoma-targeted therapy based on nanotechnology: pHRE-Egr1-HSV-TK/131I-antiAFPMcAb-GCV/MFH

NASA Astrophysics Data System (ADS)

Lin, Mei; Huang, Junxing; Jiang, Xingmao; Zhang, Jia; Yu, Hong; Ye, Jun; Zhang, Dongsheng

2016-09-01

Combination targeted therapy is a promising cancer therapeutic strategy. Here, using PEI-Mn0.5Zn0.5Fe2O4 nanoparticles (PEI-MZF-NPs) as magnetic media for MFH (magnetic fluid hyperthermia) and gene transfer vector for gene-therapy, a combined therapy, pHRE-Egr1-HSV-TK/131I-antiAFPMcAb-GCV/MFH, for hepatoma is developed. AntiAFPMcAb (Monoclonal antibody AFP) is exploited for targeting. The plasmids pHRE-Egr1-HSV-TK are achieved by incorporation of pEgr1-HSV-TK and pHRE-Egr1-EGFP. Restriction enzyme digestion and PCR confirm the recombinant plasmids pHRE-Egr1-HSV-TK are successfully constructed. After exposure to the magnetic field, PEI-MZF-NPs/pHRE-Egr1-EGFP fluid is warmed rapidly and then the temperature is maintained at 43 °C or so, which is quite appropriate for cancer treatment. The gene expression reaches the peak when treated with 200 μCi 131I for 24 hours, indicating that the dose of 200 μCi might be the optimal dose for irradiation and 24 h irradiation later is the best time to initiate MFH. The in vitro and in vivo experiments demonstrate that pHRE-Egr1-HSV-TK/131I-antiAFPMcAb-GCV/MFH can greatly suppress hepatic tumor cell proliferation and induce cell apoptosis and necrosis and effectively inhibit the tumor growth, much better than any monotherapy does alone. Furthermore, the combination therapy has few or no adverse effects. It might be applicable as a strategy to treat hepatic cancer.

Biotransformation of β‐hydroxypyruvate and glycolaldehyde to l‐erythrulose by Pichia pastoris strain GS115 overexpressing native transketolase

PubMed Central

Wei, Yu‐Chia; Braun‐Galleani, Stephanie; Henríquez, Maria José; Bandara, Sahan

2017-01-01

Transketolase is a proven biocatalytic tool for asymmetric carbon‐carbon bond formation, both as a purified enzyme and within bacterial whole‐cell biocatalysts. The performance of Pichia pastoris as a host for transketolase whole‐cell biocatalysis was investigated using a transketolase‐overexpressing strain to catalyze formation of l‐erythrulose from β‐hydroxypyruvic acid and glycolaldehyde substrates. Pichia pastoris transketolase coding sequence from the locus PAS_chr1‐4_0150 was subcloned downstream of the methanol‐inducible AOX1 promoter in a plasmid for transformation of strain GS115, generating strain TK150. Whole and disrupted TK150 cells from shake flasks achieved 62% and 65% conversion, respectively, under optimal pH and methanol induction conditions. In a 300 μL reaction, TK150 samples from a 1L fed‐batch fermentation achieved a maximum l‐erythrulose space time yield (STY) of 46.58 g L−1 h−1, specific activity of 155 U gCDW−1, product yield on substrate (Yp/s) of 0.52 mol mol−1 and product yield on catalyst (Yp/x) of 2.23g gCDW−1. We have successfully exploited the rapid growth and high biomass characteristics of Pichia pastoris in whole cell biocatalysis. At high cell density, the engineered TK150 Pichia pastoris strain tolerated high concentrations of substrate and product to achieve high STY of the chiral sugar l‐erythrulose. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:99–106, 2018 PMID:29086489

Evaluating the potential genotoxicity of phthalates esters (PAEs) in perfumes using in vitro assays.

PubMed

Al-Saleh, Iman; Al-Rajudi, Tahreer; Al-Qudaihi, Ghofran; Manogaran, Pulicat

2017-10-01

We previously reported high levels of phthalate esters (PAEs) added as solvents or fixatives in 47 brands of perfumes. Diethyl phthalate was the most abundant compound (0.232-23,649 ppm), and 83.3% of the perfumes had levels >1 ppm, the threshold limit cited by a Greenpeace investigation. All samples had dimethyl phthalate levels higher than its threshold limit of 0.1 ppm, and 88, 38, and 7% of the perfumes had benzyl butyl phthalate, di(2-ethylhexyl) phthalate, and dibutyl phthalate levels, respectively, above their threshold limits. The role of PAEs as endocrine disruptors has been well documented, but their effect on genotoxic behavior has received little attention. We used in vitro single-cell gel electrophoresis (comet) and micronucleus (MN) assays with human lymphoblastoid TK6 cells to evaluate the genotoxic potency of 42 of the same perfumes and to determine its association with PAEs. All perfumes induced more DNA damage than a negative control (NEG), ≥ 90% of the samples caused more damage than cells treated with the vehicles possibly used in perfume's preparations such as methanol (ME) and ethanol (ET), and 11.6% of the perfumes caused more DNA damage than a positive control (hydrogen peroxide). Chromosome breakage expressed as MN frequency was higher in cells treated with 71.4, 64.3, 57.1, and 4.8% of the perfumes than in NEG, cells treated with ME or ET, and another positive control (x-rays), respectively. The genotoxic responses in the comet and MN assays were not correlated. The comet assay indicated that the damage in TK6 cells treated with five PAEs at concentrations of 0.05 and 0.2 ppm either individually or as a mixture did not differ significantly from the damage in cells treated with the perfumes. Unlike the comet assay, the sensitivity of the MN assay to PAEs was weak at both low and high concentrations, and MN frequencies were generally low. This study demonstrates for the first time the possible contribution of PAEs in perfumes to DNA damage and suggests that their use as solvents or fixatives should be regulated. Other ingredients with mutagenic/genotoxic properties, however, may also have contributed to the DNA damage. Future studies should focus on applying a series of assays that use different cellular models with various endpoints to identify the spectrum of genotoxic mechanisms involved.

Predictive Toxicology of cobalt ferrite nanoparticles: comparative in-vitro study of different cellular models using methods of knowledge discovery from data

PubMed Central

2013-01-01

Background Cobalt-ferrite nanoparticles (Co-Fe NPs) are attractive for nanotechnology-based therapies. Thus, exploring their effect on viability of seven different cell lines representing different organs of the human body is highly important. Methods The toxicological effects of Co-Fe NPs were studied by in-vitro exposure of A549 and NCIH441 cell-lines (lung), precision-cut lung slices from rat, HepG2 cell-line (liver), MDCK cell-line (kidney), Caco-2 TC7 cell-line (intestine), TK6 (lymphoblasts) and primary mouse dendritic-cells. Toxicity was examined following exposure to Co-Fe NPs in the concentration range of 0.05 -1.2 mM for 24 and 72 h, using Alamar blue, MTT and neutral red assays. Changes in oxidative stress were determined by a dichlorodihydrofluorescein diacetate based assay. Data analysis and predictive modeling of the obtained data sets were executed by employing methods of Knowledge Discovery from Data with emphasis on a decision tree model (J48). Results Different dose–response curves of cell viability were obtained for each of the seven cell lines upon exposure to Co-Fe NPs. Increase of oxidative stress was induced by Co-Fe NPs and found to be dependent on the cell type. A high linear correlation (R2=0.97) was found between the toxicity of Co-Fe NPs and the extent of ROS generation following their exposure to Co-Fe NPs. The algorithm we applied to model the observed toxicity belongs to a type of supervised classifier. The decision tree model yielded the following order with decrease of the ranking parameter: NP concentrations (as the most influencing parameter), cell type (possessing the following hierarchy of cell sensitivity towards viability decrease: TK6 > Lung slices > NCIH441 > Caco-2 = MDCK > A549 > HepG2 = Dendritic) and time of exposure, where the highest-ranking parameter (NP concentration) provides the highest information gain with respect to toxicity. The validity of the chosen decision tree model J48 was established by yielding a higher accuracy than that of the well-known “naive bayes” classifier. Conclusions The observed correlation between the oxidative stress, caused by the presence of the Co-Fe NPs, with the hierarchy of sensitivity of the different cell types towards toxicity, suggests that oxidative stress is one possible mechanism for the toxicity of Co-Fe NPs. PMID:23895432

Microglia used as vehicles for both inducible thymidine kinase gene therapy and MRI contrast agents for glioma therapy.

PubMed

Ribot, E; Bouzier-Sore, A-K; Bouchaud, V; Miraux, S; Delville, M-H; Franconi, J-M; Voisin, P

2007-08-01

Microglia are phagocytic cells that are chemoattracted by brain tumors and can represent up to 70% of the tumor cell population. To get insight into gene therapy against glioma, we decided to take advantage of those microglia properties and to use those cells as vehicles to transport simultaneously a suicide gene (under the control of a heat-sensitive promoter) and contrast agents to localize them by magnetic resonance imaging before applying any therapeutic treatment. Thymidine kinase (TK) expression and its functionality after gancyclovir administration were investigated. After the heat shock (44 degrees C and 20 min), TK was expressed in 50% of the cells. However, after gancyclovir treatment, 90% of the cells died by apoptosis, showing an important bystander effect. Then, the cells were incubated with new lanthanide contrast agents to check both their potential toxicity and their MR properties. Results indicate that the nanoparticles did not induce any cell toxicity and yield a hypersignal on MR images at 4.7 T. These in vitro experiments indicate that microglia are good candidates as vectors in gene therapy against brain tumors. Finally, microglia containing gadolinium-grafted nanoparticles were injected in the close vicinity of C6 tumor, in a mouse. The hyperintensive signal obtained on in vivo images as well as its retention time show the potential of the novel contrast agents for cellular imaging.

Heat capacity and entropy at the temperatures 5 K to 720 K and thermal expansion from the temperatures 298 K to 573 K of synthetic enargite (Cu3AsS4)

USGS Publications Warehouse

Seal, R.R.; Robie, R.A.; Hemingway, B.S.; Evans, H.T.

1996-01-01

The heat capacity of synthetic Cu3AsS4 (enargite) was measured by quasi-adiabatic calorimetry from the temperatures 5 K to 355 K and by differential scanning calorimetry from T = 339 K to T = 720 K. Heat-capacity anomalies were observed at T = (58.5 ?? 0.5) K (??trsHom = 1.4??R??K; ??trsSom = 0.02??R) and at T = (66.5 ?? 0.5) K (??trsHom = 4.6??R??K; ??trsSom = 0.08??R), where R = 8.31451 J??K-1??mol-1. The causes of the anomalies are unknown. At T = 298.15 K, Cop,m and Som(T) are (190.4 ?? 0.2) J??K-1??mol-1 and (257.6 ?? 0.6) J??K-1??mol-1, respectively. The superambient heat capacities are described from T = 298.15 K to T = 944 K by the least-squares regression equation: Cop,m/(J??K-1??mol-1) = (196.7 ?? 1.2) + (0.0499 ?? 0.0016)??(T/K) -(1918 000 ?? 84 000)??(T/K)-2. The thermal expansion of synthetic enargite was measured from T = 298.15 K to T = 573 K by powder X-ray diffraction. The thermal expansion of the unit-cell volume (Z = 2) is described from T = 298.15 K to T = 573 K by the least-squares equation: V/pm3 = 106??(288.2 ?? 0.2) + 104??(1.49 ?? 0.04)??(T/K). ?? 1996 Academic Press Limited.

Engineering of Systematic Elimination of a Targeted Chromosome in Human Cells.

PubMed

Sato, Hiroshi; Kato, Hiroki; Yamaza, Haruyoshi; Masuda, Keiji; Nguyen, Huong Thi Nguyen; Pham, Thanh Thi Mai; Han, Xu; Hirofuji, Yuta; Nonaka, Kazuaki

2017-01-01

Embryonic trisomy leads to abortion or congenital genetic disorders in humans. The most common autosomal chromosome abnormalities are trisomy of chromosomes 13, 18, and 21. Although alteration of gene dosage is thought to contribute to disorders caused by extra copies of chromosomes, genes associated with specific disease phenotypes remain unclear. To generate a normal cell from a trisomic cell as a means of etiological analysis or candidate therapy for trisomy syndromes, we developed a system to eliminate a targeted chromosome from human cells. Chromosome 21 was targeted by integration of a DNA cassette in HeLa cells that harbored three copies of chromosome 21. The DNA cassette included two inverted loxP sites and a herpes simplex virus thymidine kinase (HSV-tk) gene. This system causes missegregation of chromosome 21 after expression of Cre recombinase and subsequently enables the selection of cells lacking the chromosome by culturing in a medium that includes ganciclovir (GCV). Cells harboring only two copies of chromosome 21 were efficiently induced by transfection of a Cre expression vector, indicating that this approach is useful for eliminating a targeted chromosome.

Gender differences in the metabolism of 1,3-butadiene to butadiene diepoxide in Sprague-Dawley rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thornton-Manning, J.R.; Dahl, A.R.; Bechtold, W.E.

1995-12-01

1,3-Butadiene (BD), a gaseous compound used in the production of rubber, is a potent carcinogen in mice and a weak carcinogen in rats. The mechanism of BD-induced carcinogenicity is thought to involve genotoxic effects of its reactive epoxide metabolites butadiene monoepoxide (BDO) and butadiene diepoxide (BDO{sub 2}). Studies in our laboratory have shown that levels of the epoxides, particularly BDO{sub 2}, are greater in mice-the more sensitive species-than rats. While both epoxides are genotoxic in a number of assays, BDO{sub 2} is mutagenic in TK6 human lymphoblastoid cells at concentrations approximately 100-fold lower than BDO. Species differences in carcinogenicity ofmore » BD have posed a dilemma to investigators deciding which animal model is most appropriate for BD risk assessment.« less

Effects of overexpressing photosynthetic carbon flux control enzymes in the cyanobacterium Synechocystis PCC 6803.

PubMed

Liang, Feiyan; Lindblad, Peter

2016-11-01

Synechocystis PCC 6803 is a model unicellular cyanobacterium used in e.g. photosynthesis and CO 2 assimilation research. In the present study we examined the effects of overexpressing Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), sedoheptulose 1,7-biphosphatase (SBPase), fructose-bisphosphate aldolase (FBA) and transketolase (TK), confirmed carbon flux control enzymes of the Calvin-Bassham-Benson (CBB) cycle in higher plants, in Synechocystis PCC 6803. Overexpressing RuBisCO, SBPase and FBA resulted in increased in vivo oxygen evolution (maximal 115%), growth rate and biomass accumulation (maximal 52%) under 100μmolphotonsm -2 s -1 light condition. Cells overexpressing TK showed a chlorotic phenotype but increased biomass by approximately 42% under 100μmolphotonsm -2 s -1 light condition. Under 15μmolphotonsm -2 s -1 light condition, cells overexpressing TK showed enhanced in vivo oxygen evolution. This study demonstrates increased growth and biomass accumulation when overexpressing selected enzymes of the CBB cycle. RuBisCO, SBPase, FBA and TK are identified as four potential targets to improve growth and subsequently also yield of valuable products from Synechocystis PCC 6803. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Pathogenicity in mice of strains of herpes simplex virus which are resistant to acyclovir in vitro and in vivo.

PubMed Central

Field, H J; Darby, G

1980-01-01

Mice infected with three different isolates of herpes simplex virus (HSV) and treated with acyclovir (acycloguanosine; ACV) showed low levels of virus replication during the acute phase of infection. However, virus isolated from such treated mice did not show increased resistance to ACV. In contrast, resistant virus was readily isolated in vitro by passaging HSV in the presence of the drug. The degree of resistance was determined, in part, by the nature of the cells used to test the virus. The majority of ACV-resistant strains induced low or undetectable levels of HSV-specified thymidine kinase (TK), the enzyme responsible for phosphorylating ACV in infected cells. The TK-resistant strains were attenuated when injected into mice as indicated by reductions in virus replication, inflammation, and establishment of latent infections in sensory ganglia. The reduced virulence of the TK- strains was most marked after intracerebral inoculation, where the lethal dose was increased more than 100-fold compared with the parental isolates. However, one mutant is described which induced high levels of TK but was highly resistant to ACV and retained virulence for mice. PMID:6247969

Synthesis and In Vitro Evaluation of 5-[18F]Fluoroalkyl Pyrimidine Nucleosides for Molecular Imaging of Herpes Simplex Virus Type-1 Thymidine Kinase Reporter Gene Expression

PubMed Central

Chacko, Ann-Marie; Qu, Wenchao; Kung, Hank F.

2014-01-01

Two novel series of 5-fluoroalkyl-2′-deoxyuridines (FPrDU, FBuDU, FPeDU) and 2′-fluoro-2′-deoxy-5-fluoroalkylarabinouridines (FFPrAU, FFBuAU, FFPeAU), having three, four or five methylene units (propyl, butyl, or pentyl) at C-5, were prepared and tested as reporter probes for imaging HSV1-tk gene expression. The Negishi coupling methodology was employed to efficiently synthesize the radiolabeling precursors. All six 5-[18F]fluoroalkyl pyrimidines were prepared readily from 3-N-benzoyl-3′,5′-di-O-benzoyl-protected 5-O-mesylate precursors in 17–35% radiochemical yield (decay-corrected). In vitro studies highlighted that all six [18F]labeled nucleosides selectively accumulated in cells expressing the HSV1-TK protein, with negligible uptake in control cells. [18F]FPrDU, [18F]FBuDU, [18F]FPeDU, and [18F]FFBuAU had the best uptake profiles. Despite selective accumulation in HSV1-tk expressing cells, all 5-fluoroalkyl pyrimidine nucleosides had low to negligible cytotoxic activity (CC50>1000–209 μM). Ultimately, results demonstrated that 5-[18F]fluoropropyl, [18F]fluorobutyl, and [18F]fluoropentyl pyrimidine nucleosides have potential as in vivo HSV1-TK PET reporter probes over a dynamic range of reporter gene expression levels. PMID:18800764

DPYD, TYMS, TYMP, TK1, and TK2 Genetic Expressions as Response Markers in Locally Advanced Rectal Cancer Patients Treated with Fluoropyrimidine-Based Chemoradiotherapy

PubMed Central

Wu, Chan-Han; Huang, Chun-Ming; Chung, Fu-Yen; Huang, Ching-Wen; Tsai, Hsiang-Lin; Chen, Chin-Fan; Wang, Jaw-Yuan

2013-01-01

This study is to investigate multiple chemotherapeutic agent- and radiation-related genetic biomarkers in locally advanced rectal cancer (LARC) patients following fluoropyrimidine-based concurrent chemoradiotherapy (CCRT) for response prediction. We initially selected 6 fluoropyrimidine metabolism-related genes (DPYD, ORPT, TYMS, TYMP, TK1, and TK2) and 3 radiotherapy response-related genes (GLUT1, HIF-1 α, and HIF-2 α) as targets for gene expression identification in 60 LARC cancer specimens. Subsequently, a high-sensitivity weighted enzymatic chip array was designed and constructed to predict responses following CCRT. After CCRT, 39 of 60 (65%) LARC patients were classified as responders (pathological tumor regression grade 2 ~ 4). Using a panel of multiple genetic biomarkers (chip), including DPYD, TYMS, TYMP, TK1, and TK2, at a cutoff value for 3 positive genes, a sensitivity of 89.7% and a specificity of 81% were obtained (AUC: 0.915; 95% CI: 0.840–0.991). Negative chip results were significantly correlated to poor CCRT responses (TRG 0-1) (P = 0.014, hazard ratio: 22.704, 95% CI: 3.055–235.448 in multivariate analysis). Disease-free survival analysis showed significantly better survival rate in patients with positive chip results (P = 0.0001). We suggest that a chip including DPYD, TYMS, TYMP, TK1, and TK2 genes is a potential tool to predict response in LARC following fluoropyrimidine-based CCRT. PMID:24455740

Genotoxicity analysis of two halonitromethanes, a novel group of disinfection by-products (DBPs), in human cells treated in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liviac, Danae; Creus, Amadeu; Marcos, Ricard

Halonitromethanes (HNMs) constitute an emerging class of disinfection by-products (DBPs) produced when chlorine and/or ozone are used for water treatment. The HNMs are structurally similar to halomethanes, but have a nitro-group in place of hydrogen bonded to the central carbon atom. Since little information exists on the genotoxic potential of HNMs, a study has been carried out with two HNM compounds, namely trichloronitromethane (TCNM) and bromonitromethane (BNM) by using human cells. Primary damage induction has been measured with the Comet assay, which is used to determine both the repair kinetics of the induced damage and the proportion of induced oxidativemore » damage. In addition, the fixed DNA damage has been evaluated by using the micronucleus (MN) assay. The results obtained indicate that both compounds are genotoxic, inducing high levels of DNA breaks in the Comet assay, and that this DNA damage repairs well over time. In addition, oxidized bases constitute a high proportion of DNA-induced damage (50-75%). Contrarily, no positive effects were observed in the frequency of micronucleus, which measures both clastogenic and aneugenic effects, neither using TK6 cells nor peripheral blood lymphocytes. This lack of fixed genetic damage would minimize the potential mutagenic risk associated with HNMs exposure.« less

INDUCTION OF CELL CYCLE ARREST AND APOPTOSIS BY ORMENIS ERIOLEPIS A MORROCAN ENDEMIC PLANT IN VARIOUS HUMAN CANCER CELL LINES

PubMed Central

Belayachi, Lamiae; Aceves-Luquero, Clara; Merghoub, Nawel; de Mattos, Silvia Fernández; Amzazi, Saaîd; Villalonga, Priam; Bakri, Youssef

2017-01-01

Background: Ormenis eriolepis Coss (Asteraceae) is an endemic Moroccan subspecies, traditionally named “Hellala” or “Fergoga”. It’s usually used for its hypoglycemic effect as well as for the treatment of stomacal pain. As far as we know, there is no scientific exploration of anti tumoral activity of Ormenis eriolepis extracts. Materials and Methods: In this regard, we performed a screening of organic extracts and fractions in a panel of both hematological and solid cancer cell lines, to evaluate the potential in vitro anti tumoral activity and to elucidate the respective mechanisms that may be responsible for growth arrest and cell death induction. The plant was extracted using organic solvents, and four different extracts were screened on Jurkat, Jeko-1, TK-6, LN229, SW620, U2OS, PC-3 and NIH3T3 cells. Results: Cell viability assays revealed that, the IC50 values were (11,63±5,37μg/ml) for Jurkat, (13,33±1,67μg/ml) for Jeko-1, (41,67±1,98μg/ml) for LN229 and (19,31±4,88μg/ml) for PC-3 cells upon treatment with Oe-DF and Oe-HE respectively. Both the fraction and extract exhibited no effects on TK6 and NIH3T3. Cytometry analysis accompanied by DNA damage signaling protein levels monitoring (p-H2A.X), showed that both the Dichloromethane Fraction and Hexanic extract induce DNA double stranded breaks (DSBs) accompanied by cell cycle arrest in G1 (Jurkat, Jeko -1 and LN22) and G2/M (PC-3) phases which is agreed with the caspase activity observed. Additional experiments with selective inhibitors of stress and survival pathways (JNK, MAPK, Rho, p53, and JAK3) indicated that none of these pathways was significantly involved in apoptosis induction. The bioactive compound analysis by CG/MS indicated that the major compounds in Oe-DF were: Linoleic Acid (15,89%), Podophyllotoxin (17,89%) and Quercetin (22,95%). For Oe-HE the major molecules were: Linoleic Acid (9,76%), α-curcumene (7,07%), α-bisabolol (5,49%), Campesterol (4,41%), Stigmasterol (14,08%) and β-sitosterol (7,49%). Conclusion: Our data suggest that bioactive compounds present in Ormenis eriolepis show significant anti proliferative activity inducing cell cycle arrest and cell death operating through apoptosis pathway. PMID:28573252

A Methodology for Modeling Confined, Temperature Sensitive Cushioning Systems

DTIC Science & Technology

1980-06-01

thickness of cushion T, and®- s temperature 0, and as a dependent variable, G, the peak acceleration. The initial model, Equation (IV-11), proved deficient ...k9) = TR * TCTH ALV(60) = Tk * TCTH AL2 V6)= Tk2 * FCTH V2 =TRk * TCrFH *AL V(6~3) =THZ * TC.TH AU! V(,34) =TRa * TCTH 141 Yj)=Tks * T(-Th * AL V(.4b

Fialuridine induces acute liver failure in chimeric TK-NOG mice: a model for detecting hepatic drug toxicity prior to human testing.

PubMed

Xu, Dan; Nishimura, Toshi; Nishimura, Sachiko; Zhang, Haili; Zheng, Ming; Guo, Ying-Ying; Masek, Marylin; Michie, Sara A; Glenn, Jeffrey; Peltz, Gary

2014-04-01

Seven of 15 clinical trial participants treated with a nucleoside analogue (fialuridine [FIAU]) developed acute liver failure. Five treated participants died, and two required a liver transplant. Preclinical toxicology studies in mice, rats, dogs, and primates did not provide any indication that FIAU would be hepatotoxic in humans. Therefore, we investigated whether FIAU-induced liver toxicity could be detected in chimeric TK-NOG mice with humanized livers. Control and chimeric TK-NOG mice with humanized livers were treated orally with FIAU 400, 100, 25, or 2.5 mg/kg/d. The response to drug treatment was evaluated by measuring plasma lactate and liver enzymes, by assessing liver histology, and by electron microscopy. After treatment with FIAU 400 mg/kg/d for 4 d, chimeric mice developed clinical and serologic evidence of liver failure and lactic acidosis. Analysis of liver tissue revealed steatosis in regions with human, but not mouse, hepatocytes. Electron micrographs revealed lipid and mitochondrial abnormalities in the human hepatocytes in FIAU-treated chimeric mice. Dose-dependent liver toxicity was detected in chimeric mice treated with FIAU 100, 25, or 2.5 mg/kg/d for 14 d. Liver toxicity did not develop in control mice that were treated with the same FIAU doses for 14 d. In contrast, treatment with another nucleotide analogue (sofosbuvir 440 or 44 mg/kg/d po) for 14 d, which did not cause liver toxicity in human trial participants, did not cause liver toxicity in mice with humanized livers. FIAU-induced liver toxicity could be readily detected using chimeric TK-NOG mice with humanized livers, even when the mice were treated with a FIAU dose that was only 10-fold above the dose used in human participants. The clinical features, laboratory abnormalities, liver histology, and ultra-structural changes observed in FIAU-treated chimeric mice mirrored those of FIAU-treated human participants. The use of chimeric mice in preclinical toxicology studies could improve the safety of candidate medications selected for testing in human participants. Please see later in the article for the Editors' Summary.

Efficiency of introns from various origins in fish cells.

PubMed

Bétancourt, O H; Attal, J; Théron, M C; Puissant, C; Houdebine, L M

1993-06-01

Several vectors containing (1) regulatory regions from Rous sarcoma virus (RSV), human cytomegalovirus (CMV), and herpes simplex thymidine kinase (TK); (2) introns from early or late SV40 genes and from trout growth hormone gene (tGH); (3) chloramphenicol acetyltransferase gene (CAT); and (4) transcription terminators from SV40 were transfected into carp EPC cells, salmon CHSE cells, tilapia TO2 cells, quail QT6 cells, and hamster CHO cells. CAT activity was measured in extracts from several cell lines 3 days after transfection and in the fish EPC stable clones. The CMV and RSV promoters were the most potent in all cell types. The intron from late SV40 genes (VP1 intron) worked properly in QT6 and CHO cells but not in EPC and very weakly in TO2 cells. The tGH intron was efficient in all cell types but preferentially in fish cells. The small t intron from SV40 was processed in all cell types. The small t and, to a lesser extent, the tGH introns amplified expression of cat gene in stable clones, in comparison to the transiently transfected cells. These results indicate that elements from mammalian genes may not be properly recognized by the fish cellular machinery and in an unpredictable manner. This finding suggests that vectors prepared to express foreign genes in transfected cultured fish cells and transgenic fish should preferably contain DNA sequences from fish genes or, alternatively, those sequences from mammalian genes that have been previously proved to be compatible with the fish cellular machinery.

Synthesis and evaluation of novel 18 F-labeled quinazoline derivatives with low lipophilicity for tumor PET imaging.

PubMed

Chong, Yan; Chang, Jin; Zhao, Wenwen; He, Yong; Li, Yuqiao; Zhang, Huabei; Qi, Chuanmin

2018-02-01

Four novel 18 F-labeled quinazoline derivatives with low lipophilicity, [ 18 F]4-(2-fluoroethoxy)-6,7-dimethoxyquinazoline ([ 18 F]I), [ 18 F]4-(3-((4-(2-fluoroethoxy)-7-methoxyquinazolin-6-yl)oxy)propyl)morpholine ([ 18 F]II), [ 18 F]4-(2-fluoroethoxy)-7-methoxy-6-(2-methoxyethoxy)quinazoline ([ 18 F]III), and [ 18 F]4-(2-fluoroethoxy)-6,7-bis(2-methoxyethoxy)quinazoline ([ 18 F]IV), were synthesized via a 2-step radiosynthesis procedure with an overall radiochemical yield of 10% to 38% (without decay correction) and radiochemical purities of >98%. The lipophilicity and stability of labeled compounds were tested in vitro. The log P values of the 4 radiotracers ranged from 0.52 to 1.07. We then performed ELISA to measure their affinities to EGFR-TK; ELISA assay results indicated that each inhibitor was specifically bounded to EGFR-TK in a dose-dependent manner. The EGFR-TK autophosphorylation IC 50 values of [ 18 F]I, [ 18 F]II, [ 18 F]III, and [ 18 F]IV were 7.732, 0.4698, 0.1174, and 0.1176 μM, respectively. All labeled compounds were evaluated via cellular uptake and blocking studies in HepG2 cell lines in vitro. Cellular uptake and blocking experiment results indicated that [ 18 F]I and [ 18 F]III had excellent cellular uptake at 120-minute postinjection in HepG2 carcinoma cells (51.80 ± 3.42%ID/mg protein and 27.31 ± 1.94%ID/mg protein, respectively). Additionally, biodistribution experiments in S180 tumor-bearing mice in vivo indicated that [ 18 F]I had a very fast clearance in blood and a relatively high uptake ratio of tumor to blood (4.76) and tumor to muscle (1.82) at 60-minute postinjection. [ 18 F]III had a quick clearance in plasma, and its highest uptake ratio of tumor to muscle was 2.55 at 15-minute postinjection. These experimental results and experiences were valuable for the further exploration of novel radiotracers of quinazoline derivatives. Copyright © 2017 John Wiley & Sons, Ltd.

Evaluating In Vitro-In Vivo Extrapolation of Toxicokinetics

PubMed Central

MacMillan, Denise K; Ford, Jermaine; Fennell, Timothy R; Black, Sherry R; Snyder, Rodney W; Sipes, Nisha S; Westerhout, Joost; Setzer, R Woodrow; Pearce, Robert G; Simmons, Jane Ellen; Thomas, Russell S

2018-01-01

Abstract Prioritizing the risk posed by thousands of chemicals potentially present in the environment requires exposure, toxicity, and toxicokinetic (TK) data, which are often unavailable. Relatively high throughput, in vitro TK (HTTK) assays and in vitro-to-in vivo extrapolation (IVIVE) methods have been developed to predict TK, but most of the in vivo TK data available to benchmark these methods are from pharmaceuticals. Here we report on new, in vivo rat TK experiments for 26 non-pharmaceutical chemicals with environmental relevance. Both intravenous and oral dosing were used to calculate bioavailability. These chemicals, and an additional 19 chemicals (including some pharmaceuticals) from previously published in vivo rat studies, were systematically analyzed to estimate in vivo TK parameters (e.g., volume of distribution [Vd], elimination rate). For each of the chemicals, rat-specific HTTK data were available and key TK predictions were examined: oral bioavailability, clearance, Vd, and uncertainty. For the non-pharmaceutical chemicals, predictions for bioavailability were not effective. While no pharmaceutical was absorbed at less than 10%, the fraction bioavailable for non-pharmaceutical chemicals was as low as 0.3%. Total clearance was generally more under-estimated for nonpharmaceuticals and Vd methods calibrated to pharmaceuticals may not be appropriate for other chemicals. However, the steady-state, peak, and time-integrated plasma concentrations of nonpharmaceuticals were predicted with reasonable accuracy. The plasma concentration predictions improved when experimental measurements of bioavailability were incorporated. In summary, HTTK and IVIVE methods are adequately robust to be applied to high throughput in vitro toxicity screening data of environmentally relevant chemicals for prioritizing based on human health risks. PMID:29385628

Design and characterization of a prototype enzyme microreactor: quantification of immobilized transketolase kinetics.

PubMed

Matosevic, S; Lye, G J; Baganz, F

2010-01-01

In this work, we describe the design of an immobilized enzyme microreactor (IEMR) for use in transketolase (TK) bioconversion process characterization. The prototype microreactor is based on a 200-microm ID fused silica capillary for quantitative kinetic analysis. The concept is based on the reversible immobilization of His(6)-tagged enzymes via Ni-NTA linkage to surface derivatized silica. For the initial microreactor design, the mode of operation is a stop-flow analysis which promotes higher degrees of conversion. Kinetics for the immobilized TK-catalysed synthesis of L-erythrulose from substrates glycolaldehyde (GA) and hydroxypyruvate (HPA) were evaluated based on a Michaelis-Menten model. Results show that the TK kinetic parameters in the IEMR (V(max(app)) = 0.1 +/- 0.02 mmol min(-1), K(m(app)) = 26 +/- 4 mM) are comparable with those measured in free solution. Furthermore, the k(cat) for the microreactor of 4.1 x 10(5) s(-1) was close to the value for the bioconversion in free solution. This is attributed to the controlled orientation and monolayer surface coverage of the His(6)-immobilized TK. Furthermore, we show quantitative elution of the immobilized TK and the regeneration and reuse of the derivatized capillary over five cycles. The ability to quantify kinetic parameters of engineered enzymes at this scale has benefits for the rapid and parallel evaluation of evolved enzyme libraries for synthetic biology applications and for the generation of kinetic models to aid bioconversion process design and bioreactor selection as a more efficient alternative to previously established microwell-based systems for TK bioprocess characterization.

Generation and characterization of koi herpesvirus recombinants lacking viral enzymes of nucleotide metabolism.

PubMed

Fuchs, Walter; Fichtner, Dieter; Bergmann, Sven M; Mettenleiter, Thomas C

2011-06-01

Koi herpesvirus (KHV) causes a fatal disease in koi and common carp, but no reliable and genetically characterized vaccines are available up to now. Therefore, we generated KHV recombinants possessing deletions within the viral ribonucleotide reductase (RNR), thymidine kinase (TK), dUTPase, or TK and dUTPase genes, and their corresponding rescuants. All KHV mutants were replication competent in cultured cells. Whereas plaque sizes and titers of RNR-negative KHV were reduced, replication of the other mutants was not affected. Experimental infection of carp indicated attenuation of TK- or dUTPase-deleted KHV, and PCR analysis of tissue samples permitted differentiation of mutant from wild-type virus.

An interaction map of small-molecule kinase inhibitors with anaplastic lymphoma kinase (ALK) mutants in ALK-positive non-small cell lung cancer.

PubMed

Ai, Xinghao; Shen, Shengping; Shen, Lan; Lu, Shun

2015-05-01

Human anaplastic lymphoma kinase (ALK) has become a well-established target for the treatment of ALK-positive non-small cell lung cancer (NSCLC). Here, we have profiled seven small-molecule inhibitors, including 2 that are approved drugs, against a panel of clinically relevant mutations in ALK tyrosine kinase (TK) domain, aiming at a comprehensive understanding of molecular mechanism and biological implication underlying inhibitor response to ALK TK mutation. We find that (i) the gatekeeper mutation L1196M causes crizotinib resistance by simultaneously increasing and decreasing the binding affinities of, respectively, ATP and inhibitor to ALK, whereas the secondary mutation C1156Y, which is located far away from the ATP-binding site of ALK TK domain, causes the resistance by inducing marked allosteric effect on the site, (ii) the 2nd and 3rd generation kinase inhibitors exhibit relatively high sensitivity towards ALK mutants as compared to 1st generation inhibitors, (iii) the pan-kinase inhibitor staurosporine is insensitive for most mutations due to its high structural compatibility, and (iv) ATP affinity to ALK is generally reduced upon most clinically relevant mutations. Furthermore, we also identify six novel mutation-inhibitor pairs that are potentially associated with drug resistance. In addition, the G1202R and C1156Y mutations are expected to generally cause resistance for many existing inhibitors, since they can address significant effect on the geometric shape and physicochemical property of ALK active pocket. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Different sensitivities of cultured mammalian cells towards aphidicolin-enhanced DNA effects in the comet assay.

PubMed

Speit, Günter; Schütz, Petra; Bausinger, Julia

2016-06-01

The comet assay in combination with the polymerase inhibitor aphidicolin (APC) has been used to measure DNA excision repair activity, DNA repair kinetics and individual DNA repair capacity. Since APC can enhance genotoxic effects of mutagens measured by the comet assay, this approach has been proposed for increasing the sensitivity of the comet assay in human biomonitoring. The APC-modified comet assay has mainly been performed with human blood and it was shown that it not only enhances the detection of DNA damage repaired by nucleotide excision repair (NER) but also damage typically repaired by base excision repair (BER). Recently, we reported that in contrast to blood leukocytes, A549 cells (a human lung adenocarcinoma cell line) seem to be insensitive towards the repair-inhibiting action of APC. To further elucidate the general usefulness of the APC-modified comet assay for studying repair in cultured mammalian cells, we comparatively investigated further cell lines (HeLa, TK6, V79). DNA damage was induced by BPDE (benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide) and MMS (methyl methanesulfonate) in the absence and presence of APC (3 or 15μM). APC was either added for 2h together with the mutagen or cells were pre-incubated for 30min with APC before the mutagen was added. The results indicate that the cell lines tested differ fundamentally with regard to their sensitivity and specificity towards the repair-inhibiting effect of APC. The actual cause for these differences is still unclear but potential molecular explanations are discussed. Irrespective of the underlying mechanism(s), our study revealed practical limitations of the use of the APC-modified comet assay. Copyright © 2016 Elsevier B.V. All rights reserved.

Issues raised by the reference doses for perfluorooctane sulfonate and perfluorooctanoic acid.

PubMed

Dong, Zhaomin; Bahar, Md Mezbaul; Jit, Joytishna; Kennedy, Bruce; Priestly, Brian; Ng, Jack; Lamb, Dane; Liu, Yanju; Duan, Luchun; Naidu, Ravi

2017-08-01

On 25th May 2016, the U.S. EPA released reference doses (RfDs) for Perfluorooctane Sulfonate (PFOS) and Perfluorooctanoic Acid (PFOA) of 20ng/kg/day, which were much more conservative than previous values. These RfDs rely on the choices of animal point of departure (PoD) and the toxicokinetics (TK) model. At this stage, considering that the human evidence is not strong enough for RfD determination, using animal data may be appropriate but with more uncertainties. In this article, the uncertainties concerning RfDs from the choices of PoD and TK models are addressed. Firstly, the candidate PoDs should include more critical endpoints (such as immunotoxicity), which may lead to lower RfDs. Secondly, the reliability of the adopted three-compartment TK model is compromised: the parameters are not non-biologically plausible; and this TK model was applied to simulate gestation and lactation exposures, while the two exposure scenarios were not actually included in the model structure. Copyright © 2017. Published by Elsevier Ltd.

Hypoxia/hepatoma dual specific suicide gene expression plasmid delivery using bio-reducible polymer for hepatocellular carcinoma therapy.

PubMed

Kim, Hyun Ah; Nam, Kihoon; Lee, Minhyung; Kim, Sung Wan

2013-10-10

Gene therapy is suggested as a promising alternative strategy of hepatocellular carcinoma (HCC, also called hepatoma) therapy. To achieve a successful and safe gene therapy, tight regulation of gene expression is required to minimize side-effects in normal tissues. In this study, we developed a novel hypoxia and hepatoma dual specific gene expression vector. The constructed vectors were transfected into various cell lines using bio-reducible polymer, PAM-ABP. First, pAFPS-Luc or pAFPL-Luc vector was constructed with the alpha-fectoprotein (AFP) promoter and enhancer for hepatoma tissue specific gene expression. Then, pEpo-AFPL-Luc was constructed by insertion of the erythropoietin (Epo) enhancer for hypoxic cancer specific gene expression. In vitro transfection assay showed that pEpo-AFPL-Luc transfected hepatoma cell increased gene expression under hypoxic condition. To confirm the therapeutic effect of dual specific vector, herpes simplex virus thymidine kinase (HSV-TK) gene was introduced for cancer cell killing. The pEpo-AFPL-TK was transfected into hepatoma cell lines in the presence of ganciclovir (GCV) pro-drug. Caspase-3/7, MTT and TUNEL assays elucidated that pEpo-AFPL-TK transfected cells showed significant increasing of death rate in hypoxic hepatoma cells compared to controls. Therefore, the hypoxia/hepatoma dual specific gene expression vector with the Epo enhancer and AFP promoter may be useful for hepatoma specific gene therapy. © 2013.

A ROS-responsive polymeric micelle with a π-conjugated thioketal moiety for enhanced drug loading and efficient drug delivery.

PubMed

Sun, Changzhen; Liang, Yan; Hao, Na; Xu, Long; Cheng, Furong; Su, Ting; Cao, Jun; Gao, Wenxia; Pu, Yuji; He, Bin

2017-11-07

As the implications of reactive oxygen species (ROS) are elucidated in many diseases, ROS-responsive nanoparticles are attracting great interest from researchers. In this work, a ROS sensitive thioketal (TK) moiety with a π-conjugated structure was introduced into biodegradable methoxy poly(ethylene glycol)-thioketal-poly(ε-caprolactone)mPEG-TK-PCL micelles as a linker, which was designed to speed up the drug release and thus enhance the therapeutic efficacy. The micelle showed a high drug loading content of 12.8% and excellent stability under physiological conditions because of the evocation of π-π stacking and hydrophobic interactions with the anticancer drug doxorubicin (DOX). The polymeric micelle presented a better drug carrier capacity and higher in vitro anticancer efficacy towards cancer cells. The in vivo study showed that DOX-loaded mPEG-TK-PCL micelles displayed lower toxicity towards normal cells and remarkably enhanced antitumor efficacy. This research provides a way to design potential drug carriers for efficient cancer chemotherapy.

Properties of Cells Carrying the Herpes Simplex Virus Type 2 Thymidine Kinase Gene: Mechanisms of Reversion to a Thymidine Kinase-Negative Phenotype

PubMed Central

Bastow, K. F.; Darby, G.; Wildy, P.; Minson, A. C.

1980-01-01

We have isolated cells with a thymidine kinase-negative (tk−) phenotype from cells which carry the herpes simplex virus type 2 tk gene by selection in 5-bromodeoxyuridine or 9-(2-hydroxyethoxymethyl)guanine. Both selection routines generated revertants with a frequency of 10−3 to 10−4, and resistance to either compound conferred simultaneous resistance to the other. tk− revertants fell into three classes: (i) cells that arose by deletion of all virus sequences, (ii) cells that had lost the virus tk gene but retained a nonselected virus-specific function and arose by deletion of part of the virus-specific sequence, and (iii) cells that retained the potential to express all of the virus-specific functions of the parental cells and retained all of the virus-specific DNA sequences. Images PMID:16789205

Two dechlorinated chlordecone derivatives formed by in situ chemical reduction are devoid of genotoxicity and mutagenicity and have lower proangiogenic properties compared to the parent compound.

PubMed

Legeay, Samuel; Billat, Pierre-André; Clere, Nicolas; Nesslany, Fabrice; Bristeau, Sébastien; Faure, Sébastien; Mouvet, Christophe

2018-05-01

Chlordecone (CLD) is a chlorinated hydrocarbon insecticide, now classified as a persistent organic pollutant. Several studies have previously reported that chronic exposure to CLD leads to hepatotoxicity, neurotoxicity, raises early child development and pregnancy complications, and increases the risk of liver and prostate cancer. In situ chemical reduction (ISCR) has been identified as a possible way for the remediation of soils contaminated by CLD. In the present study, the objectives were (i) to evaluate the genotoxicity and the mutagenicity of two CLD metabolites formed by ISCR, CLD-5a-hydro, or CLD-5-hydro (5a- or 5- according to CAS nomenclature; CLD-1Cl) and tri-hydroCLD (CLD-3Cl), and (ii) to explore the angiogenic properties of these molecules. Mutagenicity and genotoxicity were investigated using the Ames's technique on Salmonella typhimurium and the in vitro micronucleus micromethod with TK6 human lymphoblastoid cells. The proangiogenic properties were evaluated on the in vitro capillary network formation of human primary endothelial cells. Like CLD, the dechlorinated derivatives of CLD studied were devoid of genotoxic and mutagenic activity. In the assay targeting angiogenic properties, significantly lower microvessel lengths formed by endothelial cells were observed for the CLD-3Cl-treated cells compared to the CLD-treated cells for two of the three tested concentrations. These results suggest that dechlorinated CLD derivatives are devoid of mutagenicity and genotoxicity and have lower proangiogenic properties than CLD.

Thymidine kinase 2 deficiency-induced mitochondrial DNA depletion causes abnormal development of adipose tissues and adipokine levels in mice.

PubMed

Villarroya, Joan; Dorado, Beatriz; Vilà, Maya R; Garcia-Arumí, Elena; Domingo, Pere; Giralt, Marta; Hirano, Michio; Villarroya, Francesc

2011-01-01

Mammal adipose tissues require mitochondrial activity for proper development and differentiation. The components of the mitochondrial respiratory chain/oxidative phosphorylation system (OXPHOS) are encoded by both mitochondrial and nuclear genomes. The maintenance of mitochondrial DNA (mtDNA) is a key element for a functional mitochondrial oxidative activity in mammalian cells. To ascertain the role of mtDNA levels in adipose tissue, we have analyzed the alterations in white (WAT) and brown (BAT) adipose tissues in thymidine kinase 2 (Tk2) H126N knockin mice, a model of TK2 deficiency-induced mtDNA depletion. We observed respectively severe and moderate mtDNA depletion in TK2-deficient BAT and WAT, showing both tissues moderate hypotrophy and reduced fat accumulation. Electron microscopy revealed altered mitochondrial morphology in brown but not in white adipocytes from TK2-deficient mice. Although significant reduction in mtDNA-encoded transcripts was observed both in WAT and BAT, protein levels from distinct OXPHOS complexes were significantly reduced only in TK2-deficient BAT. Accordingly, the activity of cytochrome c oxidase was significantly lowered only in BAT from TK2-deficient mice. The analysis of transcripts encoding up to fourteen components of specific adipose tissue functions revealed that, in both TK2-deficient WAT and BAT, there was a consistent reduction of thermogenesis related gene expression and a severe reduction in leptin mRNA. Reduced levels of resistin mRNA were found in BAT from TK2-deficient mice. Analysis of serum indicated a dramatic reduction in circulating levels of leptin and resistin. In summary, our present study establishes that mtDNA depletion leads to a moderate impairment in mitochondrial respiratory function, especially in BAT, causes substantial alterations in WAT and BAT development, and has a profound impact in the endocrine properties of adipose tissues. © 2011 Villarroya et al.

Thymidine Kinase 2 Deficiency-Induced Mitochondrial DNA Depletion Causes Abnormal Development of Adipose Tissues and Adipokine Levels in Mice

PubMed Central

Villarroya, Joan; Dorado, Beatriz; Vilà, Maya R.; Garcia-Arumí, Elena; Domingo, Pere; Giralt, Marta; Hirano, Michio; Villarroya, Francesc

2011-01-01

Mammal adipose tissues require mitochondrial activity for proper development and differentiation. The components of the mitochondrial respiratory chain/oxidative phosphorylation system (OXPHOS) are encoded by both mitochondrial and nuclear genomes. The maintenance of mitochondrial DNA (mtDNA) is a key element for a functional mitochondrial oxidative activity in mammalian cells. To ascertain the role of mtDNA levels in adipose tissue, we have analyzed the alterations in white (WAT) and brown (BAT) adipose tissues in thymidine kinase 2 (Tk2) H126N knockin mice, a model of TK2 deficiency-induced mtDNA depletion. We observed respectively severe and moderate mtDNA depletion in TK2-deficient BAT and WAT, showing both tissues moderate hypotrophy and reduced fat accumulation. Electron microscopy revealed altered mitochondrial morphology in brown but not in white adipocytes from TK2-deficient mice. Although significant reduction in mtDNA-encoded transcripts was observed both in WAT and BAT, protein levels from distinct OXPHOS complexes were significantly reduced only in TK2-deficient BAT. Accordingly, the activity of cytochrome c oxidase was significantly lowered only in BAT from TK2-deficient mice. The analysis of transcripts encoding up to fourteen components of specific adipose tissue functions revealed that, in both TK2-deficient WAT and BAT, there was a consistent reduction of thermogenesis related gene expression and a severe reduction in leptin mRNA. Reduced levels of resistin mRNA were found in BAT from TK2-deficient mice. Analysis of serum indicated a dramatic reduction in circulating levels of leptin and resistin. In summary, our present study establishes that mtDNA depletion leads to a moderate impairment in mitochondrial respiratory function, especially in BAT, causes substantial alterations in WAT and BAT development, and has a profound impact in the endocrine properties of adipose tissues. PMID:22216345

Comparative biology approaches for charged particle exposures and cancer development processes

NASA Astrophysics Data System (ADS)

Kronenberg, Amy; Gauny, Stacey; Kwoh, Ely; Sudo, Hiroko; Wiese, Claudia; Dan, Cristian; Turker, Mitchell

Comparative biology studies can provide useful information for the extrapolation of results be-tween cells in culture and the more complex environment of the tissue. In other circumstances, they provide a method to guide the interpretation of results obtained for cells from differ-ent species. We have considered several key cancer development processes following charged particle exposures using comparative biology approaches. Our particular emphases have been mutagenesis and genomic instability. Carcinogenesis requires the accumulation of mutations and most of htese mutations occur on autosomes. Two loci provide the greatest avenue for the consideration of charged particle-induced mutation involving autosomes: the TK1 locus in human cells and the APRT locus in mouse cells. Each locus can provide information on a wide variety of mutational changes, from small intragenic mutations through multilocus dele-tions and extensive tracts of mitotic recombination. In addition, the mouse model can provide a direct measurement of chromosome loss which cannot be accomplished in the human cell system. Another feature of the mouse APRT model is the ability to examine effects for cells exposed in vitro with those obtained for cells exposed in situ. We will provide a comparison of the results obtained for the TK1 locus following 1 GeV/amu Fe ion exposures to the human lymphoid cells with those obtained for the APRT locus for mouse kidney epithelial cells (in vitro or in situ). Substantial conservation of mechanisms is found amongst these three exposure scenarios, with some differences attributable to the specific conditions of exposure. A similar approach will be applied to the consideraiton of proton-induced autosomal mutations in the three model systems. A comparison of the results obtained for Fe ions vs. protons in each case will highlight LET-specificc differences in response. Another cancer development process that is receiving considerable interest is genomic instability. We have examined this process following exposure to sparsely and densely ionizing charged particles in human lymphoid cells and in human epithelial cells. A comparison of the results in these systems can reveal similari-ties and differences as a function of cell type and LET. Last, we will approach the question of the relevance of genomic instability in the context of charged particle mutagenesis. In many models, it has been difficult to link these two processes. We will present data regarding the mechanistic associations between these processes. Taken together, these studies will allow the definition of conserved pathways that are likely to contribute strongly to the cancer risks for astronauts exposed to charged particle radiations. Supported by NASA grant NNJ07HC721 to A. Kronenberg and NASA grant NNX10AC12G to M. Turker.

Induction of genomic instability in TK6 human lymphoblasts exposed to 137Cs gamma radiation: comparison to the induction by exposure to accelerated 56Fe particles

NASA Technical Reports Server (NTRS)

Evans, Helen H.; Horng, Min-Fen; Ricanati, Marlene; Diaz-Insua, M.; Jordan, Robert; Schwartz, Jeffrey L.

2003-01-01

The induction of genomic instability in TK6 human lymphoblasts by exposure to (137)Cs gamma radiation was investigated by measuring the frequency and characteristics of unstable clones isolated approximately 36 generations after exposure. Clones surviving irradiation and control clones were analyzed for 17 characteristics including chromosomal aberrations, growth defects, alterations in response to a second irradiation, and mutant frequencies at the thymidine kinase and Na(+)/K(+) ATPase loci. Putative unstable clones were defined as those that exhibited a significant alteration in one or more characteristics compared to the controls. The frequency and characteristics of the unstable clones were compared in clones exposed to (137)Cs gamma rays or (56)Fe particles. The majority of the unstable clones isolated after exposure to either gamma rays or (56)Fe particles exhibited chromosomal instability. Alterations in growth characteristics, radiation response and mutant frequencies occurred much less often than cytogenetic alterations in these unstable clones. The frequency and complexity of the unstable clones were greater after exposure to (56)Fe particles than to gamma rays. Unstable clones that survived 36 generations after exposure to gamma rays exhibited increases in the incidence of dicentric chromosomes but not of chromatid breaks, whereas unstable clones that survived 36 generations after exposure to (56)Fe particles exhibited increases in both chromatid and chromosome aberrations.

Safety and immunogenicity of a gE/gI/TK gene-deleted pseudorabies virus variant expressing the E2 protein of classical swine fever virus in pigs.

PubMed

Lei, Jian-Lin; Xia, Shui-Li; Wang, Yimin; Du, Mingliang; Xiang, Guang-Tao; Cong, Xin; Luo, Yuzi; Li, Lian-Feng; Zhang, Lingkai; Yu, Jiahui; Hu, Yonghao; Qiu, Hua-Ji; Sun, Yuan

2016-06-01

Classical swine fever (CSF) and pseudorabies (PR) are both major infectious diseases of pigs, causing enormous economic losses to the swine industry in many countries. A marker vaccine that enables differentiation of infected from vaccinated animals (DIVA) is highly desirable for control and eradication of these two diseases in endemic areas. Since late 2011, PR outbreaks have been frequently reported in many Bartha-K61-vaccinated pig farms in China. It has been demonstrated that a pseudorabies virus (PRV) variant with altered antigenicity and increased pathogenicity was responsible for the outbreaks. Previously, we showed that rPRVTJ-delgE/gI/TK, a gE/gI/TK-deleted PRV variant, was safe for susceptible animals and provided a complete protection against lethal PRV variant challenge, indicating that rPRVTJ-delgE/gI/TK can be used as an attractive vaccine vector. To develop a safe bivalent vaccine against CSF and PR, we generated a recombinant virus rPRVTJ-delgE/gI/TK-E2 expressing the E2 protein of classical swine fever virus (CSFV) based on rPRVTJ-delgE/gI/TK and evaluated its safety and immunogenicity in pigs. The results indicated that pigs (n=5) immunized with rPRVTJ-delgE/gI/TK-E2 of different doses did not exhibit clinical signs or viral shedding following immunization, the immunized pigs produced anti-PRV or anti-CSFV neutralizing antibodies and the pigs immunized with 10(6) or 10(5) TCID50 rPRVTJ-delgE/gI/TK-E2 were completely protected against the lethal challenge with either CSFV Shimen strain or variant PRV TJ strain. These findings suggest that rPRVTJ-delgE/gI/TK-E2 is a promising bivalent DIVA vaccine candidate against CSFV and PRV coinfections. Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

The efficacy of 9-cis retinoic acid in experimental models of cancer.

PubMed

Gottardis, M M; Lamph, W W; Shalinsky, D R; Wellstein, A; Heyman, R A

1996-01-01

9-cis retinoic acid (9-cis RA) is a retinoid receptor pan-agonist that binds with high affinity to both retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Using a variety of in vivo and in vitro cancer models, we present experimental data that 9-cis RA has activity as a potential chemotherapeutic agent. Treatment of the human promyelocytic leukemia cell line HL-60 with 9-cis RA decreases cell proliferation, increases cell differentiation, and increases apoptosis. Induction of apoptosis correlates with an increase in tissue transglutaminase (type II) activity. In vivo, 9-cis RA induces complete tumor regression of an early passage human lip squamous cell carcinoma xenograft. Finally, 9-cis RA inhibits the anchorage-independent growth of the human breast cancer cell lines MCF-7 and LY2 (an antiestrogen-resistant MCF-7 variant). Transient co-transfection assays indicate that 9-cis RA inhibits estrogen receptor transcription of an ERE-tk-LUC reporter through RAR or RXR receptors. These data suggest that retinoid receptors can antagonize estrogen-dependent transcription and provides one possible mechanism for the inhibition of cell growth by 9-cis RA in breast cancer cell lines. In summary, these findings present evidence that 9-cis RA has a wide range of activities in human cancer models.

Refined human artificial chromosome vectors for gene therapy and animal transgenesis

PubMed Central

Kazuki, Y; Hoshiya, H; Takiguchi, M; Abe, S; Iida, Y; Osaki, M; Katoh, M; Hiratsuka, M; Shirayoshi, Y; Hiramatsu, K; Ueno, E; Kajitani, N; Yoshino, T; Kazuki, K; Ishihara, C; Takehara, S; Tsuji, S; Ejima, F; Toyoda, A; Sakaki, Y; Larionov, V; Kouprina, N; Oshimura, M

2011-01-01

Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance, and the ability to carry large gene inserts. We previously developed HAC vectors from the normal human chromosomes using a chromosome engineering technique. However, endogenous genes were remained in these HACs, limiting their therapeutic applications. In this study, we refined a HAC vector without endogenous genes from human chromosome 21 in homologous recombination-proficient chicken DT40 cells. The HAC was physically characterized using a transformation-associated recombination (TAR) cloning strategy followed by sequencing of TAR-bacterial artificial chromosome clones. No endogenous genes were remained in the HAC. We demonstrated that any desired gene can be cloned into the HAC using the Cre-loxP system in Chinese hamster ovary cells, or a homologous recombination system in DT40 cells. The HAC can be efficiently transferred to other type of cells including mouse ES cells via microcell-mediated chromosome transfer. The transferred HAC was stably maintained in vitro and in vivo. Furthermore, tumor cells containing a HAC carrying the suicide gene, herpes simplex virus thymidine kinase (HSV-TK), were selectively killed by ganciclovir in vitro and in vivo. Thus, this novel HAC vector may be useful not only for gene and cell therapy, but also for animal transgenesis. PMID:21085194

Genetic toxicity of a standardized mixture of citrus polymethoxylated flavones.

PubMed

Delaney, B; Phillips, K; Vasquez, C; Wilson, A; Cox, D; Wang, H-B; Manthey, J

2002-05-01

Flavonoids are a ubiquitous family of phytochemicals that display a variety of biological effects, both beneficial and adverse depending on the individual compound. Certain flavonoids are genotoxic while others inhibit the genotoxicity of other mutagens. In the present studies, the mutagenicity of a mixture of polymethoxylated flavones (PMFs) purified from citrus peel oil was evaluated. The mixture consisted of nobiletin (32.5%), 3,3',4',5,6,7,8-heptamethoxyflavone (25.0%), tangeretin (14.0%), trimethylscutellarein (9.1%), sinensetin (3.9%), 5-demethyl-nobiletin (2.8%), hexa-O-methylquercetagetin (3.3%), 5-demethyl-tetramethylscutellarein (0.7%), 5-hydroxy-3,3',4',6,7,8-hexamethoxyflavone (0.7%), and a small quantity of unidentified flavonoid compounds (3.9%). In vitro addition of the PMF mixture over a concentration range that spanned four log doses (0.0005-5.0 mg/plate) did not reveal any evidence of mutagenicity in five bacterial tester strains (Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537) either in the absence or presence of S9 activation. The PMF mixture exhibited a statistically significant increase in mutagenicity of L5178Y tk(+/-) mouse lymphoma cells at 0.05 (38.5 x 10(-6); P<0.05) and 0.1 mg/ml (61 x 10(-6); P<0.01) compared with vehicle-treated controls (mutation frequency=19.7 x 10(-6)). However, these responses were within historical values observed in negative control cultures and extremely small compared to the positive control (EMS 0.5 microl/ml; 1685.3 x 10(-6)). Furthermore, in the presence of S9 there was no indication of genetic toxicity in L5178Y tk(+/-) cells. These results demonstrate that the PMF mixture is not genotoxic in in vitro assay systems.

Hyperthermophilic Archaeon Thermococcus kodakarensis Utilizes a Four-Step Pathway for NAD+ Salvage through Nicotinamide Deamination.

PubMed

Hachisuka, Shin-Ichi; Sato, Takaaki; Atomi, Haruyuki

2018-06-01

Many organisms possess pathways that regenerate NAD + from its degradation products, and two pathways are known to salvage NAD + from nicotinamide (Nm). One is a four-step pathway that proceeds through deamination of Nm to nicotinic acid (Na) by Nm deamidase and phosphoribosylation to nicotinic acid mononucleotide (NaMN), followed by adenylylation and amidation. Another is a two-step pathway that does not involve deamination and directly proceeds with the phosphoribosylation of Nm to nicotinamide mononucleotide (NMN), followed by adenylylation. Judging from genome sequence data, the hyperthermophilic archaeon Thermococcus kodakarensis is supposed to utilize the four-step pathway, but the fact that the adenylyltransferase encoded by TK0067 recognizes both NMN and NaMN also raises the possibility of a two-step salvage mechanism. Here, we examined the substrate specificity of the recombinant TK1676 protein, annotated as nicotinic acid phosphoribosyltransferase. The TK1676 protein displayed significant activity toward Na and phosphoribosyl pyrophosphate (PRPP) and only trace activity with Nm and PRPP. We further performed genetic analyses on TK0218 (quinolinic acid phosphoribosyltransferase) and TK1650 (Nm deamidase), involved in de novo biosynthesis and four-step salvage of NAD + , respectively. The ΔTK0218 mutant cells displayed growth defects in a minimal synthetic medium, but growth was fully restored with the addition of Na or Nm. The ΔTK0218 ΔTK1650 mutant cells did not display growth in the minimal medium, and growth was restored with the addition of Na but not Nm. The enzymatic and genetic analyses strongly suggest that NAD + salvage in T. kodakarensis requires deamination of Nm and proceeds through the four-step pathway. IMPORTANCE Hyperthermophiles must constantly deal with increased degradation rates of their biomolecules due to their high growth temperatures. Here, we identified the pathway that regenerates NAD + from nicotinamide (Nm) in the hyperthermophilic archaeon Thermococcus kodakarensis The organism utilizes a four-step pathway that initially hydrolyzes the amide bond of Nm to generate nicotinic acid (Na), followed by phosphoribosylation, adenylylation, and amidation. Although the two-step pathway, consisting of only phosphoribosylation of Nm and adenylylation, seems to be more efficient, Nm mononucleotide in the two-step pathway is much more thermolabile than Na mononucleotide, the corresponding intermediate in the four-step pathway. Although NAD + itself is thermolabile, this may represent an example of a metabolism that has evolved to avoid the use of thermolabile intermediates. Copyright © 2018 American Society for Microbiology.

Polyphenolic Profile and Targeted Bioactivity of Methanolic Extracts from Mediterranean Ethnomedicinal Plants on Human Cancer Cell Lines.

PubMed

Pollio, Antonino; Zarrelli, Armando; Romanucci, Valeria; Di Mauro, Alfredo; Barra, Federica; Pinto, Gabriele; Crescenzi, Elvira; Roscetto, Emanuela; Palumbo, Giuseppe

2016-03-23

The methanol extracts of the aerial part of four ethnomedicinal plants of Mediterranean region, two non-seed vascular plants, Equisetum hyemale L. and Phyllitis scolopendrium (L.) Newman, and two Spermatophyta, Juniperus communis L. (J. communis) and Cotinus coggygria Scop. (C. coggygria), were screened against four human cells lines (A549, MCF7, TK6 and U937). Only the extracts of J. communis and C. coggygria showed marked cytotoxic effects, affecting both cell morphology and growth. A dose-dependent effect of these two extracts was also observed on the cell cycle distribution. Incubation of all the cell lines in a medium containing J. communis extract determined a remarkable accumulation of cells in the G2/M phase, whereas the C. coggygria extract induced a significant increase in the percentage of G1 cells. The novelty of our findings stands on the observation that the two extracts, consistently, elicited coherent effects on the cell cycle in four cell lines, independently from their phenotype, as two of them have epithelial origin and grow adherent and two are lymphoblastoid and grow in suspension. Even the expression profiles of several proteins regulating cell cycle progression and cell death were affected by both extracts. LC-MS investigation of methanol extract of C. coggygria led to the identification of twelve flavonoids (compounds 1-11, 19) and eight polyphenols derivatives (12-18, 20), while in J. communis extract, eight flavonoids (21-28), a α-ionone glycoside (29) and a lignin (30) were found. Although many of these compounds have interesting individual biological activities, their natural blends seem to exert specific effects on the proliferation of cell lines either growing adherent or in suspension, suggesting potential use in fighting cancer.

Indirect viscosimetric method is less accurate than ektacytometry for the measurement of red blood cell deformability.

PubMed

Vent-Schmidt, Jens; Waltz, Xavier; Pichon, Aurélien; Hardy-Dessources, Marie-Dominique; Romana, Marc; Connes, Philippe

2015-01-01

The aim of this study was to test the accuracy of viscosimetric method to estimate the red blood cell (RBC) deformability properties. Thirty-three subjects were enrolled in this study: 6 healthy subjects (AA), 11 patients with sickle cell-hemoglobin C disease (SC) and 16 patients with sickle cell anemia (SS). Two methods were used to assess RBC deformability: 1) indirect viscosimetric method and 2) ektacytometry. The indirect viscosimetric method was based on the Dintenfass equation where blood viscosity, plasma viscosity and hematocrit are measured and used to calculate an index of RBC rigidity (Tk index). The RBC deformability/rigidity of the three groups was compared using the two methods. Tk index was not different between SS and SC patients and the two groups had higher values than AA group. When ektacytometry was used, RBC deformability was lower in SS and SC groups compared to the AA group and SS and SC patients were different. Although the two measures of RBC deformability were correlated, the association was not very high. Bland and Altman analysis demonstrated a 3.25 bias suggesting a slight difference between the two methods. In addition, the limit of agreement represented 28% (>15%) of the mean values of RBC deformability, showing no interchangeability between the two methods. In conclusion, measuring RBC deformability by indirect viscosimetry is less accurate than by ektacytometry, which is considered the gold standard.

[C–H···anion] interactions mediate the templation and anion binding properties of topologically non-trivial metal–organic structures in aqueous solutions† †Electronic supplementary information (ESI) available: For general methods, further details of synthesis and characterization, anions binding, and DFT and PM6 calculations. CCDC 1409618. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c5sc04246a Click here for additional data file. Click here for additional data file.

PubMed Central

Bilbeisi, Rana A.; Prakasam, Thirumurugan; Lusi, Matteo; El Khoury, Roberto; Platas-Iglesias, Carlos; Charbonnière, Loïc J.; Olsen, John-Carl

2016-01-01

Two synthetic approaches—temperature variation and anion templation—allowed for the selective formation of a [2]catenane ([2]C4+) or a trefoil knot (TK6+), or for the enhanced formation of a Solomon link (SL8+), all from a simple set of starting materials (Zn(ii) acetate, diformylpyridine (DFP) and a diamino-2,2′-bipyridine (DAB)) in mixed aqueous solutions. The catenane formed exclusively at 90 °C in a 1 : 1 mixed solvent of D2O and MeOD. In the presence of bromide ion as template, TK6+ formed exclusively at 50 °C in the same solvent. In the solid state, TK6+ hosts two bromide ions in its central cavity by forming six Csp2–H hydrogen bonds. In D2O, TK6+, which was originally prepared as a trifluoroacetate (TFA) salt, was found to exchange two TFA counterions for two monovalent anions of different sizes and shapes, which lodged within the knot's central cavity. In contrast to bromide, the larger triflate anion (CF3SO3 –) promoted the formation of SL8+, which was characterized by 1H NMR spectroscopy and mass spectrometry. Two dimensional heteronuclear 19F-1H-HOSEY NMR experiments detected CH···F interactions inside the cavity of SL8+. Thus, the product distribution of this dynamic link forming system is sensitive to temperature and the size and shape of the anion template, and one of the products, TK6+, is capable of binding a variety of monovalent anions in D2O with high affinity (with log β 2 values of 4 to 6 being typical). PMID:28660022

Structural insights of a self-assembling 9-residue peptide from the C-terminal tail of the SARS corona virus E-protein in DPC and SDS micelles: A combined high and low resolution spectroscopic study.

PubMed

Ghosh, Anirban; Bhattacharyya, Dipita; Bhunia, Anirban

2018-02-01

In recent years, several studies based on the interaction of self-assembling short peptides derived from viroporins with model membranes, have improved our understanding of the molecular mechanism of corona virus (CoV) infection under physiological conditions. In this study, we have characterized the mechanism of membrane interaction of a short, 9-residue peptide TK9 (T 55 VYVYSRVK 63 ) that had been derived from the carboxyl terminal of the Severe Acute Respiratory Syndrome (SARS) corona virus (SARS CoV) envelope (E) protein. The peptide has been studied for its physical changes in the presence of both zwitterionic DPC and negatively charged SDS model membrane micelles, respectively, with the help of a battery of biophysical techniques including two-dimensional solution state NMR spectroscopy. Interestingly, in both micellar environments, TK9 adopted an alpha helical conformation; however, the helical propensities were much higher in the case of DPC compared to those of SDS micelle, suggesting that TK9 has more specificity towards eukaryotic cell membrane than the bacterial cell membrane. The orientation of the peptide TK9 also varies in the different micellar environments. The peptide's affinity was further manifested by its pronounced membrane disruption ability towards the mammalian compared to the bacterial membrane mimic. Collectively, the in-depth structural information on the interaction of TK9 with different membrane environments explains the host specificity and membrane orientation owing to subsequent membrane disruption implicated in the viral pathogenesis. Copyright © 2017 Elsevier B.V. All rights reserved.

Heavy ion mutagenesis: linear energy transfer effects and genetic linkage

NASA Technical Reports Server (NTRS)

Kronenberg, A.; Gauny, S.; Criddle, K.; Vannais, D.; Ueno, A.; Kraemer, S.; Waldren, C. A.; Chatterjee, A. (Principal Investigator)

1995-01-01

We have characterized a series of 69 independent mutants at the endogenous hprt locus of human TK6 lymphoblasts and over 200 independent S1-deficient mutants of the human x hamster hybrid cell line AL arising spontaneously or following low-fluence exposures to densely ionizing Fe ions (600 MeV/amu, linear energy transfer = 190 keV/microns). We find that large deletions are common. The entire hprt gene (> 44 kb) was missing in 19/39 Fe-induced mutants, while only 2/30 spontaneous mutants lost the entire hprt coding sequence. When the gene of interest (S1 locus = M1C1 gene) is located on a nonessential human chromosome 11, multilocus deletions of several million base pairs are observed frequently. The S1 mutation frequency is more than 50-fold greater than the frequency of hprt mutants in the same cells. Taken together, these results suggest that low-fluence exposures to Fe ions are often cytotoxic due to their ability to create multilocus deletions that may often include the loss of essential genes. In addition, the tumorigenic potential of these HZE heavy ions may be due to the high potential for loss of tumor suppressor genes. The relative insensitivity of the hprt locus to mutation is likely due to tight linkage to a gene that is required for viability.

Linking transgene expression of engineered mesenchymal stem cells and angiopoietin-1-induced differentiation to target cancer angiogenesis.

PubMed

Conrad, Claudius; Hüsemann, Yves; Niess, Hanno; von Luettichau, Irene; Huss, Ralf; Bauer, Christian; Jauch, Karl-Walter; Klein, Christoph A; Bruns, Christiane; Nelson, Peter J

2011-03-01

To specifically target tumor angiogenesis by linking transgene expression of engineered mesenchymal stem cells to angiopoietin-1-induced differentiation. Mesenchymal stem cells (MSCs) have been used to deliver therapeutic genes into solid tumors. These strategies rely on their homing mechanisms only to deliver the therapeutic agent. We engineered murine MSC to express reporter genes or therapeutic genes under the selective control of the Tie2 promoter/enhancer. This approach uses the differentiative potential of MSCs induced by the tumor microenvironment to drive therapeutic gene expression only in the context of angiogenesis. When injected into the peripheral circulation of mice with either, orthotopic pancreatic or spontaneous breast cancer, the engineered MSCs were actively recruited to growing tumor vasculature and induced the selective expression of either reporter red florescent protein or suicide genes [herpes simplex virus-thymidine kinase (TK) gene] when the adoptively transferred MSC developed endothelial-like characteristics. The TK gene product in combination with the prodrug ganciclovir (GCV) produces a potent toxin, which affects replicative cells. The homing of engineered MSC with selective induction of TK in concert with GCV resulted in a toxic tumor-specific environment. The efficacy of this approach was demonstrated by significant reduction in primary tumor growth and prolongation of life in both tumor models. This "Trojan Horse" combined stem cell/gene therapy represents a novel treatment strategy for tailored therapy of solid tumors.

Discovery of novel 4-anilinoquinazoline derivatives as potent inhibitors of epidermal growth factor receptor with antitumor activity.

PubMed

Xu, Yun-Yun; Li, Si-Ning; Yu, Gao-Jian; Hu, Qing-Hua; Li, Huan-Qiu

2013-10-01

Two new series of new compounds containing a 6-amino-substituted group or 6-acrylamide-substituted group linked to a 4-anilinoquinazoline nucleus have been discovered as potential EGFR inhibitors. These compounds proved efficient effects on antiproliferative activity and EGFR-TK inhibitory activity. Especially, N(6)-((5-bromothiophen-2-yl)methyl)-N(4)-(3-chlorophenyl)quinazoline-4,6-diamine (5e), showed the most potent inhibitory activity (IC50=3.11μM for Hep G2, IC50=0.82μM for A549). The EGFR molecular docking model suggested that the new compound is nicely bound to the region of EGFR, and cell morphology by Hoechst stain experiment suggested that these compounds efficiently induced apoptosis of A549 cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

Bioethanol production by heterologous expression of Pdc and AdhII in Streptomyces lividans.

PubMed

Lee, Jae Sun; Chi, Won-Jae; Hong, Soon-Kwang; Yang, Ji-Won; Chang, Yong Keun

2013-07-01

Two genes from Zymomonas mobilis that are responsible for ethanol production, pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhII), were heterologously expressed in the Gram-positive bacterium Streptomyces lividans TK24. An examination of carbon distribution revealed that a significant portion of carbon metabolism was switched from biomass and organic acid biosynthesis to ethanol production upon the expression of pdc and adhII. The recombinant S. lividans TK24 produced ethanol from glucose with a yield of 23.7% based on the carbohydrate consumed. The recombinant was able to produce ethanol from xylose, L-arabinose, mannose, L-rhamnose, galactose, ribose, and cellobiose with yields of 16.0, 25.6, 21.5, 33.6, 30.6, 14.6, and 33.3%, respectively. Polymeric substances such as starch and xylan were directly converted to ethanol by the recombinant with ethanol yields of 18.9 and 8.8%, respectively. The recombinant S. lividans TK24/Tpet developed in this study is potentially a useful microbial resource for ethanol production from various sources of biomasses, especially microalgae.

Genetically engineered theranostic mesenchymal stem cells for the evaluation of the anticancer efficacy of enzyme/prodrug systems.

PubMed

Nouri, Faranak Salman; Wang, Xing; Hatefi, Arash

2015-02-28

Over the past decade, various enzyme/prodrug systems such as thymidine kinase/ganciclovir (TK/GCV), yeast cytosine deaminase/5-fluorocytosine (yCD/5-FC) and nitroreductase/CB1954 (NTR/CB1954) have been used for stem cell mediated suicide gene therapy of cancer. Yet, no study has been conducted to compare and demonstrate the advantages and disadvantages of using one system over another. Knowing that each enzyme/prodrug system has its own strengths and weaknesses, we utilized mesenchymal stem cells (MSCs) as a medium to perform for the first time a comparative study that illustrated the impact of subtle differences among these systems on the therapeutic outcome. For therapeutic purposes, we first genetically modified MSCs to stably express a panel of four suicide genes including TK (TK007 and TK(SR39) mutants), yeast cytosine deaminase:uracil phosphoribosyltransferase (yCD:UPRT) and nitroreductase (NTR). Then, we evaluated the anticancer efficacies of the genetically engineered MSCs in vitro and in vivo by using SKOV3 cell line which is sensitive to all four enzyme/prodrug systems. In addition, all MSCs were engineered to stably express luciferase gene making them suitable for quantitative imaging and dose-response relationship studies in animals. Considering the limitations imposed by the prodrugs' bystander effects, our findings show that yCD:UPRT/5-FC is the most effective enzyme/prodrug system among the ones tested. Our findings also demonstrate that theranostic MSCs are a reliable medium for the side-by-side evaluation and screening of the enzyme/prodrug systems at the preclinical level. The results of this study could help scientists who utilize cell-based, non-viral or viral vectors for suicide gene therapy of cancer make more informed decisions when choosing enzyme/prodrug systems. Copyright © 2015 Elsevier B.V. All rights reserved.

A comparison of the cytogenetic alterations and global DNA hypomethylation induced by the benzene metabolite, hydroquinone, with those induced by melphalan and etoposide

PubMed Central

Ji, Z; Zhang, L; Peng, V.; Ren, X; McHale, CM; Smith, MT

2015-01-01

Specific cytogenetic alterations and changes in DNA methylation are involved in leukemogenesis. Benzene, an established human leukemogen, is known to induce cytogenetic changes through its active metabolites including hydroquinone (HQ), but the specific alterations have not been fully characterized. Global DNA hypomethylation was reported in a population exposed to benzene, but has not been confirmed in vitro. In this study, we examined cytogenetic changes in chromosomes 5, 7, 8, 11 and 21, and global DNA methylation in human TK6 lymphoblastoid cells treated with HQ for 48 h, and compared the HQ-induced alterations with those induced by two well-known leukemogens, melphalan, an alkylating agent, and etoposide, a DNA topoisomerase II inhibitor. We found that rather than inducing cytogenetic alterations distinct from those induced by melphalan and etoposide, HQ induced alterations characteristic of each agent. HQ induced global DNA hypomethylation at a level intermediate to melphalan (no effect) and etoposide (potent effect). These results suggest that HQ may act similar to an alkylating agent and also similar to a DNA topoisomerase II inhibitor in living cells, both of which may be potential mechanisms of benzene toxicity. In addition to cytogenetic changes, global DNA hypomethylation may be another mechanism underlying the leukemogenicity of benzene. PMID:20339439

Different Types of Luciferase Reporters Show Distinct Susceptibility to T3-Evoked Downregulation.

PubMed

Kollár, Anna; Kvárta-Papp, Zsuzsanna; Egri, Péter; Gereben, Balázs

2016-01-01

The firefly luciferase reporter protein is a crucial tool for studies targeting a broad range of biological questions. Importantly, luciferase assays are also widely used to explore mechanisms underlying thyroid hormone dependent regulation of gene expression. However, it was demonstrated that the firefly luciferase reporter is subject to triiodothyronine (T3)-evoked, promoter independent downregulation that is mediated by the thyroid hormone receptor. Since this effect can interfere with readout accuracy, the study aimed to find luciferase reporters that are not susceptible to this phenomenon. Luciferase reporter constructs were generated under the control of a minimal thymidine kinase (TK) promoter and transiently transfected into JEG-3 cells to test their activity upon T3 treatment. Activity of the TK-(dCpG)Luc encoding a synthetic (dCpG)Luciferase and TK-NanoLuc expressing the NanoLuc reporter was not significantly changed by T3 treatment while the firefly luciferase control was suppressed by ∼2.6-fold. T3 also downregulated the activity of Renilla luciferase by ∼30%. Novel types of luciferase reporters, especially the synthetic (dCpG)Luciferase, can be more accurate to study T3-regulated gene expression than the classical firefly luciferase reporter. Renilla luciferase, a popular transfection control of dual luciferase assays, should be used with caution in conditions with T3 treatment.

Effect of NK cell immunotherapy on immune function in patients with hepatic carcinoma: A preliminary clinical study

PubMed Central

Qin, Zilin; Chen, Jibing; Zeng, Jianying; Niu, Lizhi; Xie, Silun; Wang, Xiaohua; Liang, Yingqing; Wu, Zhenyi; Zhang, Mingjie

2017-01-01

ABSTRACT We investigated the effectiveness of adoptive transfer of KIR ligand-mismatched highly activated nature killer (HANK) cells in patients with hepatic carcinoma. Peripheral blood mononuclear cells were obtained and cultured in vitro to induce expansion and activation of HANK cells. After 12 d of culture, the cells were divided into 3 parts and infused intravenously on days 13 to 15. The patients (n = 16) were given one to 6 courses of immunotherapy. No side effects were observed. The lymphocyte subsets and cytokine, thymidine kinase 1 (TK1) and circulating tumor cell (CTC) levels were measured 1 day before treatment and 1 month after the final infusion: the absolute number of total T cells and NK cells and the IL-2 and TNF-β levels were significantly higher, and the TK1 and CTC levels were significantly lower at 1 month after treatment. The percentage of patients who experienced partial response, disease stabilization, and disease progression at 3 months after treatment was 18.8%, 50.0% and 31.2%, respectively. The total follow-up period was 2–12 months. The median progression-free survival from treatment was 7.5 months. This is the first study on the benefits of HANK cell immunotherapy for hepatic carcinoma These encouraging preliminary observations imply that HANK cell immunotherapy is safe, can improve the immune function of patients with liver cancer, and may even reduce the rate of tumor metastasis and recurrence. However, further studies on larger samples of patients with a longer follow-up period are required to confirm these findings. PMID:28353401

Threshold oxygen levels in Na(I) for the formation of NaCrO 2(s) on 18-8 stainless steels from accurate thermodynamic measurements

NASA Astrophysics Data System (ADS)

Sreedharan, O. M.; Madan, B. S.; Gnanamoorthy, J. B.

1983-12-01

The compound NaCrO 2(s) is an important corrosion product in sodium-cooled LMFBRs. The standard Gibbs energy of formation of NaCrO 2(s) is required for the computation of threshold oxygen levels in Na(1) for the formation of NaCrO 2(s) on 18-8 stainless steels. For this purpose the emf of the galvanic cell: Pt, NaCrO 2, Cr 2O 3, Na 2CrO 4/15 YSZ/O 2 ( P O 2 = 0.21 atm, air), Pt was measured over 784-1012 K to be: (E±4.4)(mV) = 483.67-0.34155 T(K). From this, the standard Gibbs energy of formation of NaCrO 2(s) from the elements ( ΔG f,T0) and from the oxides ( ΔG f,OX,T0) was calculated to be: [ΔG f,T0(NaCrO 2, s)±1.86] (kJ/mol) =-869.98 + 0.18575 T(K) , [ΔG f,OX,T0(NaCr0 2, s)±4.8] (kJ/mol) = -104.25-0.00856 T(K) . The molar heat capacity, C P0, of NaCrO 2(s) was measured by DSC to be (350-600 K): C P0(NaCrO 2, s) (J/K mol) = 27.15 + 0.1247 T (K) , From these data, values of -99.3 kJ/mol and 91.6 J/K mol were computed for ΔH f,2980 and S 2980 of NaCrO 2(s). The internal consistency was checked with the use of enthalpy data on Na 2CrO 4(s). From the standard Gibbs energy of formation of NaCrO 2(s) the equation logC 0(wppm) = 3.9905-3147.6 T(K) was derived, where C 0 is the threshold oxygen level for the formation of NaCrO 2(s) on 18-8 stainless steels.

Evaluation of scopadulciol-related molecules for their stimulatory effect on the cytotoxicity of acyclovir and ganciclovir against Herpes simplex virus type 1 thymidine kinase gene-transfected HeLa cells.

PubMed

Hayashi, Kyoko; Rahman, S M Abdur; Ohno, Hiroaki; Tanaka, Tetsuaki; Toyooka, Naoki; Nemoto, Hideo; Hayashi, Toshimitsu

2004-08-01

Herpes simplex virus type 1 thymidine kinase (HSV TK) is involved in both antiherpetic therapy and cancer gene therapy with acyclovir (ACV) and ganciclovir (GCV). Enhanced sensitivity to these drugs is advantageous in their clinical use. In the present study, scopadulciol (SDC) and its related compounds were evaluated for their stimulatory effect on the cytotoxicity of ACV and GCV by determination of selective toxicities against HSV TK-expressing HeLa cells. Although SDC remarkably potenciated the cytotoxicity of ACV and GCV, the other tested compounds showed only weak selectivity, except for compound 34.

Immunosuppressive Myeloid Cells' Blockade in the Glioma Microenvironment Enhances the Efficacy of Immune-Stimulatory Gene Therapy.

PubMed

Kamran, Neha; Kadiyala, Padma; Saxena, Meghna; Candolfi, Marianela; Li, Youping; Moreno-Ayala, Mariela A; Raja, Nicholas; Shah, Diana; Lowenstein, Pedro R; Castro, Maria G

2017-01-04

Survival of glioma (GBM) patients treated with the current standard of care remains dismal. Immunotherapeutic approaches that harness the cytotoxic and memory potential of the host immune system have shown great benefit in other cancers. GBMs have developed multiple strategies, including the accumulation of myeloid-derived suppressor cells (MDSCs) to induce immunosuppression. It is therefore imperative to develop multipronged approaches when aiming to generate a robust anti-tumor immune response. Herein, we tested whether combining MDSC depletion or checkpoint blockade would augment the efficacy of immune-stimulatory herpes simplex type-I thymidine kinase (TK) plus Fms-like tyrosine kinase ligand (Flt3L)-mediated immune stimulatory gene therapy. Our results show that MDSCs constitute >40% of the tumor-infiltrating immune cells. These cells express IL-4Rα, inducible nitric oxide synthase (iNOS), arginase, programmed death ligand 1 (PDL1), and CD80, molecules that are critically involved in antigen-specific T cell suppression. Depletion of MDSCs strongly enhanced the TK/Flt3L gene therapy-induced tumor-specific CD8 T cell response, which lead to increased median survival and percentage of long-term survivors. Also, combining PDL1 or CTLA-4 immune checkpoint blockade greatly improved the efficacy of TK/Flt3L gene therapy. Our results, therefore, indicate that blocking MDSC-mediated immunosuppression holds great promise for increasing the efficacy of gene therapy-mediated immunotherapies for GBM. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Cloning of the koi herpesvirus genome as an infectious bacterial artificial chromosome demonstrates that disruption of the thymidine kinase locus induces partial attenuation in Cyprinus carpio koi.

PubMed

Costes, B; Fournier, G; Michel, B; Delforge, C; Raj, V Stalin; Dewals, B; Gillet, L; Drion, P; Body, A; Schynts, F; Lieffrig, F; Vanderplasschen, A

2008-05-01

Koi herpesvirus (KHV) is the causative agent of a lethal disease in koi and common carp. In the present study, we describe the cloning of the KHV genome as a stable and infectious bacterial artificial chromosome (BAC) clone that can be used to produce KHV recombinant strains. This goal was achieved by the insertion of a loxP-flanked BAC cassette into the thymidine kinase (TK) locus. This insertion led to a BAC plasmid that was stably maintained in bacteria and was able to regenerate virions when permissive cells were transfected with the plasmid. Reconstituted virions free of the BAC cassette but carrying a disrupted TK locus (the FL BAC-excised strain) were produced by the transfection of Cre recombinase-expressing cells with the BAC. Similarly, virions with a wild-type revertant TK sequence (the FL BAC revertant strain) were produced by the cotransfection of cells with the BAC and a DNA fragment encoding the wild-type TK sequence. Reconstituted recombinant viruses were compared to the wild-type parental virus in vitro and in vivo. The FL BAC revertant strain and the FL BAC-excised strain replicated comparably to the parental FL strain. The FL BAC revertant strain induced KHV infection in koi carp that was indistinguishable from that induced by the parental strain, while the FL BAC-excised strain exhibited a partially attenuated phenotype. Finally, the usefulness of the KHV BAC for recombination studies was demonstrated by the production of an ORF16-deleted strain by using prokaryotic recombination technology. The availability of the KHV BAC is an important advance that will allow the study of viral genes involved in KHV pathogenesis, as well as the production of attenuated recombinant candidate vaccines.

Synthesis and In Vitro Cytotoxicity of the 4-(Halogenoanilino)-6-bromoquinazolines and Their 6-(4-Fluorophenyl) Substituted Derivatives as Potential Inhibitors of Epidermal Growth Factor Receptor Tyrosine Kinase

PubMed Central

Paumo, Hugues K.

2017-01-01

Series of the 2-unsubstituted and 2-(4-chlorophenyl)–substituted 4-anilino-6-bromoquinazolines and their 6-(4-fluorophenyl)–substituted derivatives were evaluated for in vitro cytotoxicity against MCF-7 and HeLa cells. The 2-unsubstituted 4-anilino-6-bromoquinazolines lacked activity, whereas most of their 2-(4-chlorophenyl) substituted derivatives were found to exhibit significant cytotoxicity and selectivity against HeLa cells. Replacement of bromine with 4-fluorophenyl group for the 2-unsubstituted 4-anilinoquinazolines resulted in superior activity against HeLa cells compared to Gefitinib. The presence of a 4-fluorophenyl group in the 2-(4-chlorophenyl) substituted derivatives led to increased cytotoxicity against HeLa cells, except for the 3-chloroanilino derivative. The most active compounds, namely, 3g, 3l, and 4l, were found to exhibit a moderate to significant inhibitory effect against epidermal growth factor receptor tyrosine kinase (EGFR-TK). The EGFR molecular docking model suggested that these compounds are nicely bound to the region of EGFR. PMID:29156606

Onset and organ specificity of Tk2 deficiency depends on Tk1 down-regulation and transcriptional compensation.

PubMed

Dorado, Beatriz; Area, Estela; Akman, Hasan O; Hirano, Michio

2011-01-01

Deficiency of thymidine kinase 2 (TK2) is a frequent cause of isolated myopathy or encephalomyopathy in children with mitochondrial DNA (mtDNA) depletion. To determine the bases of disease onset, organ specificity and severity of TK2 deficiency, we have carefully characterized Tk2 H126N knockin mice (Tk2-/-). Although normal until postnatal day 8, Tk2-/- mice rapidly develop fatal encephalomyopathy between postnatal days 10 and 13. We have observed that wild-type Tk2 activity is constant in the second week of life, while Tk1 activity decreases significantly between postnatal days 8 and 13. The down-regulation of Tk1 activity unmasks Tk2 deficiency in Tk2-/- mice and correlates with the onset of mtDNA depletion in the brain and the heart. Resistance to pathology in Tk2 mutant organs depends on compensatory mechanisms to the reduced mtDNA level. Our analyses at postnatal day 13 have revealed that Tk2-/- heart significantly increases mitochondrial transcript levels relative to the mtDNA content. This transcriptional compensation allows the heart to maintain normal levels of mtDNA-encoded proteins. The up-regulation in mitochondrial transcripts is not due to increased expression of the master mitochondrial biogenesis regulators peroxisome proliferator-activated receptor-gamma coactivator 1 alpha and nuclear respiratory factors 1 and 2, or to enhanced expression of the mitochondrial transcription factors A, B1 or B2. Instead, Tk2-/- heart compensates for mtDNA depletion by down-regulating the expression of the mitochondrial transcriptional terminator transcription factor 3 (MTERF3). Understanding the molecular mechanisms that allow Tk2 mutant organs to be spared may help design therapies for Tk2 deficiency.

Onset and organ specificity of Tk2 deficiency depends on Tk1 down-regulation and transcriptional compensation

PubMed Central

Dorado, Beatriz; Area, Estela; Akman, Hasan O.; Hirano, Michio

2011-01-01

Deficiency of thymidine kinase 2 (TK2) is a frequent cause of isolated myopathy or encephalomyopathy in children with mitochondrial DNA (mtDNA) depletion. To determine the bases of disease onset, organ specificity and severity of TK2 deficiency, we have carefully characterized Tk2 H126N knockin mice (Tk2−/−). Although normal until postnatal day 8, Tk2−/− mice rapidly develop fatal encephalomyopathy between postnatal days 10 and 13. We have observed that wild-type Tk2 activity is constant in the second week of life, while Tk1 activity decreases significantly between postnatal days 8 and 13. The down-regulation of Tk1 activity unmasks Tk2 deficiency in Tk2−/− mice and correlates with the onset of mtDNA depletion in the brain and the heart. Resistance to pathology in Tk2 mutant organs depends on compensatory mechanisms to the reduced mtDNA level. Our analyses at postnatal day 13 have revealed that Tk2−/− heart significantly increases mitochondrial transcript levels relative to the mtDNA content. This transcriptional compensation allows the heart to maintain normal levels of mtDNA-encoded proteins. The up-regulation in mitochondrial transcripts is not due to increased expression of the master mitochondrial biogenesis regulators peroxisome proliferator-activated receptor-gamma coactivator 1 alpha and nuclear respiratory factors 1 and 2, or to enhanced expression of the mitochondrial transcription factors A, B1 or B2. Instead, Tk2−/− heart compensates for mtDNA depletion by down-regulating the expression of the mitochondrial transcriptional terminator transcription factor 3 (MTERF3). Understanding the molecular mechanisms that allow Tk2 mutant organs to be spared may help design therapies for Tk2 deficiency. PMID:20940150

Correction of glucocerebrosidase deficiency after retroviral-mediated gene transfer into hematopoietic progenitor cells from patients with Gaucher disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fink, J.K.; Correll, P.H.; Perry, L.K.

1990-03-01

Retroviral gene transfer has been used successfully to correct the glucocerebrosidase (GCase) deficiency in primary hematopoietic cells from patients with Gaucher disease. For this model of somatic gene therapy, the authors developed a high-titer, amphotropic retroviral vector designated NTG in which the human GCase gene was driven by the mutant polyoma virus enhancer/herpesvirus thymidine kinase gene (tk) promoter (Py{sup +}/Htk). NTG normalized GCase activity in transduced Gaucher fibroblasts and efficiently infected human monocytic and erythroleukemic cell lines. RNA blot-hybridization (Northern blot) analysis of these hemaptopoietic cell lines showed unexpectedly high-level expression from the Moloney murine leukemia virus long terminal repeatmore » (Mo-MLV LTR) and levels of Py{sup +}/Htk enhancer/promoter-initiated human GCase RNA that approximated endogenous GCase RNA levels. Furthermore, NTG efficiently infected human hematopoietic progenitor cells. Detection of the provirus in approximately one-third of NTG-infected progenitor colonies that had not been selected in G418-containing medium indicates that relative resistance to G418 underestimated the actual gene transfer efficiency. Northern blot analysis of NTG-infected, progenitor-derived cells showed expression from both the Mo-MLV LTR and the Py{sup +}/Htk enhancer/promoter. NTG-transduced hematopoietic progenitor cells from patients with Gaucher disease generated progeny in which GCase activity has been normalized.« less

The Gas6/TAM System and Multiple Sclerosis.

PubMed

Bellan, Mattia; Pirisi, Mario; Sainaghi, Pier Paolo

2016-10-28

Growth arrest specific 6 (Gas6) is a multimodular circulating protein, the biological actions of which are mediated by the interaction with three transmembrane tyrosine kinase receptors: Tyro3, Axl, and MerTK, collectively named TAM. Over the last few decades, many progresses have been done in the understanding of the biological activities of this highly pleiotropic system, which plays a role in the regulation of immune response, inflammation, coagulation, cell growth, and clearance of apoptotic bodies. Recent findings have further related Gas6 and TAM receptors to neuroinflammation in general and, specifically, to multiple sclerosis (MS). In this paper, we review the biology of the Gas6/TAM system and the current evidence supporting its potential role in the pathogenesis of MS.

Assessment of diurnal systemic dose of agrochemicals in regulatory toxicity testing--an integrated approach without additional animal use.

PubMed

Saghir, Shakil A; Bartels, Michael J; Rick, David L; McCoy, Alene T; Rasoulpour, Reza J; Ellis-Hutchings, Robert G; Sue Marty, M; Terry, Claire; Bailey, Jason P; Billington, Richard; Bus, James S

2012-07-01

Integrated toxicokinetics (TK) data provide information on the rate, extent and duration of systemic exposure across doses, species, strains, gender, and life stages within a toxicology program. While routine for pharmaceuticals, TK assessments of non-pharmaceuticals are still relatively rare, and have never before been included in a full range of guideline studies for a new agrochemical. In order to better understand the relationship between diurnal systemic dose (AUC(24h)) and toxicity of agrochemicals, TK analyses in the study animals is now included in all short- (excluding acute), medium- and long-term guideline mammalian toxicity studies including reproduction/developmental tests. This paper describes a detailed procedure for the implementation of TK in short-, medium- and long-term regulatory toxicity studies, without the use of satellite animals, conducted on three agrochemicals (X11422208, 2,4-D and X574175). In these studies, kinetically-derived maximum doses (KMD) from short-term studies instead of, or along with, maximum tolerated doses (MTD) were used for the selection of the high dose in subsequent longer-term studies. In addition to leveraging TK data to guide dose level selection, the integrated program was also used to select the most appropriate method of oral administration (i.e., gavage versus dietary) of test materials for rat and rabbit developmental toxicity studies. The integrated TK data obtained across toxicity studies (without the use of additional/satellite animals) provided data critical to understanding differences in response across doses, species, strains, sexes, and life stages. Such data should also be useful in mode of action studies and to improve human risk assessments. Copyright © 2012 Elsevier Inc. All rights reserved.

Automatic analysis of the micronucleus test in primary human lymphocytes using image analysis.

PubMed

Frieauff, W; Martus, H J; Suter, W; Elhajouji, A

2013-01-01

The in vitro micronucleus test (MNT) is a well-established test for early screening of new chemical entities in industrial toxicology. For assessing the clastogenic or aneugenic potential of a test compound, micronucleus induction in cells has been shown repeatedly to be a sensitive and a specific parameter. Various automated systems to replace the tedious and time-consuming visual slide analysis procedure as well as flow cytometric approaches have been discussed. The ROBIAS (Robotic Image Analysis System) for both automatic cytotoxicity assessment and micronucleus detection in human lymphocytes was developed at Novartis where the assay has been used to validate positive results obtained in the MNT in TK6 cells, which serves as the primary screening system for genotoxicity profiling in early drug development. In addition, the in vitro MNT has become an accepted alternative to support clinical studies and will be used for regulatory purposes as well. The comparison of visual with automatic analysis results showed a high degree of concordance for 25 independent experiments conducted for the profiling of 12 compounds. For concentration series of cyclophosphamide and carbendazim, a very good correlation between automatic and visual analysis by two examiners could be established, both for the relative division index used as cytotoxicity parameter, as well as for micronuclei scoring in mono- and binucleated cells. Generally, false-positive micronucleus decisions could be controlled by fast and simple relocation of the automatically detected patterns. The possibility to analyse 24 slides within 65h by automatic analysis over the weekend and the high reproducibility of the results make automatic image processing a powerful tool for the micronucleus analysis in primary human lymphocytes. The automated slide analysis for the MNT in human lymphocytes complements the portfolio of image analysis applications on ROBIAS which is supporting various assays at Novartis.

Life-stage-, sex-, and dose-dependent dietary toxicokinetics and relationship to toxicity of 2,4-dichlorophenoxyacetic acid (2,4-D) in rats: implications for toxicity test dose selection, design, and interpretation.

PubMed

Saghir, Shakil A; Marty, Mary S; Zablotny, Carol L; Passage, Julie K; Perala, Adam W; Neal, Barbara H; Hammond, Larry; Bus, James S

2013-12-01

Life-stage-dependent toxicity and dose-dependent toxicokinetics (TK) were evaluated in Sprague Dawley rats following dietary exposure to 2,4-dichlorophenoxyacetic acid (2,4-D). 2,4-D renal clearance is impacted by dose-dependent saturation of the renal organic anion transporter; thus, this study focused on identifying inflection points of onset of dietary nonlinear TK to inform dose selection decisions for toxicity studies. Male and female rats were fed 2,4-D-fortified diets at doses to 1600 ppm for 4-weeks premating, <2 weeks during mating, and to test day (TD) 71 to parental (P1) males and to P1 females through gestation/lactation to TD 96. F1 offspring were exposed via milk with continuing diet exposure until postnatal day (PND) 35. As assessed by plasma area under the curve for the time-course plasma concentration, nonlinear TK was observed ≥ 1200 ppm (63 mg/kg/day) for P1 males and between 200 and 400 ppm (14-27 mg/kg/day) for P1 females. Dam milk and pup plasma levels were higher on lactation day (LD) 14 than LD 4. Relative to P1 adults, 2,4-D levels were higher in dams during late gestation/lactation and postweaning pups (PND 21-35) and coincided with elevated intake of diet/kg body weight. Using conventional maximum tolerated dose (MTD) criteria based on body weight changes for dose selection would have resulted in excessive top doses approximately 2-fold higher than those identified incorporating critical TK data. These data indicate that demonstration of nonlinear TK, if present at dose levels substantially above real-world human exposures, is a key dose selection consideration for improving the human relevance of toxicity studies compared with studies employing conventional MTD dose selection strategies.

Life-Stage-, Sex-, and Dose-Dependent Dietary Toxicokinetics and Relationship to Toxicity of 2,4-Dichlorophenoxyacetic Acid (2,4-D) in Rats: Implications for Toxicity Test Dose Selection, Design, and Interpretation

PubMed Central

Marty, Mary S.

2013-01-01

Life-stage-dependent toxicity and dose-dependent toxicokinetics (TK) were evaluated in Sprague Dawley rats following dietary exposure to 2,4-dichlorophenoxyacetic acid (2,4-D). 2,4-D renal clearance is impacted by dose-dependent saturation of the renal organic anion transporter; thus, this study focused on identifying inflection points of onset of dietary nonlinear TK to inform dose selection decisions for toxicity studies. Male and female rats were fed 2,4-D-fortified diets at doses to 1600 ppm for 4-weeks premating, <2 weeks during mating, and to test day (TD) 71 to parental (P1) males and to P1 females through gestation/lactation to TD 96. F1 offspring were exposed via milk with continuing diet exposure until postnatal day (PND) 35. As assessed by plasma area under the curve for the time-course plasma concentration, nonlinear TK was observed ≥1200 ppm (63mg/kg/day) for P1 males and between 200 and 400 ppm (14–27mg/kg/day) for P1 females. Dam milk and pup plasma levels were higher on lactation day (LD) 14 than LD 4. Relative to P1 adults, 2,4-D levels were higher in dams during late gestation/lactation and postweaning pups (PND 21–35) and coincided with elevated intake of diet/kg body weight. Using conventional maximum tolerated dose (MTD) criteria based on body weight changes for dose selection would have resulted in excessive top doses approximately 2-fold higher than those identified incorporating critical TK data. These data indicate that demonstration of nonlinear TK, if present at dose levels substantially above real-world human exposures, is a key dose selection consideration for improving the human relevance of toxicity studies compared with studies employing conventional MTD dose selection strategies. PMID:24105888

Effect of age on toxicokinetics among human volunteers exposed to propylene glycol methyl ether (PGME).

PubMed

Hopf, Nancy B; Vernez, David; Berthet, Aurelie; Charriere, Nicole; Arnoux, Christine; Tomicic, Catherine

2012-05-20

Aging adults represent the fastest growing population segment in many countries. Physiological and metabolic changes in the aging process may alter how aging adults biologically respond to pollutants. In a controlled human toxicokinetic study (exposure chamber; 12 m³), aging volunteers (n=10; >58 years) were exposed to propylene glycol monomethyl ether (PGME, CAS no. 107-98-2) at 50 ppm for 6 h. The dose-dependent renal excretion of oxidative metabolites, conjugated and free PGME could potentially be altered by age. (1) Compare PGME toxicokinetic profiles between aging and young volunteers (20-25 years) and gender; (2) test the predictive power of a compartmental toxicokinetic (TK) model developed for aging persons against urinary PGME concentrations found in this study. Urine samples were collected before, during, and after the exposure. Urinary PGME was quantified by capillary GC/FID. Differences in urinary PGME profiles were not noted between genders but between age groups. Metabolic parameters had to be changed to fit the age adjusted TK model to the experimental results, implying a slower enzymatic pathway in the aging volunteers. For an appropriate exposure assessment, urinary total PGME should be quantified. Age is a factor that should be considered when biological limit values are developed. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Diagnostic Algorithm to Reflect Regressive Changes of Human Papilloma Virus in Tissue Biopsies

PubMed Central

Lhee, Min Jin; Cha, Youn Jin; Bae, Jong Man; Kim, Young Tae

2014-01-01

Purpose Landmark indicators have not yet to be developed to detect the regression of cervical intraepithelial neoplasia (CIN). We propose that quantitative viral load and indicative histological criteria can be used to differentiate between atypical squamous cells of undetermined significance (ASCUS) and a CIN of grade 1. Materials and Methods We collected 115 tissue biopsies from women who tested positive for the human papilloma virus (HPV). Nine morphological parameters including nuclear size, perinuclear halo, hyperchromasia, typical koilocyte (TK), abortive koilocyte (AK), bi-/multi-nucleation, keratohyaline granules, inflammation, and dyskeratosis were examined for each case. Correlation analyses, cumulative logistic regression, and binary logistic regression were used to determine optimal cut-off values of HPV copy numbers. The parameters TK, perinuclear halo, multi-nucleation, and nuclear size were significantly correlated quantitatively to HPV copy number. Results An HPV loading number of 58.9 and AK number of 20 were optimal to discriminate between negative and subtle findings in biopsies. An HPV loading number of 271.49 and AK of 20 were optimal for discriminating between equivocal changes and obvious koilocytosis. Conclusion We propose that a squamous epithelial lesion with AK of >20 and quantitative HPV copy number between 58.9-271.49 represents a new spectrum of subtle pathological findings, characterized by AK in ASCUS. This can be described as a distinct entity and called "regressing koilocytosis". PMID:24532500

A high-resolution whole genome radiation hybrid map of human chromosome 17q22-q25.3 across the genes for GH and TK

DOE Office of Scientific and Technical Information (OSTI.GOV)

Foster, J.W.; Schafer, A.J.; Critcher, R.

1996-04-15

We have constructed a whole genome radiation hybrid (WG-RH) map across a region of human chromosome 17q, from growth hormone (GH) to thymidine kinase (TK). A panel of 128 WG-RH hybrid cell lines generated by X-irradiation and fusion has been tested for the retention of 39 sequence-tagged site (STS) markers by the polymerase chain reaction. This genome mapping technique has allowed the integration of existing VNTR and microsatellite markers with additional new markers and existing STS markers previously mapped to this region by other means. The WG-RH map includes eight expressed sequence tag (EST) and three anonymous markers developed formore » this study, together with 23 anonymous microsatellites and five existing ESTs. Analysis of these data resulted in a high-density comprehensive map across this region of the genome. A subset of these markers has been used to produce a framework map consisting of 20 loci ordered with odds greater than 1000:1. The markers are of sufficient density to build a YAC contig across this region based on marker content. We have developed sequence tags for both ends of a 2.1-Mb YAC and mapped these using the WG-RH panel, allowing a direct comparison of cRay{sub 6000} to physical distance. 31 refs., 3 figs., 2 tabs.« less

Tropospheric GOM at the Pic du Midi Observatory-Correcting Bias in Denuder Based Observations.

PubMed

Marusczak, Nicolas; Sonke, Jeroen E; Fu, Xuewu; Jiskra, Martin

2017-01-17

Gaseous elemental mercury (GEM, Hg) emissions are transformed to divalent reactive Hg (RM) forms throughout the troposphere and stratosphere. RM is often operationally quantified as the sum of particle bound Hg (PBM) and gaseous oxidized Hg (GOM). The measurement of GOM and PBM is challenging and under mounting criticism. Here we intercompare six months of automated GOM and PBM measurements using a Tekran (TK) KCl-coated denuder and quartz regenerable particulate filter method (GOM TK , PBM TK , and RM TK ) with RM CEM collected on cation exchange membranes (CEMs) at the high altitude Pic du Midi Observatory. We find that RM TK is systematically lower by a factor of 1.3 than RM CEM . We observe a significant relationship between GOM TK (but not PBM TK ) and Tekran flush TK blanks suggesting significant loss (36%) of labile GOM TK from the denuder or inlet. Adding the flush TK blank to RM TK results in good agreement with RM CEM (slope = 1.01, r 2 = 0.90) suggesting we can correct bias in RM TK and GOM TK . We provide a bias corrected (*) Pic du Midi data set for 2012-2014 that shows GOM* and RM* levels in dry free tropospheric air of 198 ± 57 and 229 ± 58 pg m -3 which agree well with in-flight observed RM and with model based GOM and RM estimates.

Cytoplasmically Retargeted HSV1-tk/GFP Reporter Gene Mutants for Optimization of Noninvasive Molecular-Genetic Imaging

PubMed Central

Ponomarev, Vladimir; Doubrovin, Michael; Serganova, Inna; Beresten, Tatiana; Vider, Jelena; Shavrin, Aleksander; Ageyeva, Ludmila; Balatoni, Julius; Blasberg, Ronald; Tjuvajev, Juri Gelovani

2003-01-01

Abstract To optimize the sensitivity of imaging HSV1-tk/GFP reporter gene expression, a series of HSV1-tk/GFP mutants was developed with altered nuclear localization and better cellular enzymatic activity, compared to that of the native HSV1-tk/GFP fusion protein (HSV1-tk/GFP). Several modifications of HSV1-tk/GFP reporter gene were performed, including targeted inactivating mutations in the nuclear localization signal (NLS), the addition of a nuclear export signal (NES), a combination of both mutation types, and a truncation of the first 135 bp of the native hsv1-tk coding sequence containing a “cryptic” testicular promoter and the NLS. A recombinant HSV1-tk/GFP protein and a highly sensitive sandwich enzyme-linked immunosorbent assay for HSV1-tk/GFP were developed to quantitate the amount of reporter gene product in different assays to allow normalization of the data. These different mutations resulted in various degrees of nuclear clearance, predominant cytoplasmic distribution, and increased total cellular enzymatic activity of the HSV1-tk/GFP mutants, compared to native HSV1-tk/GFP when expressed at the same levels. This appears to be the result of improvedmetabolic bioavailability of cytoplasmically retargeted mutant HSV1-tk/GFP enzymes for reaction with the radiolabeled probe (e.g., FIAU). The analysis of enzymatic properties of different HSV1-tk/GFP mutants using FIAU as a substrate revealed no significant differences from that of the native HSV1-tk/GFP. Improved total cellular enzymatic activity of cytoplasmically retargeted HSV1-tk/GFP mutants observed in vitro was confirmed by noninvasive imaging of transduced subcutaneous tumor xenografts bearing these reporters using [131I]FIAU and a γ-camera. PMID:12869307

Lumbar Lordosis Minus Thoracic Kyphosis: Remain Constant in Adolescent Idiopathic Scoliosis Patients Before and After Correction Surgery.

PubMed

Yang, Mingyuan; Yang, Changwei; Chen, Ziqiang; Wei, Xianzhao; Chen, Yuanyuan; Zhao, Jian; Shao, Jie; Zhu, Xiaodong; Li, Ming

2016-03-01

A retrospective study. To explore the relationship between the change of lumbar lordosis (LL) and thoracic kyphosis (TK) in AIS patients after correction surgery. TK tends to decrease in Lenke 1 and Lenke 2 AIS patients after correction surgery using pedicle screws, with the compensation of LL decrease. We hypothesize that lumbar lordosis minus thoracic kyphosis (LL-TK) remains constant after correction surgery to achieve the sagittal balance in AIS patients. Medical records of Lenke 1 or Lenke 2 AIS patients who received posterior correction surgery using pedicle screws in our hospital from January 2010 to January 2013 were reviewed. General characters of patients and radiological parameters were evaluated before the surgery and at two years' follow-up. Correlation analysis between TK and LL was conducted. LL-TK and the change of LL and TK were analyzed at preoperation and final follow-up. A total of 76 Lenke 1 and Lenke 2 AIS patients were included. Both TK and LL decreased significantly after correction surgery (P = 0.019 and P = 0.040, respectively). There were significant correlations between TK and LL before and after surgery, respectively (preoperative: r = 0.234, P = 0.042; postoperative: r = 0.310, P = 0.006). Preoperative and postoperative LL-TK was 23.80° and 25.09°, respectively, and no significant difference of LL-TK was observed (P = 0.372). The same tendency was observed in the change of LL and TK, and significant correlation was also found between the change of TK and LL (r = 0.626, P = 0.002). The same change of LL and TK and no significant difference in LL-TK indicated that LL-TK might be an important compensatory mechanism in keeping sagittal balance.

Cloning and heterologous expression of blasticidin S biosynthetic genes from Streptomyces griseochromogenes.

PubMed

Cone, M C; Petrich, A K; Gould, S J; Zabriskie, T M

1998-06-01

Two small chromosomal DNA fragments (2.6 and 4.8 kb) from the blasticidin S producer Streptomyces griseochromogenes were cloned in the high copy number vector pIJ702 and shown to confer increased resistance to blasticidin S upon S. lividans TK24. These fragments were used to screen a library of S. griseochromogenes DNA prepared in the cosmid shuttle vector pOJ446. Cosmids containing DNA inserts of at least 23 kb were identified which hybridized to one or the other resistance fragment, but not to both. Transformation of S. lividans TK24 with several cosmids hybridizing with the 4.8 kb resistance fragment resulted in clones that produced cytosylglucuronic acid, the first intermediate of the blasticidin S biosynthetic pathway, and other blasticidin-related metabolites. A strain of S. lividans TK24 harboring both the 4.8 kb-hybridizing cosmid and the 2.6 kb resistance fragment cloned in pIJ702 produced 12.5 times as much demethylblasticidin S as the transformant harboring the cosmid alone.

Analysis of a cis-Acting Element Involved in Regulation by Estrogen of Human Angiotensinogen Gene Expression.

PubMed

Zhao, Yan-Yan; Sun, Kai-Lai; Ashok, Kumar

1998-01-01

The work was aimed to identify the estrogen responsive element in the human angiotensinogen gene. The nucleotide sequence between the transcription initiation site and TATA box in angiotensinogen gene promoter was found to be strongly homologous with the consensus estrogen responsive element. This sequence was confirmed as the estrogen responsive element (HAG ERE) by electrophoretic mobility shift assay. The recombinant expression vectors were constructed in which chloramphenicol acetyltransferase (CAT) reporter gene was driven by angiotensinogen core promoter with HAG ERE of by TK core promoter with multiplied HAG ERE, and were used in cotransfection with the human estrogen receptor expression vector into HepG(2) cells; CAT assays showed an increase of the CAT activity on 17beta-estradiol treatment in those transfectants. These results suggest that the human angiotensinogen gene is transcriptionally up-regulated by estrogen through the estrogen responsive element near TATA box of the promoter.

Reporter gene imaging of targeted T cell immunotherapy in recurrent glioma.

PubMed

Keu, Khun Visith; Witney, Timothy H; Yaghoubi, Shahriar; Rosenberg, Jarrett; Kurien, Anita; Magnusson, Rachel; Williams, John; Habte, Frezghi; Wagner, Jamie R; Forman, Stephen; Brown, Christine; Allen-Auerbach, Martin; Czernin, Johannes; Tang, Winson; Jensen, Michael C; Badie, Behnam; Gambhir, Sanjiv S

2017-01-18

High-grade gliomas are aggressive cancers that often become rapidly fatal. Immunotherapy using CD8 + cytotoxic T lymphocytes (CTLs), engineered to express both herpes simplex virus type 1 thymidine kinase (HSV1-TK) and interleukin-13 (IL-13) zetakine chimeric antigen receptor (CAR), is a treatment strategy with considerable potential. To optimize this and related immunotherapies, it would be helpful to monitor CTL viability and trafficking to glioma cells. We show that noninvasive positron emission tomography (PET) imaging with 9-[4-[ 18 F]fluoro-3-(hydroxymethyl)butyl]guanine ([ 18 F]FHBG) can track HSV1-tk reporter gene expression present in CAR-engineered CTLs. [ 18 F]FHBG imaging was safe and enabled the longitudinal imaging of T cells stably transfected with a PET reporter gene in patients. Further optimization of this imaging approach for monitoring in vivo cell trafficking should greatly benefit various cell-based therapies for cancer. Copyright © 2017, American Association for the Advancement of Science.

TP53-dependent chromosome instability is associated with transient reductions in telomere length in immortal telomerase-positive cell lines

NASA Technical Reports Server (NTRS)

Schwartz, J. L.; Jordan, R.; Liber, H.; Murnane, J. P.; Evans, H. H.

2001-01-01

Telomere shortening in telomerase-negative somatic cells leads to the activation of the TP53 protein and the elimination of potentially unstable cells. We examined the effect of TP53 gene expression on both telomere metabolism and chromosome stability in immortal, telomerase-positive cell lines. Telomere length, telomerase activity, and chromosome instability were measured in multiple clones isolated from three related human B-lymphoblast cell lines that vary in TP53 expression; TK6 cells express wild-type TP53, WTK1 cells overexpress a mutant form of TP53, and NH32 cells express no TP53 protein. Clonal variations in both telomere length and chromosome stability were observed, and shorter telomeres were associated with higher levels of chromosome instability. The shortest telomeres were found in WTK1- and NH32-derived cells, and these cells had 5- to 10-fold higher levels of chromosome instability. The primary marker of instability was the presence of dicentric chromosomes. Aneuploidy and other stable chromosome alterations were also found in clones showing high levels of dicentrics. Polyploidy was found only in WTK1-derived cells. Both telomere length and chromosome instability fluctuated in the different cell populations with time in culture, presumably as unstable cells and cells with short telomeres were eliminated from the growing population. Our results suggest that transient reductions in telomere lengths may be common in immortal cell lines and that these alterations in telomere metabolism can have a profound effect on chromosome stability. Copyright 2000 Wiley-Liss, Inc.

Efficient repair of DNA double-strand breaks in malignant cells with structural instability

PubMed Central

Cheng, Yue; Zhang, Zhenhua; Keenan, Bridget; Roschke, Anna V.; Nakahara, Kenneth; Aplan, Peter D.

2009-01-01

Aberrant repair of DNA double strand breaks (DSBs) is thought to be important in the generation of gross chromosomal rearrangements (GCRs). To examine how DNA DSBs might lead to GCRs, we investigated the repair of a single DNA DSB in a structurally unstable cell line. An I-SceI recognition site was introduced into OVCAR-8 cells between a constitutive promoter (EF1α) and the Herpes simplex virus thymidine kinase (TK) gene, which confers sensitivity to gancyclovir (GCV). Expression of I-SceI in these cells caused a single DSB. Clones with aberrant repair could acquire resistance to GCV by separation of the EF1α promoter from the TK gene, or deletion of either the EF1α promoter or the TK gene. All mutations that we identified were interstitial deletions. Treatment of cells with etoposide or bleomycin, agents known to produce DNA DSBs following expression of I-SceI also did not generate GCRs. Because we identified solely interstitial deletions using the aforementioned negative selection system, we developed a positive selection system to produce GCR. A construct containing an I-SceI restriction site immediately followed by a hygromycin phosphotransferase cDNA, with no promoter, was stably integrated into OVCAR-8 cells. DNA DSBs were produced by an I-SceI expression vector. None of the hygromycin resistant clones recovered had linked the hygromycin phosphotransferase cDNA to an endogenous promoter, but had instead captured a portion of the I-SceI expression vector. These results indicate that even in a structurally unstable malignant cell line, the majority of DNA DSBs are repaired by religation of the two broken chromosome ends, without the introduction of a GCR. PMID:19909760

Efficient repair of DNA double-strand breaks in malignant cells with structural instability.

PubMed

Cheng, Yue; Zhang, Zhenhua; Keenan, Bridget; Roschke, Anna V; Nakahara, Kenneth; Aplan, Peter D

2010-01-05

Aberrant repair of DNA double-strand breaks (DSBs) is thought to be important in the generation of gross chromosomal rearrangements (GCRs). To examine how DNA DSBs might lead to GCRs, we investigated the repair of a single DNA DSB in a structurally unstable cell line. An I-SceI recognition site was introduced into OVCAR-8 cells between a constitutive promoter (EF1alpha) and the Herpes simplex virus thymidine kinase (TK) gene, which confers sensitivity to gancyclovir (GCV). Expression of I-SceI in these cells caused a single DSB. Clones with aberrant repair could acquire resistance to GCV by separation of the EF1alpha promoter from the TK gene, or deletion of either the EF1alpha promoter or the TK gene. All mutations that we identified were interstitial deletions. Treatment of cells with etoposide or bleomycin, agents known to produce DNA DSBs following expression of I-SceI also did not generate GCRs. Because we identified solely interstitial deletions using the aforementioned negative selection system, we developed a positive selection system to produce GCR. A construct containing an I-SceI restriction site immediately followed by a hygromycin phosphotransferase cDNA, with no promoter, was stably integrated into OVCAR-8 cells. DNA DSBs were produced by an I-SceI expression vector. None of the hygromycin resistant clones recovered had linked the hygromycin phosphotransferase cDNA to an endogenous promoter, but had instead captured a portion of the I-SceI expression vector. These results indicate that even in a structurally unstable malignant cell line, the majority of DNA DSBs are repaired by religation of the two broken chromosome ends, without the introduction of a GCR.

Effects of Laser Printer–Emitted Engineered Nanoparticles on Cytotoxicity, Chemokine Expression, Reactive Oxygen Species, DNA Methylation, and DNA Damage: A Comprehensive in Vitro Analysis in Human Small Airway Epithelial Cells, Macrophages, and Lymphoblasts

PubMed Central

Pirela, Sandra V.; Miousse, Isabelle R.; Lu, Xiaoyan; Castranova, Vincent; Thomas, Treye; Qian, Yong; Bello, Dhimiter; Kobzik, Lester; Koturbash, Igor; Demokritou, Philip

2015-01-01

Background Engineered nanomaterials (ENMs) incorporated into toner formulations of printing equipment become airborne during consumer use. Although information on the complex physicochemical and toxicological properties of both toner powders and printer-emitted particles (PEPs) continues to grow, most toxicological studies have not used the actual PEPs but rather have primarily used raw toner powders, which are not representative of current exposures experienced at the consumer level during printing. Objectives We assessed the biological responses of a panel of human cell lines to PEPs. Methods Three physiologically relevant cell lines—small airway epithelial cells (SAECs), macrophages (THP-1 cells), and lymphoblasts (TK6 cells)—were exposed to PEPs at a wide range of doses (0.5–100 μg/mL) corresponding to human inhalation exposure durations at the consumer level of 8 hr or more. Following treatment, toxicological parameters reflecting distinct mechanisms were evaluated. Results PEPs caused significant membrane integrity damage, an increase in reactive oxygen species (ROS) production, and an increase in pro-inflammatory cytokine release in different cell lines at doses equivalent to exposure durations from 7.8 to 1,500 hr. Furthermore, there were differences in methylation patterns that, although not statistically significant, demonstrate the potential effects of PEPs on the overall epigenome following exposure. Conclusions The in vitro findings obtained in this study suggest that laser printer–emitted engineered nanoparticles may be deleterious to lung cells and provide preliminary evidence of epigenetic modifications that might translate to pulmonary disorders. Citation Pirela SV, Miousse IR, Lu X, Castranova V, Thomas T, Qian Y, Bello D, Kobzik L, Koturbash I, Demokritou P. 2016. Effects of laser printer–emitted engineered nanoparticles on cytotoxicity, chemokine expression, reactive oxygen species, DNA methylation, and DNA damage: a comprehensive in vitro analysis in human small airway epithelial cells, macrophages, and lymphoblasts. Environ Health Perspect 124:210–219; http://dx.doi.org/10.1289/ehp.1409582 PMID:26080392

A vaccine strain of pseudorabies virus with deletions in the thymidine kinase and glycoprotein X genes.

PubMed

Marchioli, C C; Yancey, R J; Wardley, R C; Thomsen, D R; Post, L E

1987-11-01

A pseudorabies virus (PRV) mutant with deletions in genes for glycoprotein X (gX) and thymidine kinase, designated delta GX delta TK, was constructed and evaluated as a vaccine for protecting swine against PRV-induced mortality. Doses greater than or equal to 10(3) plaque-forming units (PFU) of this strain given to mice provided protection from challenge exposure with virulent PRV. Sera tested from mice inoculated with delta GX delta TK had high titers of neutralizing antibody to PRV, but reactivity in the same sera was not significantly different from that in sera from noninoculated mice (controls) when sera from both groups were evaluated by use of an ELISA with gX antigen produced in Escherichia coli. Compared with noninoculated pigs (controls), those given delta GX delta TK (greater than or equal to 10(2) PFU) were protected completely from lethal challenge exposure, without experiencing adverse effects on weight gain and with reduction of shedding of virulent challenge virus. Serotest results indicated that, although inoculated pigs responded with strong neutralizing antibody titers, the response of delta GX delta TK-inoculated pigs to gX, as determined by ELISA before challenge exposure, was not significantly greater than the ELISA values obtained from control pigs. The ELISA values from a group of pigs inoculated with a commercially available vaccine were significantly (P less than 0.05) higher than those of control pigs. The experimental vaccine, delta GX delta TK, was avirulent for mice, swine, and sheep, but was mildly virulent for calves (mortality, 1 of 12) and more virulent for dogs (mortality, 3 of 6) and cats (mortality, 2 of 6).(ABSTRACT TRUNCATED AT 250 WORDS)

Thymidine kinase 1 as a molecular target for boron neutron capture therapy of brain tumors

PubMed Central

Barth, Rolf F.; Yang, Weilian; Wu, Gong; Swindall, Michele; Byun, Youngjoo; Narayanasamy, Sureshbabu; Tjarks, Werner; Tordoff, Kevin; Moeschberger, Melvin L.; Eriksson, Staffan; Binns, Peter J.; Riley, Kent J.

2008-01-01

The purpose of the present study was to evaluate the effectiveness of a 3-carboranyl thymidine analogue (3CTA), 3-[5-{2-(2,3-dihydroxyprop-1-yl)-o-carboran-1-yl}pentan-1-yl] thymidine, designated N5–2OH, for boron neutron capture therapy (BNCT) of brain tumors using the RG2 rat glioma model. Target validation was established using the thymidine kinase (TK) 1(+) wild-type, murine L929 cell line and its TK1(−) mutant counterpart, which were implanted s.c. (s.c.) into nude mice. Two intratumoral (i.t.) injections of 10B-enriched N5–2OH were administered to tumor-bearing mice at 2-hour intervals, after which BNCT was carried out at the Massachusetts Institute of Technology (MIT) Research Reactor. Thirty days after BNCT, mice bearing TK1(+) L929 tumors had a 15× reduction in tumor volume compared with TK1(−) controls. Based on these favorable results, BNCT studies were then initiated in rats bearing intracerebral (i.c.) RG2 gliomas, after i.c. administration of N5–2OH by Alzet osmotic pumps, either alone or in combination with i.v. (i.v.) boronophenylalanine (BPA), a drug that has been used clinically. The mean survival times (MSTs) of RG2 glioma bearing rats were 45.6 ± 7.2 days, 35.0 ± 3.3days, and 52.9 ± 8.9 days, respectively, for animals that received N5–2OH, BPA, or both. The differences between the survival plots of rats that received N5–2OH and BPA alone were highly significant (P = 0.0003). These data provide proof-of-principle that a 3CTA can function as a boron delivery agent for NCT. Further studies are planned to design and synthesize 3CTAs with enhanced chemical and biological properties, and increased therapeutic efficacy. PMID:18981415

Establishment and characterization of a human gastrointestinal stromal tumour (GIST) xenograft in athymic nude mice.

PubMed

Revheim, Mona-Elisabeth; Seierstad, Therese; Berner, Jeanne-Marie; Bruland, Oyvind Sverre; Røe, Kathrine; Ohnstad, Hege Oma; Bjerkehagen, Bodil; Bach-Gansmo, Tore

2009-11-01

The majority of gastrointestinal stromal tumours (GISTs) contain oncogenic KIT (v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog) or platelet-derived growth factor-alpha (PDGFRA) receptor tyrosine kinase (TK) mutations and are initially, but only temporarily sensitive to TK inhibitors. The aim of this study was to establish and characterize a human GIST xenograft that could be used for evaluating various molecularly targeted therapies. GIST tissue from four patients was implanted under the skin of athymic nude mice. In one case a tumour line was established. The xenograft showed characteristic GIST morphology and exhibited the same mutation profile as that of the patient. A human GIST xenograft with mutation in KIT exons 11 and 17 has been established and maintained in nude mice for 3 years (13 passages). This model will enable further studies on mechanisms of resistance, combination therapies and allow testing of novel targeted therapies.

Alpharetroviral self-inactivating vectors produced by a superinfection-resistant stable packaging cell line allow genetic modification of primary human T lymphocytes.

PubMed

Labenski, Verena; Suerth, Julia D; Barczak, Elke; Heckl, Dirk; Levy, Camille; Bernadin, Ornellie; Charpentier, Emmanuelle; Williams, David A; Fehse, Boris; Verhoeyen, Els; Schambach, Axel

2016-08-01

Primary human T lymphocytes represent an important cell population for adoptive immunotherapies, including chimeric-antigen and T-cell receptor applications, as they have the capability to eliminate non-self, virus-infected and tumor cells. Given the increasing numbers of clinical immunotherapy applications, the development of an optimal vector platform for genetic T lymphocyte engineering, which allows cost-effective high-quality vector productions, remains a critical goal. Alpharetroviral self-inactivating vectors (ARV) have several advantages compared to other vector platforms, including a more random genomic integration pattern and reduced likelihood for inducing aberrant splicing of integrated proviruses. We developed an ARV platform for the transduction of primary human T lymphocytes. We demonstrated functional transgene transfer using the clinically relevant herpes-simplex-virus thymidine kinase variant TK.007. Proof-of-concept of alpharetroviral-mediated T-lymphocyte engineering was shown in vitro and in a humanized transplantation model in vivo. Furthermore, we established a stable, human alpharetroviral packaging cell line in which we deleted the entry receptor (SLC1A5) for RD114/TR-pseudotyped ARVs to prevent superinfection and enhance genomic integrity of the packaging cell line and viral particles. We showed that superinfection can be entirely prevented, while maintaining high recombinant virus titers. Taken together, this resulted in an improved production platform representing an economic strategy for translating the promising features of ARVs for therapeutic T-lymphocyte engineering. Copyright © 2016 Elsevier Ltd. All rights reserved.

Phagocytosis of microparticles by alveolar macrophages during acute lung injury requires MerTK.

PubMed

Mohning, Michael P; Thomas, Stacey M; Barthel, Lea; Mould, Kara J; McCubbrey, Alexandria L; Frasch, S Courtney; Bratton, Donna L; Henson, Peter M; Janssen, William J

2018-01-01

Microparticles are a newly recognized class of mediators in the pathophysiology of lung inflammation and injury, but little is known about the factors that regulate their accumulation and clearance. The primary objective of our study was to determine whether alveolar macrophages engulf microparticles and to elucidate the mechanisms by which this occurs. Alveolar microparticles were quantified in bronchoalveolar fluid of mice with lung injury induced by LPS and hydrochloric acid. Microparticle numbers were greatest at the peak of inflammation and declined as inflammation resolved. Isolated, fluorescently labeled particles were placed in culture with macrophages to evaluate ingestion in the presence of endocytosis inhibitors. Ingestion was blocked with cytochalasin D and wortmannin, consistent with a phagocytic process. In separate experiments, mice were treated intratracheally with labeled microparticles, and their uptake was assessed though microscopy and flow cytometry. Resident alveolar macrophages, not recruited macrophages, were the primary cell-ingesting microparticles in the alveolus during lung injury. In vitro, microparticles promoted inflammatory signaling in LPS primed epithelial cells, signifying the importance of microparticle clearance in resolving lung injury. Microparticles were found to have phosphatidylserine exposed on their surfaces. Accordingly, we measured expression of phosphatidylserine receptors on macrophages and found high expression of MerTK and Axl in the resident macrophage population. Endocytosis of microparticles was markedly reduced in MerTK-deficient macrophages in vitro and in vivo. In conclusion, microparticles are released during acute lung injury and peak in number at the height of inflammation. Resident alveolar macrophages efficiently clear these microparticles through MerTK-mediated phagocytosis.

Immunohistochemical localization of cardio-active neuropeptides in the heart of a living fossil, Nautilus pompilius L. (Cephalopoda, Tetrabranchiata).

PubMed

Springer, J; Ruth, P; Beuerlein, K; Westermann, B; Schipp, R

2004-01-01

Neuropeptides play an important role in modulating the effects of neurotransmitters such as acetylcholine and noradrenaline in the heart and the vascular system of vertebrates and invertebrates. Various neuropeptides, including substance P (SP), vasoactive intestinal polypeptide (VIP) and FMRFamide, have been localized in the brain in cephalopods and the neurosecretory system of the vena cava. Previous studies involving cephalopods have mainly focussed on the modern, coleoid cephalopods, whereas little attention was paid to the living fossil Nautilus. In this study, the distributions of the peptides related to tachykinins (TKs) and the high affinity receptor for the best characterized TK substance P (tachykinin NK-1), VIP, as well as FMRFamide were investigated in the heart of Nautilus pompilius L. by immunohistochemistry. TK-like immunoreactivity (TK-LI) was seen associated to a sub-population of hemocytes, VIP-LI glial cells in larger nerves entering the heart, whereas FMRFamide immunoreactivity was distributed throughout the entire heart, including the semilunar atrioventricular valves. The pattern of FMRFamide immunoreactivity matched that of Bodian silver staining for nervous tissue. The NK-1-LI receptor was located on endothelial cells, which were also positive for endothelial nitric oxide synthase-LI (eNOS). The results indicate that neuropeptides may be involved in the regulation of the Nautilus heart via different mechanisms, (1) by direct interaction with myocardial receptors (FMRFamide), (2) by interacting with the nervus cardiacus (VIP-related peptides) and (3) indirectly by stimulating eNOS in the endothelium throughout the heart (TK-related peptides).

Effects of Modeled Microgravity on Expression Profiles of Micro RNA in Human Lymphoblastoid Cells

NASA Technical Reports Server (NTRS)

Mangala, Lingegowda S.; Emami, Kamal; Story, Michael; Ramesh, Govindarajan; Rohde, Larry; Wu, Honglu

2010-01-01

Among space radiation and other environmental factors, microgravity or an altered gravity is undoubtedly the most significant stress experienced by living organisms during flight. In comparison to the static 1g, microgravity has been shown to alter global gene expression patterns and protein levels in cultured cells or animals. Micro RNA (miRNA) has recently emerged as an important regulator of gene expression, possibly regulating as many as one-third of all human genes. miRNA represents a class of single-stranded noncoding regulatory RNA molecules ( 22 nt) that control gene expressions by inhibiting the translation of mRNA to proteins. However, very little is known on the effect of altered gravity on miRNA expression. We hypothesized that the miRNA expression profile will be altered in zero gravity resulting in regulation of the gene expression and functional changes of the cells. To test this hypothesis, we cultured TK6 human lymphoblastoid cells in Synthecon s Rotary cell culture system (bioreactors) for 72 h either in the rotating (10 rpm) to model the microgravity in space or in the static condition. The cell viability was determined before and after culturing the cells in the bioreactor using both trypan blue and guava via count. Expressions of a panel of 352 human miRNA were analyzed using the miRNA PCRarray. Out of 352 miRNAs, expressions of 75 were significantly altered by a change of greater than 1.5 folds and seven miRNAs were altered by a fold change greater than 2 under the rotating culture condition. Among these seven, miR-545 and miR-517a were down regulated by 2 folds, whereas miR-150, miR-302a, miR-139-3p, miR-515-3p and miR-564 were up regulated by 2 to 8 folds. To confirm whether this altered miRNA expression correlates with gene expression and functional changes of the cells, we performed DNA Illumina Microarray Analysis and validated the related genes using q-RT PCR.

Molecular cloning, subcelluar location and expression profile of signal transducer and activator of transcription 2 (STAT2) from turbot, Scophthalmus maximus.

PubMed

Wang, Na; Wang, Xian-Li; Yang, Chang-Geng; Chen, Song-Lin

2013-10-01

Signal transducer and activator of transcription 2 (STAT2) is an important molecule involved in the type I interferon signalling pathway. To date, little STAT2 homologue is available in fish except Atlantic salmon and goldfish. In this paper, STAT2 was firstly cloned and characterized from turbot, a marine flatfish with high economic value. Briefly, turbot STAT2 cDNA is 3206 bp in length encoding a predicted protein of 793 amino acids. The phylogenetic tree shows that turbot STAT2 protein shared the closest relationship with Atlantic salmon. Analysis of subcellular distribution indicates that STAT2 is mainly present in the cytoplasm of TK cells. Stat2 mRNA is constitutively expressed in widespread tissues and induced by several folds in turbot tissues and TK cells after stimulation with Vibrio anguillarum and lymphocystis disease virus (LCDV). Unlike the higher vertebrate STAT2, turbot STAT2 nuclear export signal (NES) exists not in the C-terminal 79 amino acids but in N-terminal 137-312 amino acids (STAT_alpha domain). The nuclear translocation of turbot STAT2 after Poly(I:C) treatment proved its transcription activity in TK cells. All these results suggested that STAT2 may be involved in the immune response in turbot as a transcription factor. Copyright © 2013 Elsevier Ltd. All rights reserved.

ZRBA1, a Mixed EGFR/DNA Targeting Molecule, Potentiates Radiation Response Through Delayed DNA Damage Repair Process in a Triple Negative Breast Cancer Model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heravi, Mitra; Department of Radiation Oncology, McGill University, Montreal; Segal Cancer Center, Jewish General Hospital, Montreal

2015-06-01

Purpose: ZRBA1 is a combi-molecule designed to induce DNA alkylating lesions and to block epidermal growth factor receptor (EGFR) TK domain. Inasmuch as ZRBA1 downregulates the EGFR TK-mediated antisurvival signaling and induces DNA damage, we postulated that it might be a radiosensitizer. The aim of this study was to further investigate the potentiating effect of ZRBA1 in combination with radiation and to elucidate the possible mechanisms of interaction between these 2 treatment modalities. Methods and Materials: The triple negative human breast MDA-MB-468 cancer cell line and mouse mammary cancer 4T1 cell line were used in this study. Clonogenic assay, Westernmore » blot analysis, and DNA damage analysis were performed at multiple time points after treatment. To confirm our in vitro findings, in vivo tumor growth delay assay was performed. Results: Our results show that a combination of ZRBA1 and radiation increases the radiation sensitivity of both cell lines significantly with a dose enhancement factor of 1.56, induces significant numbers of DNA strand breaks, prolongs higher DNA damage up to 24 hours after treatment, and significantly increases tumor growth delay in a syngeneic mouse model. Conclusions: Our data suggest that the higher efficacy of this combination could be partially due to increased DNA damage and delayed DNA repair process and to the inhibition of EGFR. The encouraging results of this combination demonstrated a significant improvement in treatment efficiency and therefore could be applicable in early clinical trial settings.« less

Effects of FSH and 17beta-estradiol on the transactivation of estrogen-regulated promoters and cell proliferation in L cells.

PubMed

Pasapera, Ana María; Jiménez-Aguilera, María del Pilar; Chauchereau, Anne; Milgrom, Edwin; Olivares, Aleida; Uribe, Aída; Gutiérrez-Sagal, Rubén; Ulloa-Aguirre, Alfredo

2005-03-01

In the present study, we analyzed human follicle-stimulating hormone (FSH)-induced cell proliferation and transactivation of estrogen-sensitive reporter genes-in L cells stably expressing the human FSH receptor [L-(hFSHR(+)) cells]. In order to dissect the signaling pathways involved in this process, L-(hFSHR(+)) cells were transiently transfected with either the 3X-ERE-TAT-Luc or the ERE-VitA2-TK-CAT reporter genes and treated with FSH or PKA activators (cholera toxin, forskolin and 8-Br-cAMP) in the presence or absence of various kinase inhibitors. We found that FSH and all PKA activators, specifically induced transactivation of both reporter genes. Transactivation of estrogen-sensitive genes by FSH or PKA activators were blocked (approximately 90%) by H89 (PKA inhibitor) and LY294002 but not by Wortmannin (PI3-K inhibitors), 4-OH-tamoxifen, ICI182,780 or SB203580 (p38 MAPK inhibitor); PD98059 (ERK1/2 inhibitor) partially (approximately 30%) blocked the FSH-mediated effect. The combination of FSH and estradiol resulted in a synergistic effect on transactivation as well as on cell proliferation, and this enhancement was attenuated by antiestrogens. We additionally analyzed the participation of the coactivators SRC-1 and cAMP response element binding protein (CREB)-binding protein (CBP) in FSH-evoked estrogen receptor (ER)-dependent transactivation; we found that CBP but not SRC-1 potentiated FSH-induced transcriptional activation of both ER-sensitive reporters, being this effect stronger on the ERE-VitA2-TK-CAT than on the 3X-ERE-TAT-Luc reporter. Thus, in L-(hFSHR(+)) cells FSH induces transcriptional activation of estrogen-sensitive genes through an A-kinase-triggered signaling pathway, using also to a lesser extent the ERK1/2 and p38 pathways. PI3-K is not apparently involved in this FSH-mediated process since LY294002, but not Wortmannin, specifically binds ERs and completely blocks estrogen action. Presumably, CBP cooperates with the ER on genes that contain estrogen responsive elements through mechanisms involving the participation of other proteins and/or basal transcription factors (e.g. CREB), which in turn mediate the transcriptional response of estrogen-sensitive reporter genes to FSH stimulation.

Tissue-Specific Expression of Herpes Simplex Virus Thymidine Kinase Gene Delivered by Adeno-Associated Virus Inhibits the Growth of Human Hepatocellular Carcinoma in Athymic Mice

NASA Astrophysics Data System (ADS)

Su, Hua; Lu, Ronghua; Chang, Judy C.; Kan, Yuet Wai

1997-12-01

About 70% of hepatocellular carcinomas are known to express α -fetoprotein, which is normally expressed in fetal but not in adult livers. To induce herpes simplex virus-thymidine kinase expression in these cancer cells, we constructed an adeno-associated viral vector containing the HSV-TK gene under the control of the α -fetoprotein enhancer and albumin promoter. We previously demonstrated in vitro that although this vector can transduce a variety of human cells, only transduced AFP and albumin-expressing hepatocellular carcinoma cell lines were sensitive to killing by ganciclovir (GCV). In the present study, we explored the effect of this vector on hepatocellular carcinoma cells in vivo. Subcutaneous tumors generated in nude mice by implanting hepatocellular carcinoma cells previously transduced with this vector shrank dramatically after treatment with GCV. Bystander effect was also observed on the tumors generated by mixing transduced and untransduced cells. To test whether the tumor cells can be transduced by the virus in vivo, we injected the recombinant adeno-associated virus into tumors generated by untransduced hepatocarcinoma cell line. Tumor growth were retarded after treatment with GCV. These experiments demonstrate the feasibility of in vivo transduction of tumor cell with rAAV.

Synthesizing a Trefoil Knotted Block Copolymer via Ring-Expansion Strategy

DOE PAGES

Cao, Pengfei; Rong, Li-Han; Mangadlao, Joey; ...

2017-02-07

We synthesized a synthetic trefoil knotted poly(e-caprolatone) block-poly(L-lactide) (TK-PLA-b-PCL) via a ring expansion strategy from a trefoil knotted tin (Sn) initiator. Ring closing reaction between the bis-copper(I) templated phenanthro line complex and dibutyldimethoxytin results in a templated trefoil knotted initiator. Furthermore, the bis-copper(I) templated trefoil knotted poly(L-lactide) (TK-PLA) can be synthesized by ring-opening polymerization of L-lactide monomer, and decomplexation reaction of the templated TK-PLA will result in a geniune TK-PLA without constraint from the copper template. Subsequent insertion of e caprolactone in the bis-copper(I) templated TK-PLA forms the templated trefoil knotted block copolymer, i.e., TK-PLA-b-PCL, and the copper-free TK-PLA-b-PCL canmore » be obtained by decomplexation reaction. Finally, both TK-PLA and TK-PLA-b-PCL are analyzed by the 1 H NMR, FT-IR, UV-vis, DLS, and GPC.« less

Somatic cell hybrid mapping on mouse chromosome 11 (MMU11): Assignment of markers relative to two breakpoints in band D

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morris, D.J.; Robinson, T.J.; Adler, I.D.

1993-02-01

Mouse [times] rat somatic cell hybrids were generated by fusing mouse cell lines that are heterozygous for reciprocal translocations involving the T42H and T9Ad breakpoints on mouse chromosome 11 (MMU11) to a thymidine kinase-negative (Tk[sup [minus

Hypoglycemic effects of Trichosanthes kirilowii and its protein constituent in diabetic mice: the involvement of insulin receptor pathway.

PubMed

Lo, Hsin-Yi; Li, Tsai-Chung; Yang, Tse-Yen; Li, Chia-Cheng; Chiang, Jen-Huai; Hsiang, Chien-Yun; Ho, Tin-Yun

2017-01-18

Diabetes is a serious chronic metabolic disorder. Trichosanthes kirilowii Maxim. (TK) is traditionally used for the treatment of diabetes in traditional Chinese medicine (TCM). However, the clinical application of TK on diabetic patients and the hypoglycemic efficacies of TK are still unclear. A retrospective cohort study was conducted to analyze the usage of Chinese herbs in patients with type 2 diabetes in Taiwan. Glucose tolerance test was performed to analyze the hypoglycemic effect of TK. Proteomic approach was performed to identify the protein constituents of TK. Insulin receptor (IR) kinase activity assay and glucose tolerance tests in diabetic mice were further used to elucidate the hypoglycemic mechanisms and efficacies of TK. By a retrospective cohort study, we found that TK was the most frequently used Chinese medicinal herb in type 2 diabetic patients in Taiwan. Oral administration of aqueous extract of TK displayed hypoglycemic effects in a dose-dependent manner in mice. An abundant novel TK protein (TKP) was further identified by proteomic approach. TKP interacted with IR by docking analysis and activated the kinase activity of IR. In addition, TKP enhanced the clearance of glucose in diabetic mice in a dose-dependent manner. In conclusion, this study applied a bed-to-bench approach to elucidate the hypoglycemic efficacies and mechanisms of TK on clinical usage. In addition, we newly identified a hypoglycemic protein TKP from TK. Our findings might provide a reasonable explanation of TK on the treatment of diabetes in TCM.

Characterization of BRCA2 Transcriptional Regulation

DTIC Science & Technology

2001-12-01

tig of BRCA2 promoter construct and 0.1 and we verify the role of USF in regulation of basal activity of jig of pRL-TK Renilla luciferase vector...Promega) with 4 1 l of Fugene-6 the promoter. was used for each transfection. The pRL-TK Renilla luciferase activity was used to control for transfection...pCMV-CREB, pCMV-Myc, BRCA2 Reporter Constructs-A BAC clone (B489G) containing the 5’ and pCMV-Max. Firefly luciferase and Renilla luciferase assays

Inter-Individual Variability in High-Throughput Risk ...

EPA Pesticide Factsheets

We incorporate realistic human variability into an open-source high-throughput (HT) toxicokinetics (TK) modeling framework for use in a next-generation risk prioritization approach. Risk prioritization involves rapid triage of thousands of environmental chemicals, most which have little or no existing TK data. Chemicals are prioritized based on model estimates of hazard and exposure, to decide which chemicals should be first in line for further study. Hazard may be estimated with in vitro HT screening assays, e.g., U.S. EPA’s ToxCast program. Bioactive ToxCast concentrations can be extrapolated to doses that produce equivalent concentrations in body tissues using a reverse TK approach in which generic TK models are parameterized with 1) chemical-specific parameters derived from in vitro measurements and predicted from chemical structure; and 2) with physiological parameters for a virtual population. Here we draw physiological parameters from realistic estimates of distributions of demographic and anthropometric quantities in the modern U.S. population, based on the most recent CDC NHANES data. A Monte Carlo approach, accounting for the correlation structure in physiological parameters, is used to estimate ToxCast equivalent doses for the most sensitive portion of the population. To quantify risk, ToxCast equivalent doses are compared to estimates of exposure rates based on Bayesian inferences drawn from NHANES urinary analyte biomonitoring data. The inclusion

Effects of Simulated Microgravity on the Expression Profile of Microrna in Human Lymphoblastoid Cells

NASA Astrophysics Data System (ADS)

Zhang, Ye; Wu, Honglu; Ramesh, Govindarajan; Rohde, Larry; Story, Michael; Mangala, Lingegowda

2012-07-01

EFFECTS OF SIMULATED MICROGRAVITY ON THE EXPRESSION PROFILE OF MICRORNA IN HUMAN LYMPHOBLASTOID CELLS Lingegowda S. Mangala1,2, Ye Zhang1,3, Zhenhua He2, Kamal Emami1, Govindarajan T. Ramesh4, Michael Story 5, Larry H. Rohde2, and Honglu Wu1 1 NASA Johnson Space Center, Houston, Texas, USA 2 University of Houston Clear Lake, Houston, Texas, USA 3 Wyle Integrated Science and Engineering Group, Houston, Texas, USA 4 Norfolk State University, Norfolk, VA, USA 5 University of Texas, Southwestern Medical Center, Dallas, Texas, USA This study explores the changes in expression of microRNA (miRNA) and related genes under simulated microgravity conditions. In comparison to static 1g, microgravity has been shown to alter global gene expression patterns and protein levels in cultured cells or animals. miRNA has recently emerged as an important regulator of gene expression, possibly regulating as many as one-third of all human genes. However, very little is known about the effect of altered gravity on miRNA expression. To test the hypothesis that the miRNA expression profile would be altered in zero gravity resulting in altered regulation of gene expression leading to metabolic or functional changes in cells, we cultured TK6 human lymphoblastoid cells in a High Aspect Ratio Vessel (HARV; bioreactor) for 72 h either in the rotating condition to model microgravity in space or in the static condition as a control. Expression of several miRNA was changed significantly in the simulated microgravity condition including miR-150, miR-34a, miR-423-5p, miR-22 and miR-141, miR-618 and miR-222. To confirm whether this altered miRNA expression correlates with gene expression and functional changes of the cells, we performed DNA microarray and validated the related genes using q-RT PCR. Network and pathway analysis of gene and miRNA expression profiles indicates that the regulation of cell communication and catalytic activities, as well as pathways involved in immune response_IL-15 signaling and NGF mediated NF-kB activation were significantly altered under the simulated microgravity condition.

Cloning of the Koi Herpesvirus Genome as an Infectious Bacterial Artificial Chromosome Demonstrates That Disruption of the Thymidine Kinase Locus Induces Partial Attenuation in Cyprinus carpio koi▿

PubMed Central

Costes, B.; Fournier, G.; Michel, B.; Delforge, C.; Raj, V. Stalin; Dewals, B.; Gillet, L.; Drion, P.; Body, A.; Schynts, F.; Lieffrig, F.; Vanderplasschen, A.

2008-01-01

Koi herpesvirus (KHV) is the causative agent of a lethal disease in koi and common carp. In the present study, we describe the cloning of the KHV genome as a stable and infectious bacterial artificial chromosome (BAC) clone that can be used to produce KHV recombinant strains. This goal was achieved by the insertion of a loxP-flanked BAC cassette into the thymidine kinase (TK) locus. This insertion led to a BAC plasmid that was stably maintained in bacteria and was able to regenerate virions when permissive cells were transfected with the plasmid. Reconstituted virions free of the BAC cassette but carrying a disrupted TK locus (the FL BAC-excised strain) were produced by the transfection of Cre recombinase-expressing cells with the BAC. Similarly, virions with a wild-type revertant TK sequence (the FL BAC revertant strain) were produced by the cotransfection of cells with the BAC and a DNA fragment encoding the wild-type TK sequence. Reconstituted recombinant viruses were compared to the wild-type parental virus in vitro and in vivo. The FL BAC revertant strain and the FL BAC-excised strain replicated comparably to the parental FL strain. The FL BAC revertant strain induced KHV infection in koi carp that was indistinguishable from that induced by the parental strain, while the FL BAC-excised strain exhibited a partially attenuated phenotype. Finally, the usefulness of the KHV BAC for recombination studies was demonstrated by the production of an ORF16-deleted strain by using prokaryotic recombination technology. The availability of the KHV BAC is an important advance that will allow the study of viral genes involved in KHV pathogenesis, as well as the production of attenuated recombinant candidate vaccines. PMID:18337580

Lemongrass essential oil and citral inhibit Src/Stat3 activity and suppress the proliferation/survival of small-cell lung cancer cells, alone or in combination with chemotherapeutic agents.

PubMed

Maruoka, Takayuki; Kitanaka, Akira; Kubota, Yoshitsugu; Yamaoka, Genji; Kameda, Tomohiro; Imataki, Osamu; Dobashi, Hiroaki; Bandoh, Shuji; Kadowaki, Norimitsu; Tanaka, Terukazu

2018-03-13

Small-cell lung cancer (SCLC) is intractable due to its high propensity for relapse. Novel agents are thus needed for SCLC treatment. Lemongrass essential oil (LG-EO) and its major constituent, citral, have been reported to inhibit the proliferation and survival of several types of cancer cells. However, the precise mechanisms through which LG-EO and citral exert their effects on SCLC cells have not been fully elucidated. SCLC cells express Src and have high levels of Src-tyrosine kinase (Src-TK) activity. In most SCLC cell lines, constitutive phosphorylation of Stat3(Y705), which is essential for its activation, has been detected. Src-TK can phosphorylate Stat3(Y705), and activated Stat3 promotes the expression of the anti-apoptotic factors Bcl-xL and Mcl-1. In the present study, LG-EO and citral prevented Src-TK from phosphorylating Stat3(Y705), resulting in decreased Bcl-xL and Mcl-1 expression, in turn suppressing the proliferation/survival of SCLC cells. To confirm these findings, the wild-type-src gene was transfected into the LU135 SCLC cell line (LU135‑wt-src), in which Src and activated phospho-Stat3(Y705) were overexpressed. The suppression of cell proliferation and the induction of apoptosis by treatment with LG-EO or citral were significantly attenuated in the LU135-wt-src cells compared with the control LU135-mock cells. The signal transducer and activator of transcription 3 (Stat3) signaling pathway is also associated with intrinsic drug resistance. LU135-wt-src cells were significantly resistant to conventional chemotherapeutic agents compared with LU135-mock cells. The combined effects of citral and each conventional chemotherapeutic agent on SCLC cells were also evaluated. The combination treatment exerted additive or more prominent effects on LU135-wt-src, LU165 and MN1112 cells, which are relatively chemoresistant SCLC cells. These findings suggest that either LG-EO or citral, alone or in combination with chemotherapeutic agents, may be a novel therapeutic option for SCLC patients.

Accumulation of Herpes Simplex Virus Type 1 Early and Leaky-Late Proteins Correlates with Apoptosis Prevention in Infected Human HEp-2 Cells

PubMed Central

Aubert, Martine; Rice, Stephen A.; Blaho, John A.

2001-01-01

We previously reported that a recombinant ICP27-null virus stimulated, but did not prevent, apoptosis in human HEp-2 cells during infection (M. Aubert and J. A. Blaho, J. Virol. 73:2803–2813, 1999). In the present study, we used a panel of 15 recombinant ICP27 mutant viruses to determine which features of herpes simplex virus type 1 (HSV-1) replication are required for the apoptosis-inhibitory activity. Each virus was defined experimentally as either apoptotic, partially apoptotic, or nonapoptotic based on infected HEp-2 cell morphologies, percentages of infected cells with condensed chromatin, and patterns of specific cellular death factor processing. Viruses d27-1, d1-5, d1-2, M11, M15, M16, n504R, n406R, n263R, and n59R are apoptotic or partially apoptotic in HEp-2 cells and severely defective for growth in Vero cells. Viruses d2-3, d3-4, d4-5, d5-6, and d6-7 are nonapoptotic, demonstrating that ICP27 contains a large amino-terminal region, including its RGG box RNA binding domain, which is not essential for apoptosis prevention. Accumulations of viral TK, VP16, and gD but not gC, ICP22, or ICP4 proteins correlated with prevention of apoptosis during the replication of these viruses. Of the nonapoptotic viruses, d4-5 did not produce gC, indicating that accumulation of true late gene products is not necessary for the prevention process. Analyses of viral DNA synthesis in HEp-2 cells indicated that apoptosis prevention by HSV-1 requires that the infection proceeds to the stage in which viral DNA replication takes place. Infections performed in the presence of the drug phosphonoacetic acid confirmed that the process of viral DNA synthesis and the accumulation of true late (γ2) proteins are not required for apoptosis prevention. Based on our results, we conclude that the accumulation of HSV-1 early (β) and leaky-late (γ1) proteins correlates with the prevention of apoptosis in infected HEp-2 cells. PMID:11134315

In vivo image analysis of BoHV-4-based vector in mice.

PubMed

Franceschi, Valentina; Stellari, Fabio Franco; Mangia, Carlo; Jacca, Sarah; Lavrentiadou, Sophia; Cavirani, Sandro; Heikenwalder, Mathias; Donofrio, Gaetano

2014-01-01

Due to its biological characteristics bovine herpesvirus 4 (BoHV-4) has been considered as an appropriate gene delivery vector. Its genomic clone, modified as a bacterial artificial chromosome (BAC), is better genetically manipulable and can be used as an efficient gene delivery and vaccine vector. Although a large amount of data have been accumulated in vitro on this specific aspect, the same cannot be asserted for the in vivo condition. Therefore, here we investigated the fate of a recombinant BoHV-4 strain expressing luciferase (BoHV-4-A-CMVlucΔTK) after intraperitoneal or intravenous inoculation in mice, by generating a novel recombinant BoHV-4 expressing luciferase (BoHV-4-A-CMVlucΔTK) and by following the virus replication through in vivo imaging analysis. BoHV-4-A-CMVlucΔTK was first characterized in vitro where it was shown, on one hand that its replication properties are identical to those of the parental virus, and on the other that the transduced/infected cells strongly express luciferase. When BoHV-4-A-CMVlucΔTK was inoculated in mice, either intraperitoneally or intravenously, BoHV-4-A-CMVlucΔTK infection/transduction was exclusively localized to the liver, as detected by in vivo image analysis, and in particular almost exclusively in the hepatocytes, as determined by immuno-histochemistry. These data, that add a new insight on the biology of BoHV-4 in vivo, provide the first indication for the potential use of a BoHV-4-based vector in gene-transfer in the liver.

Synthesis and evaluation of thymidine kinase 1-targeting carboranyl pyrimidine nucleoside analogs for boron neutron capture therapy of cancer.

PubMed

Agarwal, Hitesh K; Khalil, Ahmed; Ishita, Keisuke; Yang, Weilian; Nakkula, Robin J; Wu, Lai-Chu; Ali, Tehane; Tiwari, Rohit; Byun, Youngjoo; Barth, Rolf F; Tjarks, Werner

2015-07-15

A library of sixteen 2nd generation amino- and amido-substituted carboranyl pyrimidine nucleoside analogs, designed as substrates and inhibitors of thymidine kinase 1 (TK1) for potential use in boron neutron capture therapy (BNCT) of cancer, was synthesized and evaluated in enzyme kinetic-, enzyme inhibition-, metabolomic-, and biodistribution studies. One of these 2nd generation carboranyl pyrimidine nucleoside analogs (YB18A [3]), having an amino group directly attached to a meta-carborane cage tethered via ethylene spacer to the 3-position of thymidine, was approximately 3-4 times superior as a substrate and inhibitor of hTK1 than N5-2OH (2), a 1st generation carboranyl pyrimidine nucleoside analog. Both 2 and 3 appeared to be 5'-monophosphorylated in TK1(+) RG2 cells, both in vitro and in vivo. Biodistribution studies in rats bearing intracerebral RG2 glioma resulted in selective tumor uptake of 3 with an intratumoral concentration that was approximately 4 times higher than that of 2. The obtained results significantly advance the understanding of the binding interactions between TK1 and carboranyl pyrimidine nucleoside analogs and will profoundly impact future design strategies for these agents. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Structure of a [NiFe] hydrogenase maturation protease HycI provides insights into its substrate selectivity.

PubMed

Kwon, Sunghark; Nishitani, Yuichi; Hirao, Yoshinori; Kanai, Tamotsu; Atomi, Haruyuki; Miki, Kunio

2018-04-15

The immature large subunit of [NiFe] hydrogenases undergoes C-terminal cleavage by a specific protease in the final step of the post-translational process before assembly with other subunits. It has been reported that the [NiFe] hydrogenase maturation protease HycI from Thermococcus kodakarensis (TkHycI) has the catalytic ability to target the membrane-bound hydrogenase large subunit MbhL from T. kodakarensis. However, the detailed mechanism of its substrate recognition remains elusive. We determined the crystal structure of TkHycI at 1.59 Å resolution to clarify how TkHycI recognizes its own substrate MbhL. Although the overall structure of TkHycI is similar to that of its homologous protease TkHybD, TkHycI adopts a larger loop than TkHybD, thereby creating a broad and deep cleft. We analyzed the structural properties of the TkHycI cleft probably involved in its substrate recognition. Our findings provide novel and profound insights into the substrate selectivity of TkHycI. Copyright © 2018 Elsevier Inc. All rights reserved.

Does repeated and heavy exercise impair blood rheology in carriers of sickle cell trait?

PubMed

Tripette, Julien; Hardy-Dessources, Marie-Dominique; Sara, Fagnété; Montout-Hedreville, Mona; Saint-Martin, Christian; Hue, Olivier; Connes, Philippe

2007-11-01

To determine if the time courses of hemorheologic parameters are different between carriers of sickle cell trait (SCT) and subjects with normal hemoglobin in response to exercise. Observational and comparative study. Testing was conducted in a laboratory of exercise physiology. Nine carriers of sickle cell trait (SCT group) and 7 subjects with normal hemoglobin (CONT group) performed an exercise protocol of the repetition of 3 successive maximal ramp exercise tests. Blood was sampled at rest (TR), at the end of each of the 3 tests (T1, T2, T3), and during the immediate (T2h) and late (T24h, T48h) recovery periods. Blood and plasma viscosity (etab and etap, respectively), hematocrit (Hct), and red blood cell (RBC) rigidity (Tk and k indexes) were determined. In both groups, etab significantly increased in response to exercise but the SCT group had significantly higher etab at T3 and T2h. etab then returned to baseline value at T2h in the CONT group and at T24h in the SCT group. Tk and k were not changed by exercise but significantly increased above baseline value in both groups at T24h and T48h. The increase in Tk and k during late recovery was higher in the SCT group than in the CONT group, indicating that SCT carriers had significantly higher RBC rigidity than the CONT group at that time. The hemorheologic changes induced by exercise in the SCT carriers could trigger microcirculatory disorders during the recovery.

Deoxycytidine and deoxythymidine treatment for thymidine kinase 2 deficiency

PubMed Central

Lopez-Gomez, Carlos; Levy, Rebecca J; Sanchez-Quintero, Maria J; Juanola-Falgarona, Marti; Barca, Emanuele; Garcia-Diaz, Beatriz; Tadesse, Saba; Garone, Caterina; Hirano, Michio

2017-01-01

Objective Thymidine kinase 2 (TK2), a critical enzyme in the mitochondrial pyrimidine salvage pathway, is essential for mitochondrial DNA (mtDNA) maintenance. Mutations in the nuclear gene TK2 cause TK2 deficiency, which manifests predominantly in children as myopathy with mtDNA depletion. Molecular bypass therapy with the TK2 products, dCMP and dTMP, prolongs the lifespan of Tk2-deficient (Tk2-/-) mice by 2-3 fold. Because we observed rapid catabolism of the deoxynucleoside monophosphates to deoxythymidine (dT) and deoxycytidine (dC), we hypothesized that: 1) deoxynucleosides might be the major active agents and 2) inhibition of deoxycytidine deamination might enhance dTMP+dCMP therapy. Methods To test these hypotheses, we assessed two therapies in Tk2-/- mice: 1) dT+dC and 2) co-administration of the deaminase inhibitor, tetrahydrouridine (THU), with dTMP+dCMP. Results We observed that dC+dT delayed disease onset, prolonged lifespan of Tk2-deficient mice, and restored mtDNA copy number as well as respiratory chain enzyme activities and levels. In contrast, dCMP+dTMP+THU therapy decreased lifespan of Tk2-/- animals compared to dCMP+dTMP. Interpretation Our studies demonstrate that deoxynucleoside substrate enhancement is a novel therapy, which may ameliorate TK2 deficiency in patients. PMID:28318037

Genetically Engineered Theranostic Mesenchymal Stem Cells for the Evaluation of the Anticancer Efficacy of Enzyme/Prodrug Systems

PubMed Central

Nouri, Faranak Salman; Wang, Xing; Hatefi, Arash

2015-01-01

Over the past decade, various enzyme/prodrug systems such as thymidine kinase/ganciclovir (TK/GCV), yeast cytosine deaminase/5-fluorocytosine (yCD/5-FC) and nitroreductase/CB1954 (NTR/CB1954) have been used for stem cell mediated suicide gene therapy of cancer. Yet, no study has been conducted to compare and demonstrate the advantages and disadvantages of using one system over another. Knowing that each enzyme/prodrug system has its own strengths and weaknesses, we utilized mesenchymal stem cells (MSCs) as a medium to perform for the first time a comparative study that illustrated the impact of subtle differences among these systems on the therapeutic outcome. For therapeutic purposes, we first genetically modified MSCs to stably express a panel of four suicide genes including TK (TK007 and TKSR39 mutants), yeast cytosine deaminase: uracil phosphoribosyltransferase (yCD:UPRT) and nitroreductase (NTR). Then, we evaluated the anticancer efficacies of the genetically engineered MSCs in vitro and in vivo by using SKOV3 cell line which is sensitive to all four enzyme/prodrug systems. In addition, all MSCs were engineered to stably express luciferase gene making them suitable for quantitative imaging and dose-response relationship studies in animals. Considering the limitations imposed by the prodrugs’ bystander effects, our findings show that yCD:UPRT/5-FC is the most effective enzyme/prodrug system among the ones tested. Our findings also demonstrate that theranostic MSCs are a reliable medium for the side-by-side evaluation and screening of the enzyme/prodrug systems at the preclinical level. The results of this study could help scientists who utilize cell-based, non-viral or viral vectors for suicide gene therapy of cancer make more informed decisions when choosing enzyme/prodrug systems. PMID:25575867

Correlation analysis between change in thoracic kyphosis and multilevel facetectomy and screw density in main thoracic adolescent idiopathic scoliosis surgery.

PubMed

Sudo, Hideki; Abe, Yuichiro; Kokabu, Terufumi; Ito, Manabu; Abumi, Kuniyoshi; Ito, Yoichi M; Iwasaki, Norimasa

2016-09-01

Controversy exists regarding the effects of multilevel facetectomy and screw density on deformity correction, especially thoracic kyphosis (TK) restoration in adolescent idiopathic scoliosis (AIS) surgery. This study aimed to evaluate the effects of multilevel facetectomy and screw density on sagittal plane correction in patients with main thoracic (MT) AIS curve. A retrospective correlation and comparative analysis of prospectively collected, consecutive, non-randomized series of patients at a single institution was undertaken. Sixty-four consecutive patients with Lenke type 1 AIS treated with posterior correction and fusion surgery using simultaneous double-rod rotation technique were included. Patient demographics and preoperative and 2-year postoperative radiographic measurements were the outcome measures for this study. Multiple stepwise linear regression analysis was conducted between change in TK (T5-T12) and the following factors: age at surgery, Risser sign, number of facetectomy level, screw density, preoperative main thoracic curve, flexibility in main thoracic curve, coronal correction rate, preoperative TK, and preoperative lumbar lordosis. Patients were classified into two groups: TK<15° group defined by preoperative TK below the mean degree of TK for the entire cohort (<15°) and the TK≥15° group, defined by preoperative TK above the mean degree of kyphosis (≥15°). Independent sample t tests were used to compare demographic data as well as radiographic outcomes between the two groups. There were no study-specific biases related to conflicts of interest. The average preoperative TK was 14.0°, which improved significantly to 23.1° (p<.0001) at the 2-year final follow-up. Greater change in TK was predicted by a low preoperative TK (p<.0001). The TK <15° group showed significant correlation between change in TK and number of facetectomy level (r=0.492, p=.002). Similarly, significant correlation was found between change in TK and screw density (r=0.333, p=.047). Conversely, in the TK ≥15° group, correlation was found neither between change in TK and number of facetectomy level (r=0.047, p=.812), nor with screw density (r=0.030, p=.880). Furthermore, in patients with preoperative TK<15°, change in TK was significantly correlated with screw density at the concave side (r=0.351, p=.036) but not at the convex side (r=0.144, p=.402). In patients with hypokyphotic thoracic spine, significant positive correlation was found between change in TK and multilevel facetectomy or screw density at the concave side. This indicates that in patients with AIS who have thoracic hypokyphosis as part of their deformity, the abovementioned factors must be considered in preoperative planning to correct hypokyphosis. Copyright © 2016 Elsevier Inc. All rights reserved.

Trifluorothymidine resistance is associated with decreased thymidine kinase and equilibrative nucleoside transporter expression or increased secretory phospholipase A2.

PubMed

Temmink, Olaf H; Bijnsdorp, Irene V; Prins, Henk-Jan; Losekoot, Nienke; Adema, Auke D; Smid, Kees; Honeywell, Richard J; Ylstra, Bauke; Eijk, Paul P; Fukushima, Masakazu; Peters, Godefridus J

2010-04-01

Trifluorothymidine (TFT) is part of the novel oral formulation TAS-102, which is currently evaluated in phase II studies. Drug resistance is an important limitation of cancer therapy. The aim of the present study was to induce resistance to TFT in H630 colon cancer cells using two different schedules and to analyze the resistance mechanism. Cells were exposed either continuously or intermittently to TFT, resulting in H630-cTFT and H630-4TFT, respectively. Cells were analyzed for cross-resistance, cell cycle, protein expression, and activity of thymidine phosphorylase (TP), thymidine kinase (TK), thymidylate synthase (TS), equilibrative nucleoside transporter (hENT), gene expression (microarray), and genomic alterations. Both cell lines were cross-resistant to 2'-deoxy-5-fluorouridine (>170-fold). Exposure to IC(75)-TFT increased the S/G(2)-M phase of H630 cells, whereas in the resistant variants, no change was observed. The two main target enzymes TS and TP remained unchanged in both TFT-resistant variants. In H630-4TFT cells, TK protein expression and activity were decreased, resulting in less activated TFT and was most likely the mechanism of TFT resistance. In H630-cTFT cells, hENT mRNA expression was decreased 2- to 3-fold, resulting in a 5- to 10-fold decreased TFT-nucleotide accumulation. Surprisingly, microarray-mRNA analysis revealed a strong increase of secretory phospholipase-A2 (sPLA2; 47-fold), which was also found by reverse transcription-PCR (RT-PCR; 211-fold). sPLA2 inhibition reversed TFT resistance partially. H630-cTFT had many chromosomal aberrations, but the exact role of sPLA2 in TFT resistance remains unclear. Altogether, resistance induction to TFT can lead to different mechanisms of resistance, including decreased TK protein expression and enzyme activity, decreased hENT expression, as well as (phospho)lipid metabolism. Mol Cancer Ther; 9(4); 1047-57. (c)2010 AACR.

17beta-estradiol promotes breast cancer cell proliferation-inducing stromal cell-derived factor-1-mediated epidermal growth factor receptor transactivation: reversal by gefitinib pretreatment.

PubMed

Pattarozzi, Alessandra; Gatti, Monica; Barbieri, Federica; Würth, Roberto; Porcile, Carola; Lunardi, Gianluigi; Ratto, Alessandra; Favoni, Roberto; Bajetto, Adriana; Ferrari, Angelo; Florio, Tullio

2008-01-01

The coordinated activity of estrogens and epidermal growth factor receptor (EGFR) family agonists represents the main determinant of breast cancer cell proliferation. Stromal cell-derived factor-1 (SDF-1) enhances extracellular signal-regulated kinases 1 and 2 (ERK1/2) activity via the transactivation of EGFR and 17beta-estradiol (E2) induces SDF-1 production to exert autocrine proliferative effects. On this basis, we evaluated whether the inhibition of the tyrosine kinase (TK) activity of EGFR may control different mitogenic stimuli in breast tumors using the EGFR-TK inhibitor gefitinib to antagonize the proliferation induced by E2 in T47D human breast cancer cells. EGF, E2, and SDF-1 induced a dose-dependent T47D cell proliferation, that being nonadditive suggested the activation of common intracellular pathways. Gefitinib treatment inhibited not only the EGF-dependent proliferation and ERK1/2 activation but also the effects of SDF-1 and E2, suggesting that these activities were mediated by EGFR transactivation. Indeed, both SDF-1 and E2 caused EGFR tyrosine phosphorylation. The molecular link between E2 and SDF-1 proliferative effects was identified because 1,1'-(1,4-phenylenebis(methylene))-bis-1,4,8,11-tetraazacyclotetradecane octahydrochloride (AMD3100), a CXCR4 antagonist, inhibited SDF-1- and E2-dependent proliferation and EGFR and ERK1/2 phosphorylation. EGFR transactivation was dependent on c-Src activation. E2 treatment caused a powerful SDF-1 release from T47D cells. Finally, in SKBR3, E2-resistant cells, EGFR was constitutively activated, and AMD3100 reduced EGFR phosphorylation and cell proliferation, whereas HER2-neu was transactivated by SDF-1 in SKBR3 but not in T47D cells. In conclusion, we show that activation of CXCR4 transduces proliferative signals from the E2 receptor to EGFR, whose inhibition is able to revert breast cancer cell proliferation induced by multiple receptor activation.

QSAR and molecular graphics analysis of N2-phenylguanines as inhibitors of herpes simplex virus thymidine kinases.

PubMed

Gaudio, A C; Richards, W G; Takahata, Y

2000-02-01

A quantitative structure-activity relationship study of N2-(substituted)-phenylguanines (PHG) as inhibitors of herpes simplex virus thymidine kinase (HSV TK) was performed. The activity of a set of PHG derivatives were analyzed against the thymidine kinase of herpes simplex virus types 1 (HSV1 TK) and 2 (HSV2 TK). Classic and calculated physicochemical parameters were included in the analysis. The results showed that there is an important difference in the activity of the meta substituted PHG derivatives against HSV1 TK and HSV2 TK. The activity of the meta derivatives against HSV2 TK is influenced by a steric effect, which is not observed against HSV1 TK. The superposition of the three-dimensional structures of the active sites of HSV1 TK (crystal structure) and HSV2 TK (homology model) revealed that the amino acid Ile97 is located near the meta position in the HSV1 TK active site, whereas the amino acid Leu97 is located near the meta position in the HSV2 TK active site. This single difference in the active sites of both enzymes can explain the source of the steric effect and serves as an indication that our previously proposed binding mode for the PHG derivatives is plausible. However, another observed mutation in the active site region, Ala168 by Ser168, suggests that an alternative binding mode, similar to that of ganciclovir, could be possible.

The Design and Development of Potent Small Molecules as Anticancer Agents Targeting EGFR TK and Tubulin Polymerization

PubMed Central

Ihmaid, Saleh; Ahmed, Hany E. A.; Zayed, Mohamed F.

2018-01-01

Some novel anthranilate diamides derivatives 4a–e, 6a–c and 9a–d were designed and synthesized to be evaluated for their in vitro anticancer activity. Structures of all newly synthesized compounds were confirmed by infra-red (IR), high-resolution mass (HR-MS) spectra, 1H nuclear magnetic resonance (NMR) and 13C nuclear magnetic resonance (NMR) analyses. Cytotoxic screening was performed according to (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium (MTT) assay method using erlotinib as a reference drug against two different types of breast cancer cells. The molecular docking study was performed for representative compounds against two targets, epidermal growth factor receptor (EGFR) and tubulin in colchicine binding site to assess their binding affinities in order to rationalize their anticancer activity in a qualitative way. The data obtained from the molecular modeling was correlated with that obtained from the biological screening. These data showed considerable anticancer activity for these newly synthesized compounds. Biological data for most of the anthranilate diamide showed excellent activity with nanomolar or sub nanomolar half maximal inhibitory concentration (IC50) values against tumor cells. EGFR tyrosine kinase (TK) inhibition assay, tubulin inhibition assay and apoptosis analysis were performed for selected compounds to get more details about their mechanism of action. Extensive structure activity relationship (SAR) analyses were also carried out. PMID:29385728

Evolution of the herpes thymidine kinase: identification and comparison of the equine herpesvirus 1 thymidine kinase gene reveals similarity to a cell-encoded thymidylate kinase.

PubMed Central

Robertson, G R; Whalley, J M

1988-01-01

We have identified the equine herpesvirus 1 (EHV-1) thymidine kinase gene (TK) by DNA-mediated transformation and by DNA sequencing. Alignment of the amino acid sequence of the EHV-1 TK with the TKs from 3 other herpesviruses revealed regions of homology, some of which correspond to the previously identified substrate binding sites, while others have as yet, no assigned function. In particular, the strict conservation of an aspartate within the proposed nucleoside binding site suggests a role in ATP binding for this residue. Comparison of 5 herpes TKs with the thymidylate kinase of yeast revealed significant similarity which was strongest in those regions important to catalytic activity of the herpes TKs, and, therefore we propose that the herpes TK may be derived from a cellular thymidylate kinase. The implications for the evolution of enzyme activities within a pathway of nucleotide metabolism are discussed. PMID:2849761

Tachykinin-Related Peptides Share a G Protein-Coupled Receptor with Ion Transport Peptide-Like in the Silkworm Bombyx mori

PubMed Central

Nagai-Okatani, Chiaki; Nagasawa, Hiromichi

2016-01-01

Recently, we identified an orphan Bombyx mori neuropeptide G protein-coupled receptor (BNGR)-A24 as an ion transport peptide-like (ITPL) receptor. BNGR-A24 belongs to the same clade as BNGR-A32 and -A33, which were recently identified as natalisin receptors. Since these three BNGRs share high similarities with known receptors for tachykinin-related peptides (TRPs), we examined whether these BNGRs can function as physiological receptors for five endogenous B. mori TRPs (TK-1–5). In a heterologous expression system, BNGR-A24 acted as a receptor for all five TRPs. In contrast, BNGR-A32 responded only to TK-5, and BNGR-A33 did not respond to any of the TRPs. These findings are consistent with recent studies on the ligand preferences for B. mori natalisins. Furthermore, we evaluated whether the binding of ITPL and TRPs to BNGR-A24 is competitive by using a Ca2+ imaging assay. Concomitant addition of a TRP receptor antagonist, spantide I, reduced the responses of BNGR-A24 not only to TK-4 but also to ITPL. The results of a binding assay using fluorescent-labeled BNGR-A24 and ligands demonstrated that the binding of ITPL to BNGR-A24 was inhibited by TK-4 as well as by spantide I, and vice versa. In addition, the ITPL-induced increase in cGMP levels of BNGR-A24-expressing BmN cells was suppressed by the addition of excess TK-4 or spantide I. The intracellular levels of cAMP and cGMP, as second messenger candidates of the TRP signaling, were not altered by the five TRPs, suggesting that these peptides act via different signaling pathways from cAMP and cGMP signaling at least in BmN cells. Taken together, the present findings suggest that ITPL and TRPs are endogenous orthosteric ligands of BNGR-A24 that may activate discrete signaling pathways. This receptor, which shares orthosteric ligands, may constitute an important model for studying ligand-biased signaling. PMID:27248837

Measuring Acquisition Workforce Quality Through Dynamic Knowledge and Performance

DTIC Science & Technology

2014-05-14

closeout • Regression: – 3 TK/EK measures (IV): PCOd , DAWIA, AXP – 7 CMMM measures (DV): 6 processes + mean 5 Summary Statistical Results Model Org T...Org R PCOd  CMMM R2 = 0.36, p = 0.15 R2 = 0.41, p = 0.25 DAWIA  CMMM R2 = 0.64, p = 0.03 R2 = 0.44, p = 0.22 AXP  CMMM R2 = 0.59, p = 0.07 R2...0.27, p = 0.37 All  CMMM R2 = 0.71, p = 0.24 R2 = 0.72, p = 0.64 6 Contributions • ID 3 TK/EK proxies: PCOd , DAWIA, AXP • Measure CMMM levels: 12 orgs

Detection and Tracking of NY-ESO-1-Specific CD8+ T Cells by High-Throughput T Cell Receptor β (TCRB) Gene Rearrangements Sequencing in a Peptide-Vaccinated Patient.

PubMed

Miyai, Manami; Eikawa, Shingo; Hosoi, Akihiro; Iino, Tamaki; Matsushita, Hirokazu; Isobe, Midori; Uenaka, Akiko; Udono, Heiichiro; Nakajima, Jun; Nakayama, Eiichi; Kakimi, Kazuhiro

2015-01-01

Comprehensive immunological evaluation is crucial for monitoring patients undergoing antigen-specific cancer immunotherapy. The identification and quantification of T cell responses is most important for the further development of such therapies. Using well-characterized clinical samples from a high responder patient (TK-f01) in an NY-ESO-1f peptide vaccine study, we performed high-throughput T cell receptor β-chain (TCRB) gene next generation sequencing (NGS) to monitor the frequency of NY-ESO-1-specific CD8+ T cells. We compared these results with those of conventional immunological assays, such as IFN-γ capture, tetramer binding and limiting dilution clonality assays. We sequenced human TCRB complementarity-determining region 3 (CDR3) rearrangements of two NY-ESO-1f-specific CD8+ T cell clones, 6-8L and 2F6, as well as PBMCs over the course of peptide vaccination. Clone 6-8L possessed the TCRB CDR3 gene TCRBV11-03*01 and BJ02-01*01 with amino acid sequence CASSLRGNEQFF, whereas 2F6 possessed TCRBV05-08*01 and BJ02-04*01 (CASSLVGTNIQYF). Using these two sequences as models, we evaluated the frequency of NY-ESO-1-specific CD8+ T cells in PBMCs ex vivo. The 6-8L CDR3 sequence was the second most frequent in PBMC and was present at high frequency (0.7133%) even prior to vaccination, and sustained over the course of vaccination. Despite a marked expansion of NY-ESO-1-specific CD8+ T cells detected from the first through 6th vaccination by tetramer staining and IFN-γ capture assays, as evaluated by CDR3 sequencing the frequency did not increase with increasing rounds of peptide vaccination. By clonal analysis using 12 day in vitro stimulation, the frequency of B*52:01-restricted NY-ESO-1f peptide-specific CD8+ T cells in PBMCs was estimated as only 0.0023%, far below the 0.7133% by NGS sequencing. Thus, assays requiring in vitro stimulation might be underestimating the frequency of clones with lower proliferation potential. High-throughput TCRB sequencing using NGS can potentially better estimate the actual frequency of antigen-specific T cells and thus provide more accurate patient monitoring.

Detection and Tracking of NY-ESO-1-Specific CD8+ T Cells by High-Throughput T Cell Receptor β (TCRB) Gene Rearrangements Sequencing in a Peptide-Vaccinated Patient

PubMed Central

Miyai, Manami; Eikawa, Shingo; Hosoi, Akihiro; Iino, Tamaki; Matsushita, Hirokazu; Isobe, Midori; Uenaka, Akiko; Udono, Heiichiro; Nakajima, Jun; Nakayama, Eiichi; Kakimi, Kazuhiro

2015-01-01

Comprehensive immunological evaluation is crucial for monitoring patients undergoing antigen-specific cancer immunotherapy. The identification and quantification of T cell responses is most important for the further development of such therapies. Using well-characterized clinical samples from a high responder patient (TK-f01) in an NY-ESO-1f peptide vaccine study, we performed high-throughput T cell receptor β-chain (TCRB) gene next generation sequencing (NGS) to monitor the frequency of NY-ESO-1-specific CD8+ T cells. We compared these results with those of conventional immunological assays, such as IFN-γ capture, tetramer binding and limiting dilution clonality assays. We sequenced human TCRB complementarity-determining region 3 (CDR3) rearrangements of two NY-ESO-1f-specific CD8+ T cell clones, 6-8L and 2F6, as well as PBMCs over the course of peptide vaccination. Clone 6-8L possessed the TCRB CDR3 gene TCRBV11-03*01 and BJ02-01*01 with amino acid sequence CASSLRGNEQFF, whereas 2F6 possessed TCRBV05-08*01 and BJ02-04*01 (CASSLVGTNIQYF). Using these two sequences as models, we evaluated the frequency of NY-ESO-1-specific CD8+ T cells in PBMCs ex vivo. The 6-8L CDR3 sequence was the second most frequent in PBMC and was present at high frequency (0.7133%) even prior to vaccination, and sustained over the course of vaccination. Despite a marked expansion of NY-ESO-1-specific CD8+ T cells detected from the first through 6th vaccination by tetramer staining and IFN-γ capture assays, as evaluated by CDR3 sequencing the frequency did not increase with increasing rounds of peptide vaccination. By clonal analysis using 12 day in vitro stimulation, the frequency of B*52:01-restricted NY-ESO-1f peptide-specific CD8+ T cells in PBMCs was estimated as only 0.0023%, far below the 0.7133% by NGS sequencing. Thus, assays requiring in vitro stimulation might be underestimating the frequency of clones with lower proliferation potential. High-throughput TCRB sequencing using NGS can potentially better estimate the actual frequency of antigen-specific T cells and thus provide more accurate patient monitoring. PMID:26291626

Molecular and clinical characterization of the myopathic form of mitochondrial DNA depletion syndrome caused by mutations in the thymidine kinase (TK2) gene.

PubMed

Chanprasert, Sirisak; Wang, Jing; Weng, Shao-Wen; Enns, Gregory M; Boué, Daniel R; Wong, Brenda L; Mendell, Jerry R; Perry, Deborah A; Sahenk, Zarife; Craigen, William J; Alcala, Francisco J Climent; Pascual, Juan M; Melancon, Serge; Zhang, Victor Wei; Scaglia, Fernando; Wong, Lee-Jun C

2013-01-01

Mitochondrial DNA (mtDNA) depletion syndromes (MDSs) are a clinically and molecularly heterogeneous group of mitochondrial cytopathies characterized by severe mtDNA copy number reduction in affected tissues. Clinically, MDSs are mainly categorized as myopathic, encephalomyopathic, hepatocerebral, or multi-systemic forms. To date, the myopathic form of MDS is mainly caused by mutations in the TK2 gene, which encodes thymidine kinase 2, the first and rate limiting step enzyme in the phosphorylation of pyrimidine nucleosides. We analyzed 9 unrelated families with 11 affected subjects exhibiting the myopathic form of MDS, by sequencing the TK2 gene. Twelve mutations including 4 novel mutations were detected in 9 families. Skeletal muscle specimens were available from 7 out of 11 subjects. Respiratory chain enzymatic activities in skeletal muscle were measured in 6 subjects, and enzymatic activities were reduced in 3 subjects. Quantitative analysis of mtDNA content in skeletal muscle was performed in 5 subjects, and marked mtDNA content reduction was observed in each. In addition, we outline the molecular and clinical characteristics of this syndrome in a total of 52 patients including those previously reported, and a total of 36 TK2 mutations are summarized. Clinically, hypotonia and proximal muscle weakness are the major phenotypes present in all subjects. In summary, our study expands the molecular and clinical spectrum associated with TK2 deficiency. © 2013.

Taxator-tk: precise taxonomic assignment of metagenomes by fast approximation of evolutionary neighborhoods

PubMed Central

Dröge, J.; Gregor, I.; McHardy, A. C.

2015-01-01

Motivation: Metagenomics characterizes microbial communities by random shotgun sequencing of DNA isolated directly from an environment of interest. An essential step in computational metagenome analysis is taxonomic sequence assignment, which allows identifying the sequenced community members and reconstructing taxonomic bins with sequence data for the individual taxa. For the massive datasets generated by next-generation sequencing technologies, this cannot be performed with de-novo phylogenetic inference methods. We describe an algorithm and the accompanying software, taxator-tk, which performs taxonomic sequence assignment by fast approximate determination of evolutionary neighbors from sequence similarities. Results: Taxator-tk was precise in its taxonomic assignment across all ranks and taxa for a range of evolutionary distances and for short as well as for long sequences. In addition to the taxonomic binning of metagenomes, it is well suited for profiling microbial communities from metagenome samples because it identifies bacterial, archaeal and eukaryotic community members without being affected by varying primer binding strengths, as in marker gene amplification, or copy number variations of marker genes across different taxa. Taxator-tk has an efficient, parallelized implementation that allows the assignment of 6 Gb of sequence data per day on a standard multiprocessor system with 10 CPU cores and microbial RefSeq as the genomic reference data. Availability and implementation: Taxator-tk source and binary program files are publicly available at http://algbio.cs.uni-duesseldorf.de/software/. Contact: Alice.McHardy@uni-duesseldorf.de Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25388150

A Comparative Study of the Aneugenic and Polyploidy-inducing Effects of Fisetin and Two Model Aurora Kinase Inhibitors

PubMed Central

Gollapudi, P.; Hasegawa, L.S.; Eastmond, D.A.

2014-01-01

Fisetin, a plant flavonol commonly found in fruits, nuts and vegetables, is frequently added to nutritional supplements due to its reported cardioprotective, anti-carcinogenic and antioxidant properties. Earlier reports from our laboratory and others have indicated that fisetin has both aneugenic and clastogenic properties in cultured cells. More recently, fisetin has also been reported to target Aurora B kinase, a Ser/Thr kinase involved in ensuring proper microtubule attachment at the spindle assembly checkpoint, and an enzyme that is overexpressed in several types of cancer. Here we have further characterized the chromosome damage caused by fisetin and compared it with that induced by two known Aurora kinase inhibitors, VX-680 and ZM-447439, in cultured TK6 cells using the micronucleus assay with CREST staining as well as a flow cytometry-based assay that measures multiple types of numerical chromosomal aberrations. The three compounds were highly effective in inducing aneuploidy and polyploidy as evidenced by increases in kinetochore-positive micronuclei, hyperdiploidy, and polyploidy. With fisetin, however, the latter two effects were most significantly observed only after cells were allowed to overcome a cell cycle delay, and occurred at higher concentrations than those induced by the other Aurora kinase inhibitors. Modest increases in kinetochore-negative micronuclei were also seen with the model Aurora kinase inhibitors. These results indicate that fisetin induces multiple types of chromosome abnormalities in human cells, and indicate a need for a thorough investigation of fisetin-augmented dietary supplements. PMID:24680981

Comparison of toxicogenomics and traditional approaches to inform mode of action and points of departure in human health risk assessment of benzo[a]pyrene in drinking water

PubMed Central

Labib, Sarah; Bourdon-Lacombe, Julie; Kuo, Byron; Buick, Julie K.; Lemieux, France; Williams, Andrew; Halappanavar, Sabina; Malik, Amal; Luijten, Mirjam; Aubrecht, Jiri; Hyduke, Daniel R.; Fornace, Albert J.; Swartz, Carol D.; Recio, Leslie; Yauk, Carole L.

2015-01-01

Toxicogenomics is proposed to be a useful tool in human health risk assessment. However, a systematic comparison of traditional risk assessment approaches with those applying toxicogenomics has never been done. We conducted a case study to evaluate the utility of toxicogenomics in the risk assessment of benzo[a]pyrene (BaP), a well-studied carcinogen, for drinking water exposures. Our study was intended to compare methodologies, not to evaluate drinking water safety. We compared traditional (RA1), genomics-informed (RA2) and genomics-only (RA3) approaches. RA2 and RA3 applied toxicogenomics data from human cell cultures and mice exposed to BaP to determine if these data could provide insight into BaP's mode of action (MOA) and derive tissue-specific points of departure (POD). Our global gene expression analysis supported that BaP is genotoxic in mice and allowed the development of a detailed MOA. Toxicogenomics analysis in human lymphoblastoid TK6 cells demonstrated a high degree of consistency in perturbed pathways with animal tissues. Quantitatively, the PODs for traditional and transcriptional approaches were similar (liver 1.2 vs. 1.0 mg/kg-bw/day; lung 0.8 vs. 3.7 mg/kg-bw/day; forestomach 0.5 vs. 7.4 mg/kg-bw/day). RA3, which applied toxicogenomics in the absence of apical toxicology data, demonstrates that this approach provides useful information in data-poor situations. Overall, our study supports the use of toxicogenomics as a relatively fast and cost-effective tool for hazard identification, preliminary evaluation of potential carcinogens, and carcinogenic potency, in addition to identifying current limitations and practical questions for future work. PMID:25605026

Deoxycytidine and Deoxythymidine Treatment for Thymidine Kinase 2 Deficiency.

PubMed

Lopez-Gomez, Carlos; Levy, Rebecca J; Sanchez-Quintero, Maria J; Juanola-Falgarona, Martí; Barca, Emanuele; Garcia-Diaz, Beatriz; Tadesse, Saba; Garone, Caterina; Hirano, Michio

2017-05-01

Thymidine kinase 2 (TK2), a critical enzyme in the mitochondrial pyrimidine salvage pathway, is essential for mitochondrial DNA (mtDNA) maintenance. Mutations in the nuclear gene, TK2, cause TK2 deficiency, which manifests predominantly in children as myopathy with mtDNA depletion. Molecular bypass therapy with the TK2 products, deoxycytidine monophosphate (dCMP) and deoxythymidine monophosphate (dTMP), prolongs the life span of Tk2-deficient (Tk2 -/- ) mice by 2- to 3-fold. Because we observed rapid catabolism of the deoxynucleoside monophosphates to deoxythymidine (dT) and deoxycytidine (dC), we hypothesized that: (1) deoxynucleosides might be the major active agents and (2) inhibition of deoxycytidine deamination might enhance dTMP+dCMP therapy. To test these hypotheses, we assessed two therapies in Tk2 -/- mice: (1) dT+dC and (2) coadministration of the deaminase inhibitor, tetrahydrouridine (THU), with dTMP+dCMP. We observed that dC+dT delayed disease onset, prolonged life span of Tk2-deficient mice and restored mtDNA copy number as well as respiratory chain enzyme activities and levels. In contrast, dCMP+dTMP+THU therapy decreased life span of Tk2 -/- animals compared to dCMP+dTMP. Our studies demonstrate that deoxynucleoside substrate enhancement is a novel therapy, which may ameliorate TK2 deficiency in patients. Ann Neurol 2017;81:641-652. © 2017 American Neurological Association.

Enzymatic activity of a subtilisin homolog, Tk-SP, from Thermococcus kodakarensis in detergents and its ability to degrade the abnormal prion protein

PubMed Central

2013-01-01

Background Tk-SP is a member of subtilisin-like serine proteases from a hyperthermophilic archaeon Thermococcus kodakarensis. It has been known that the hyper-stable protease, Tk-SP, could exhibit enzymatic activity even at high temperature and in the presence of chemical denaturants. In this work, the enzymatic activity of Tk-SP was measured in the presence of detergents and EDTA. In addition, we focused to demonstrate that Tk-SP could degrade the abnormal prion protein (PrPSc), a protease-resistant isoform of normal prion protein (PrPC). Results Tk-SP was observed to maintain its proteolytic activity with nonionic surfactants and EDTA at 80°C. We optimized the condition in which Tk-SP functions efficiently, and demonstrated that the enzyme is highly stable in the presence of 0.05% (w/v) nonionic surfactants and 0.01% (w/v) EDTA, retaining up to 80% of its activity. Additionally, we also found that Tk-SP can degrade PrPSc to a level undetectable by western-blot analysis. Conclusions Our results indicate that Tk-SP has a great potential for technological applications, such as thermo-stable detergent additives. In addition, it is also suggested that Tk-SP-containing detergents can be developed to decrease the secondary infection risks of transmissible spongiform encephalopathies (TSE). PMID:23448268

Promoter-Based Theranostics for Prostate Cancer

DTIC Science & Technology

2016-06-01

diagnosis vector consists of the tumor-specific PEG-promoter (PEG-Prom) and cDNA of human chorionic gonadotropin β chain (βhCG) as a reporter. We...transfection efficiency. We also used CpG-free cDNA of Figure 5. pCpGfree-PEGwt-HSV1-tk-neo vector expressed functional thymidine kinase in human

Fire Support Requirements Methodology Study, Phase 2 Proceedings of the Fire Support Methodology Workshop

DTIC Science & Technology

1975-12-18

It was not immediatei- clear that the -approach- would- succeed in overcoming the deficiencies of present fire support methodologies which demand- an...support require analysis up to Level 6. They also felt that deficiencies in f technique were most serious at Levels 3, 4 and 5. It was accepted that...defined as: Tk2 = _Tkl ilk2 kl (2) Tkt = Tk,t-l - ’lMktMk,t-l + 𔃼kt ,t-2 I t > (3. where Mt refers to the-number of type k targets killed in time

Reversion of mtDNA depletion in a patient with TK2 deficiency.

PubMed

Vilà, M R; Segovia-Silvestre, T; Gámez, J; Marina, A; Naini, A B; Meseguer, A; Lombès, A; Bonilla, E; DiMauro, S; Hirano, M; Andreu, A L

2003-04-08

Mutations in the thymidine kinase 2 (TK2) gene cause a myopathic form of the mitochondrial DNA depletion syndrome (MDS). Here, the authors report the unusual clinical, biochemical, and molecular findings in a 14-year-old patient in whom pathogenic mutations were identified in the TK2 gene. This report extends the phenotypic expression of primary TK2 deficiency and suggests that factors other than TK2 may modify expression of the clinical phenotype in patients with MDS syndrome.

Evaluation of [(18)F]gefitinib as a molecular imaging probe for the assessment of the epidermal growth factor receptor status in malignant tumors.

PubMed

Su, Helen; Seimbille, Yann; Ferl, Gregory Z; Bodenstein, Claudia; Fueger, Barbara; Kim, Kevin J; Hsu, Yu-Tien; Dubinett, Steven M; Phelps, Michael E; Czernin, Johannes; Weber, Wolfgang A

2008-06-01

Gefitinib, an inhibitor of the epidermal growth factor receptor-tyrosine kinase (EGFR-TK), has shown potent effects in a subset of patients carrying specific EGFR-TK mutations in advanced non-small-cell lung cancer. In this study, we asked whether PET with [(18)F]gefitinib may be used to study noninvasively the pharmacokinetics of gefitinib in vivo and to image the EGFR status of cancer cells. Synthesis of [(18)F]gefitinib has been previously described. The biodistribution and metabolic stability of [(18)F]gefitinib was assessed in mice and vervet monkeys for up to 2 h post injection by both micropositron emission tomography (PET)/computed tomography (CT) scans and postmortem ex vivo tissue harvesting. Uptake levels of radiolabeled gefitinib in EGFR-expressing human cancer cell lines with various levels of EGFR expression or mutation status were evaluated both in vivo and in vitro. MicroPET/CT scans in two species demonstrated a rapid and predominantly hepatobiliary clearance of [(18)F]gefitinib in vivo. However, uptake levels of radiolabeled gefitinib, both in vivo and in vitro, did not correlate with EGFR expression levels or functional status. This unexpected observation was due to high nonspecific, nonsaturable cellular uptake of gefitinib. The biodistribution of the drug analogue [(18)F]gefitinib suggests that it may be used to assess noninvasively the pharmacokinetics of gefitinib in patients by PET imaging. This is of clinical relevance, as insufficient intratumoral drug concentrations are considered to be a factor for resistance to gefitinib therapy. However, the highly nonspecific cellular binding of [(18)F]gefitinib may preclude the use of this imaging probe for noninvasive assessment of EGFR receptor status in patients.

The effect of first ballet classes in the community on various postural parameters in young girls.

PubMed

Moller, Anna; Masharawi, Youssef

2011-11-01

To examine the effect of first season ballet classes in the community on the thoracic kyphosis (TK), lumbar lordosis (LL), hip external rotation (ER) and joint flexibility in young girls. Longitudinal single blinded cohort control study. Institutional. 30 girls aged 6-9, recruited from the same primary school were divided equally into 2 groups: a group bi-weekly community ballet class and a sedentary control group. All girls were assessed prior to ballet classes (t(0)), at the conclusion (t(1)) (6 months), and approximately one year later (t(fu)). Beighton score for joint hyper-flexibility, peak of TK and LL, range of hip ER, ratio TK/LL, and individual's height, weight and BMI. LL at t1 became greater in the ballet girls' group (23.7°± 6) as opposed to the controls (19.5°±3.9) due to a decrease in LL in the controls from t0 to t1 (mean difference = -16.5°) (cut-off score = 3.45°) (p < 0.05). TK decreased from t0 to t1 in the ballet girls' group (mean difference = -26.1°) and controls (mean difference = -31.3°) (cut-off score = 4.85°) (p < 0.05). Left hip ER decreased only in the controls from t0 to t1 (mean difference = -13.8°) (cut-off score = 5.43°) (p < 0.05). Beighton score at tfu was greater in the ballet girls' group (6.1 ± 2.3) as opposed to the controls (4.4 ± 1.5) (p < 0.05). First season ballet classes for young girls in the community can be associated with relatively greater LL, and left hip ER and joint hyper-flexibility. Copyright © 2011 Elsevier Ltd. All rights reserved.

A tracer kinetic model for 18F-FHBG for quantitating herpes simplex virus type 1 thymidine kinase reporter gene expression in living animals using PET.

PubMed

Green, Leeta Alison; Nguyen, Khoi; Berenji, Bijan; Iyer, Meera; Bauer, Eileen; Barrio, Jorge R; Namavari, Mohammad; Satyamurthy, Nagichettiar; Gambhir, Sanjiv S

2004-09-01

Reporter probe 9-(4-18F-fluoro-3-[hydroxymethyl]butyl)guanine (18F-FHBG) and reporter gene mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) have been used for imaging reporter gene expression with PET. Current methods for quantitating the images using the percentage injected dose per gram of tissue do not distinguish between the effects of probe transport and subsequent phosphorylation. We therefore investigated tracer kinetic models for 18F-FHBG dynamic microPET data and noninvasive methods for determining blood time-activity curves in an adenoviral gene delivery model in mice. 18F-FHBG (approximately 7.4 MBq [approximately 200 microCi]) was injected into 4 mice; 18F-FHBG concentrations in plasma and whole blood were measured from mouse heart left ventricle (LV) direct sampling. Replication-incompetent adenovirus (0-2 x 10(9) plaque-forming units) with the E1 region deleted (n = 8) or replaced by HSV1-sr39tk (n = 18) was tail-vein injected into mice. Mice were dynamically scanned using microPET (approximately 7.4 MBq [approximately 200 microCi] 18F-FHBG) over 1 h; regions of interest were drawn on images of the heart and liver. Serial whole blood 18F-FHBG concentrations were measured in 6 of the mice by LV sampling, and 1 least-squares ratio of the heart image to the LV time-activity curve was calculated for all 6 mice. For 2 control mice and 9 mice expressing HSV1-sr39tk, heart image (input function) and liver image time-activity curves (tissue curves) were fit to 2- and 3-compartment models using Levenberg-Marquardt nonlinear regression. The models were compared using an F statistic. HSV1-sr39TK enzyme activity was determined from liver samples and compared with model parameter estimates. For another 3 control mice and 6 HSV1-sr39TK-positive mice, the model-predicted relative percentage of metabolites was compared with high-performance liquid chromatography analysis. The ratio of 18F-FHBG in plasma to whole blood was 0.84 +/- 0.05 (mean +/- SE) by 30 s after injection. The least-squares ratio of the heart image time-activity curve to the LV time-activity curve was 0.83 +/- 0.02, consistent with the recovery coefficient for the partial-volume effect (0.81) based on independent measures of heart geometry. A 3-compartment model best described 18F-FHBG kinetics in mice expressing HSV1-sr39tk in the liver; a 2-compartment model best described the kinetics in control mice. The 3-compartment model parameter, k3, correlated well with the HSV1-sr39TK enzyme activity (r2 = 0.88). 18F-FHBG equilibrates rapidly between plasma and whole blood in mice. Heart image time-activity curves corrected for partial-volume effects well approximate LV time-activity curves and can be used as input functions for 2- and 3-compartment models. The model parameter k3 from the 3-compartment model can be used as a noninvasive estimate for HSV1-sr39TK reporter protein activity and can predict the relative percentage of metabolites.

Development of a sufficient and effective procedure for transformation of an oleaginous yeast, Rhodosporidium toruloides DMKU3-TK16.

PubMed

Tsai, Yung-Yu; Ohashi, Takao; Kanazawa, Takenori; Polburee, Pirapan; Misaki, Ryo; Limtong, Savitree; Fujiyama, Kazuhito

2017-05-01

Rhodosporidium toruloides DMKU3-TK16 (TK16), a basidiomycetous yeast isolated in Thailand, can produce a large amount of oil corresponding to approximately 70 % of its dry cell weight. However, lack of a sufficient and efficient transformation method makes further genetic manipulation of this organism difficult. We here developed a new transformation system for R. toruloides using a lithium acetate method with the Sh ble gene as a selective marker under the control of the R. toruloides ATCC 10657 GPD1 promoter. A linear DNA fragment containing the Sh ble gene expression cassette was integrated into the genome, and its integration was confirmed by colony PCR and Southern blot. Then, we further optimized the parameters affecting the transformation efficiency, such as the amount of linear DNA, the growth phase, the incubation time in the transformation mixture, the heat shock treatment temperature, the addition of DMSO and carrier DNA, and the recovery incubation time. With the developed method, the transformation efficiency of approximately 25 transformants/μg DNA was achieved. Compared with the initial trial, transformation efficiency was enhanced 417-fold. We further demonstrated the heterologous production of EGFP in TK16 by microscopic observation and immunoblot analysis, and use the technique to disrupt the endogenous URA3 gene. The newly developed method is thus simple and time saving, making it useful for efficient introduction of an exogenous gene into R. toruloides strains. Accordingly, this new practical approach should facilitate the molecular manipulation, such as target gene introduction and deletion, of TK16 and other R. toruloides strains as a major source of biodiesel.

QIVIVE Approaches to Evaluate Inter-individual Toxicokinetic Variability

EPA Science Inventory

Manifestation of inter-individual variability in toxicokinetics (TK) will result in identical external exposure concentrations yielding differing blood or tissue concentrations. As efforts to incorporate in vitro testing strategies into human health assessment continue to grow, a...

Functional Diversity of the Schistosoma mansoni Tyrosine Kinases

PubMed Central

Avelar, Lívia G. A.; Nahum, Laila A.; Andrade, Luiza F.; Oliveira, Guilherme

2011-01-01

Schistosoma mansoni, one of the causative agents of schistosomiasis, has a complex life cycle infecting over 200 million people worldwide. Such a successful and prolific parasite life cycle has been shown to be dependent on the adaptive interaction between the parasite and hosts. Tyrosine kinases (TKs) play a key role in signaling pathways as demonstrated by a large body of experimental work in eukaryotes. Furthermore, comparative genomics have allowed the identification of TK homologs and provided insights into the functional role of TKs in several biological systems. Finally, TK structural biology has provided a rational basis for obtaining selective inhibitors directed to the treatment of human diseases. This paper covers the important aspects of the phospho-tyrosine signaling network in S. mansoni, Caenorhabditis elegans, and humans, the main process of functional diversification of TKs, that is, protein-domain shuffling, and also discusses TKs as targets for the development of new anti-schistosome drugs. PMID:21776387

A pseudoreceptor modelling study of the varicella-zoster virus and human thymidine kinase binding sites

NASA Astrophysics Data System (ADS)

Greenidge, Paulette A.; Merz, Alfred; Folkers, Gerd

1995-12-01

A representative range of pyrimidine nucleoside analogues that are known to inhibit herpes simplex virus (HSV) replication have been used to construct receptor binding site models for the varicella-zoster virus (VZV), thymidine kinase (TK) and human TK1. Given a set of interacting ligands, superimposed in such a manner as to define a pharmacophore, the pseudoreceptor modelling technique Yak provides a means of building binding site models of macromolecules for which no three-dimensional experimental structures are available. Once the models have been evaluated by their ability to reproduce experimental binding data [Vedani et al., J. Am. Chem. Soc., 117 (1995) 4987], they can be used for predictive purposes. Calculated and experimental values of relative binding affinity are compared. Our models suggest that the substitution of one residue may be sufficient to determine ligand subtype affinity.

Serum TK levels in CLL identify Binet stage A patients within biologically defined prognostic subgroups most likely to undergo disease progression.

PubMed

Matthews, Christine; Catherwood, Mark A; Morris, T C M; Kettle, Paul J; Drake, Mary B; Gilmore, William S; Alexander, H Denis

2006-10-01

Serum thymidine kinase (TK) levels have been shown to be correlated with survival in many malignancies, including chronic lymphocytic leukaemia (CLL). This study was designed to investigate associations between TK levels and other prognostic markers, in newly and previously diagnosed Binet stage A patients. Furthermore, the use of serum TK measurement to identify subcategories of disease within those defined by IgV(H) mutational status, gene usage and chromosomal aberrations was investigated. Ninety-one CLL patients were enrolled. Serum TK levels were measured using a radioenzyme assay. IgV(H) mutational status and V(H) gene usage were determined using BIOMED-2 primers and protocol. Recurring chromosomal abnormalities were detected by interphase fluorescent in situ hybridisation (FISH). Flow cytometry and reverse transcriptase polymerase chain reaction (RT-PCR) determined CD38 and Zap-70 expression, respectively. Significantly higher serum TK levels were found in IgV(H) unmutated, compared with IgV(H) mutated, patients (P < 0.001). Elevated TK levels were also found in patients with CD38 and Zap-70 positivity (P = 0.004, P < 0.001, respectively), short lymphocyte doubling time (LDT) (P = 0.044) and poor or intermediate prognosis chromosomal aberrations (P < 0.001). A TK level of >8.5 U/L best identified patients with progressive disease. Elevated TK levels could identify patients categorised, at diagnosis, into good prognosis subgroups by the various biological markers (mutated IgV(H), good prognosis chromosomal aberrations, Zap-70(-) and CD38(-)) who subsequently showed disease progression. Additionally, patients with V(H)3-21 gene usage showed high TK levels, irrespective of mutational status, and serum TK measurement retained predictive power as disease progressed in all subcategories studied.

Structure-based pharmacophore design and virtual screening for novel potential inhibitors of epidermal growth factor receptor as an approach to breast cancer chemotherapy.

PubMed

Mahernia, Shabnam; Hassanzadeh, Malihe; Sharifi, Niusha; Mehravi, Bita; Paytam, Fariba; Adib, Mehdi; Amanlou, Massoud

2018-02-01

Cancer cells are described with features of uncontrolled growth, invasion and metastasis. The epidermal growth factor receptor subfamily of tyrosine kinases (EGFR-TK) plays a crucial regulatory role in the control of cellular proliferation and progression of various cancers. Therefore, its inhibition might lead to the discovery of a new generation of anticancer drugs. In the present study, structure-based pharmacophore modeling, molecular docking and molecular dynamics simulations were applied to identify potential hits, which exhibited good inhibition on the proliferation of MCF-7 breast cancer cell line and favorable binding interactions on EGFR-TK. Selected compounds were examined for their anticancer activity against the Michigan Cancer Foundation-7 (MCF-7) breast cancer cell line which overexpresses EGFR using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay. Compounds 1 and 2, with an isoindoline-1-one core, induced significant inhibition of breast cancer cells proliferation with IC[Formula: see text] values 327 and 370 nM, respectively.

PlGF/VEGFR-1 Signaling Promotes Macrophage Polarization and Accelerated Tumor Progression in Obesity.

PubMed

Incio, Joao; Tam, Josh; Rahbari, Nuh N; Suboj, Priya; McManus, Dan T; Chin, Shan M; Vardam, Trupti D; Batista, Ana; Babykutty, Suboj; Jung, Keehoon; Khachatryan, Anna; Hato, Tai; Ligibel, Jennifer A; Krop, Ian E; Puchner, Stefan B; Schlett, Christopher L; Hoffmman, Udo; Ancukiewicz, Marek; Shibuya, Masabumi; Carmeliet, Peter; Soares, Raquel; Duda, Dan G; Jain, Rakesh K; Fukumura, Dai

2016-06-15

Obesity promotes pancreatic and breast cancer progression via mechanisms that are poorly understood. Although obesity is associated with increased systemic levels of placental growth factor (PlGF), the role of PlGF in obesity-induced tumor progression is not known. PlGF and its receptor VEGFR-1 have been shown to modulate tumor angiogenesis and promote tumor-associated macrophage (TAM) recruitment and activity. Here, we hypothesized that increased activity of PlGF/VEGFR-1 signaling mediates obesity-induced tumor progression by augmenting tumor angiogenesis and TAM recruitment/activity. We established diet-induced obese mouse models of wild-type C57BL/6, VEGFR-1 tyrosine kinase (TK)-null, or PlGF-null mice, and evaluated the role of PlGF/VEGFR-1 signaling in pancreatic and breast cancer mouse models and in human samples. We found that obesity increased TAM infiltration, tumor growth, and metastasis in pancreatic cancers, without affecting vessel density. Ablation of VEGFR-1 signaling prevented obesity-induced tumor progression and shifted the tumor immune environment toward an antitumor phenotype. Similar findings were observed in a breast cancer model. Obesity was associated with increased systemic PlGF, but not VEGF-A or VEGF-B, in pancreatic and breast cancer patients and in various mouse models of these cancers. Ablation of PlGF phenocopied the effects of VEGFR-1-TK deletion on tumors in obese mice. PlGF/VEGFR-1-TK deletion prevented weight gain in mice fed a high-fat diet, but exacerbated hyperinsulinemia. Addition of metformin not only normalized insulin levels but also enhanced antitumor immunity. Targeting PlGF/VEGFR-1 signaling reprograms the tumor immune microenvironment and inhibits obesity-induced acceleration of tumor progression. Clin Cancer Res; 22(12); 2993-3004. ©2016 AACR. ©2016 American Association for Cancer Research.

DHFR and MSH3 co-amplification in childhood acute lymphoblastic leukaemia, in vitro and in vivo.

PubMed

Matheson, Elizabeth C; Hogarth, Linda A; Case, Marian C; Irving, Julie A E; Hall, Andrew G

2007-06-01

The MSH3 and dihydrofolate reductase (DHFR) genes, located on chromosome 5, share a common promoter but are divergently transcribed. Dysregulation of the mismatch repair (MMR) pathway has been found to occur in cell line models due to co-amplification of MSH3 as a coincident effect of DHFR amplification, acquired as a mechanism generating resistance to methotrexate (MTX). The increased levels of MSH3 perturbed MutSalpha function resulting in hypermutability and increased resistance to thiopurines, drugs whose cytotoxic effects are triggered by MutSalpha. The relevance of this phenomenon in clinical samples is unknown but is extremely pertinent in childhood acute lymphoblastic leukaemia (ALL) in which children are exposed for prolonged periods to both MTX and thiopurines such that a single amplification event involving both the DHFR and the MSH3 genes may cause chemotherapeutic resistance to both agents. Thus, we have generated a leukaemic cell line (PreB697) and a normal human lymphoblastoid cell line (TK6) that are resistant to a pharmacologically relevant dose of MTX and show that while increased DHFR levels result in MTX resistance, the associated increased levels of MSH3 are insufficient to perturb MutSalpha functionality, in terms of MMR capacity or 6-thioguanine sensitivity. In addition, we show that although low-level DHFR amplification occurs alone in a significant number of samples, both at disease onset and relapse, co-amplification of both MSH3 and DHFR is rarely found in primary ALL samples, even after prolonged MTX therapy and is not at a sufficiently high level to perturb MMR function.

¹¹¹In-DOTA-Annexin V for imaging of apoptosis during HSV1-tk/GCV prodrug activation gene therapy in mice with NG4TL4 sarcoma.

PubMed

Lin, Ming-Hsien; Wu, Shih-Yen; Wang, Hsin-Ell; Liu, Ren-Shyan; Chen, Jyh-Cheng

2016-02-01

Apoptosis has been suggested as a cytocidal mechanism of the HSV1-tk-expressing cells when exposed to ganciclovir (GCV). This study evaluated the efficacy of (111)In-labeled Annexin V for monitoring tumor responses during prodrug activation gene therapy with HSV1-tk and GCV. Annexin V was conjugated to DOTA using N-hydroxysulfosuccinimide (sulfo-NHS) and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), labeled with (111)In-InCl3 and purified using size exclusion chromatography to give (111)In-DOTA-Annexin V conjugate. The radiochemical yield and the radiochemical purity of (111)In-DOTA-Annexin V were 74±12% and 98±3%, respectively (n=10). (111)In-DOTA-BSA was prepared similarly. An in vitro study to demonstrate the apoptosis of NG4TL4-STK cells after GCV treatment has been performed. Mice bearing NG4TL4-STK and NG4TL4-WT tumors were treated with GCV (10 mg/kg daily) by i.p. injection for 7 consecutive days. Before and during the GCV treatment, biodistribution studies and scintigraphic imaging were performed at 2h post injection of the radiotracers. The uptake of (111)In-DOTA-Annexin V in treated cells (13.41±1.30%) was 4.1 times higher than that in untreated cells (3.21±0.37%). The GCV-induced cell apoptosis in NG4TL4-STK tumor resulted in a significantly increasing accumulation of (111)In-DOTA-Annexin V (1.92±0.32%ID/g at day 0, 4.79±0.86%ID/g at day 2, 4.56±0.58%ID/g at day 4) was observed, but not for that of (111)In-DOTA-BSA. During consecutive GCV treatment, scintigraphic imaging with (111)In-DOTA-Annexin V revealed high uptake in NG4TL4-STK tumor compared with that in NG4TL4-WT tumor. However, no specific (111)In-DOTA-BSA accumulation in NG4TL4-STK and NG4TL4-WT tumors was observed throughout the course of GCV treatment. This study demonstrated that (111)In-DOTA-Annexin V can be used for monitoring tumor cell apoptosis during prodrug activation gene therapy with HSV1-tk and GCV for cancer treatment. Copyright © 2015 Elsevier Ltd. All rights reserved.

Antiglioma Immunological Memory in Response to Conditional Cytotoxic/Immune-Stimulatory Gene Therapy: Humoral and Cellular Immunity Lead to Tumor Regression

PubMed Central

Muhammad, A.K.M. Ghulam; Candolfi, Marianela; King, Gwendalyn D.; Yagiz, Kader; Foulad, David; Mineharu, Yohei; Kroeger, Kurt M.; Treuer, Katherine A.; Nichols, W. Stephen; Sanderson, Nicholas S.; Yang, Jieping; Khayznikov, Maksim; Van Rooijen, Nico; Lowenstein, Pedro R.; Castro, Maria G.

2009-01-01

Purpose Glioblastoma multiforme is a deadly primary brain cancer. Because the tumor kills due to recurrences, we tested the hypothesis that a new treatment would lead to immunological memory in a rat model of recurrent glioblastoma multiforme. Experimental Design We developed a combined treatment using an adenovirus (Ad) expressing fms-like tyrosine kinase-3 ligand (Flt3L), which induces the infiltration of immune cells into the tumor microenvironment, and an Ad expressing herpes simplex virus-1–thymidine kinase (TK), which kills proliferating tumor cells in the presence of ganciclovir. Results This treatment induced immunological memory that led to rejection of a second glioblastoma multiforme implanted in the contralateral hemisphere and of an extracranial glioblastoma multiforme implanted intradermally. Rechallenged long-term survivors exhibited anti-glioblastoma multiforme–specific T cells and displayed specific delayed-type hypersensitivity. Using depleting antibodies, we showed that rejection of the second tumor was dependent on CD8+ T cells. Circulating anti-glioma antibodies were observed when glioblastoma multiforme cells were implanted intradermally in naïve rats or in long-term survivors. However, rats bearing intracranial glioblastoma multiforme only exhibited circulating antitumoral antibodies upon treatment with Ad-Flt3L + Ad-TK. This combined treatment induced tumor regression and release of the chromatin-binding protein high mobility group box 1 in two further intracranial glioblastoma multiforme models, that is, Fisher rats bearing intracranial 9L and F98 glioblastoma multiforme cells. Conclusions Treatment with Ad-Flt3L + Ad-TK triggered systemic anti–glioblastoma multiforme cellular and humoral immune responses, and anti–glioblastoma multiforme immunological memory. Release of the chromatin-binding protein high mobility group box 1 could be used as a noninvasive biomarker of therapeutic efficacy for glioblastoma multiforme. The robust treatment efficacy lends further support to its implementation in a phase I clinical trial. PMID:19789315

Cities, Crowding and Crime

ERIC Educational Resources Information Center

Trotter, Robert J.

1974-01-01

This article considers the effects of human crowding in light of recent tests and observations. Factors such as sex, age, culture, socio-economic standing, frustration, and interpersonal physical distance are examined. Results indicate that crowding contributes to social problems and crime. (TK)

Publications - PDF 91-6 | Alaska Division of Geological & Geophysical

Science.gov Websites

content DGGS PDF 91-6 Publication Details Title: Summary of Alaska's mineral industry for 1990 Authors ., Bundtzen, T.K., and Wood, J.E., 1991, Summary of Alaska's mineral industry for 1990: Alaska Division of

Genotoxic capacity of Cd/Se semiconductor quantum dots with differing surface chemistries

PubMed Central

Manshian, Bella B.; Soenen, Stefaan J.; Brown, Andy; Hondow, Nicole; Wills, John; Jenkins, Gareth J. S.; Doak, Shareen H.

2016-01-01

Quantum dots (QD) have unique electronic and optical properties promoting biotechnological advances. However, our understanding of the toxicological structure–activity relationships remains limited. This study aimed to determine the biological impact of varying nanomaterial surface chemistry by assessing the interaction of QD with either a negative (carboxyl), neutral (hexadecylamine; HDA) or positive (amine) polymer coating with human lymphoblastoid TK6 cells. Following QD physico-chemical characterisation, cellular uptake was quantified by optical and electron microscopy. Cytotoxicity was evaluated and genotoxicity was characterised using the micronucleus assay (gross chromosomal damage) and the HPRT forward mutation assay (point mutagenicity). Cellular damage mechanisms were also explored, focusing on oxidative stress and mitochondrial damage. Cell uptake, cytotoxicity and genotoxicity were found to be dependent on QD surface chemistry. Carboxyl-QD demonstrated the smallest agglomerate size and greatest cellular uptake, which correlated with a dose dependent increase in cytotoxicity and genotoxicity. Amine-QD induced minimal cellular damage, while HDA-QD promoted substantial induction of cell death and genotoxicity. However, HDA-QD were not internalised by the cells and the damage they caused was most likely due to free cadmium release caused by QD dissolution. Oxidative stress and induced mitochondrial reactive oxygen species were only partially associated with cytotoxicity and genotoxicity induced by the QD, hence were not the only mechanisms of importance. Colloidal stability, nanoparticle (NP) surface chemistry, cellular uptake levels and the intrinsic characteristics of the NPs are therefore critical parameters impacting genotoxicity induced by QD. PMID:26275419

Deoxypyrimidine monophosphate bypass therapy for thymidine kinase 2 deficiency

PubMed Central

Garone, Caterina; Garcia-Diaz, Beatriz; Emmanuele, Valentina; Lopez, Luis C; Tadesse, Saba; Akman, Hasan O; Tanji, Kurenai; Quinzii, Catarina M; Hirano, Michio

2014-01-01

Autosomal recessive mutations in the thymidine kinase 2 gene (TK2) cause mitochondrial DNA depletion, multiple deletions, or both due to loss of TK2 enzyme activity and ensuing unbalanced deoxynucleotide triphosphate (dNTP) pools. To bypass Tk2 deficiency, we administered deoxycytidine and deoxythymidine monophosphates (dCMP+dTMP) to the Tk2 H126N (Tk2−/−) knock-in mouse model from postnatal day 4, when mutant mice are phenotypically normal, but biochemically affected. Assessment of 13-day-old Tk2−/− mice treated with dCMP+dTMP 200 mg/kg/day each (Tk2−/−200dCMP/dTMP) demonstrated that in mutant animals, the compounds raise dTTP concentrations, increase levels of mtDNA, ameliorate defects of mitochondrial respiratory chain enzymes, and significantly prolong their lifespan (34 days with treatment versus 13 days untreated). A second trial of dCMP+dTMP each at 400 mg/kg/day showed even greater phenotypic and biochemical improvements. In conclusion, dCMP/dTMP supplementation is the first effective pharmacologic treatment for Tk2 deficiency. Subject Categories Genetics, Gene Therapy & Genetic Disease; Metabolism PMID:24968719

Deoxypyrimidine monophosphate bypass therapy for thymidine kinase 2 deficiency.

PubMed

Garone, Caterina; Garcia-Diaz, Beatriz; Emmanuele, Valentina; Lopez, Luis C; Tadesse, Saba; Akman, Hasan O; Tanji, Kurenai; Quinzii, Catarina M; Hirano, Michio

2014-08-01

Autosomal recessive mutations in the thymidine kinase 2 gene (TK2) cause mitochondrial DNA depletion, multiple deletions, or both due to loss of TK2 enzyme activity and ensuing unbalanced deoxynucleotide triphosphate (dNTP) pools. To bypass Tk2 deficiency, we administered deoxycytidine and deoxythymidine monophosphates (dCMP+dTMP) to the Tk2 H126N (Tk2(-/-)) knock-in mouse model from postnatal day 4, when mutant mice are phenotypically normal, but biochemically affected. Assessment of 13-day-old Tk2(-/-) mice treated with dCMP+dTMP 200 mg/kg/day each (Tk2(-/-200dCMP/) (dTMP)) demonstrated that in mutant animals, the compounds raise dTTP concentrations, increase levels of mtDNA, ameliorate defects of mitochondrial respiratory chain enzymes, and significantly prolong their lifespan (34 days with treatment versus 13 days untreated). A second trial of dCMP+dTMP each at 400 mg/kg/day showed even greater phenotypic and biochemical improvements. In conclusion, dCMP/dTMP supplementation is the first effective pharmacologic treatment for Tk2 deficiency. © 2014 The Authors. Published under the terms of the CC BY 4.0 license.

[Soil fertility characteristics under different land use patterns in depressions between karst hills].

PubMed

Liu, Yan; Song, Tong-Qing; Cai, De-Suo; Zeng, Fu-Ping; Peng, Wan-Xia; Du, Hu

2014-06-01

Soil samples were collected from the depressions between karst hills by grid sampling method (5 m x 5 m), soil pH, soil organic carbon (SOC), total nitrogen (TN), total phosphorus (TP), total potassium (TK), available nitrogen (AN), available phosphorus (AP), and available potassium (AK) in surface layer (0-20 cm) under different land use patterns (burning, cutting, cutting plus root removal, enclosure, maize plantation, and pasture plantation) were measured, the main factors of influencing the soil fertility was identified by principal component analysis (PCA), and the relationships between soil nutrients and microorganisms were demonstrated by canonical correlation analysis (CCA). The results showed that the soil was slightly alkaline (pH 7.83-7.98), and the soil fertility differed under the different land use patterns, with 76.78-116.05 g x kg(-1) of SOC, 4.29-6.23 g x kg(-1) of TN, 1.15-1.47 g x kg(-1) of TP, 3.59-6.05 g x kg(-1) of TK, 331.49-505.49 mg x kg(-1) of AN), 3.92-10.91 mg x kg(-1) of AP, and 136.28-198.10 mg x kg(-1) of AK. These soil indexes except pH showed moderate or strong variation. Different land use patterns had various impacts on soil fertility: Soil nutrients such as SOC, TN, TP, and AN were most significantly influenced by land use patterns in the depressions between karst hills; Followed by soil microorganisms, especially soil actinomycetes, and the effect decreased with the increasing gradient of human disturbance from enclosure, burning, cutting, cutting plus root removal, pasture plantation, and maize plantation. CCA elucidated that considerable interactions existed in soil TP with MBP (microbial biomass phosphorus), TK with MBC (microbial biomass carbon), TN with actinomycetes in the burned area, while TN and MBC in the cutting treatment, AP and MBN (microbial biomass nitrogen) in the treatment of cutting plus root removal, pH with MBC and fungus in the enclosure treatment, TN and TK with MBP in the maize plantation, pH with fungi and actinomycetes in the pasture plantation. Land use patterns changed the soil fertility in the depressions between karst hills; therefore, in the ecological restoration and reconstruction of karst region with fragmented landforms and shallow soil, rational land use patterns should be adopted to improve the soil quality of degraded ecosystems.

Topological transformation of a trefoil knot into a [2]catenane.

PubMed

Prakasam, Thirumurugan; Bilbeisi, Rana A; El-Khoury, Roberto; Charbonnière, Loïc J; Elhabiri, Mourad; Esposito, Gennaro; Olsen, John-Carl; Trabolsi, Ali

2017-12-21

Topological transformation of a zinc-templated trefoil knot, Zn-TK, into a zinc-templated [2]catenane, Zn-[2]C, was studied. The net reaction 2 Zn-TK→3 Zn-[2]C was accomplished in 89% yield by heating a solution of Zn-TK in D 2 O. Kinetic investigation by 1 H NMR spectroscopy and high resolution mass spectrometry revealed that the mechanism is complex, involving a large pool of intermediates that form after imine bond cleavage. Bromide ions, which can occupy the central cavity of Zn-TK, inhibited the reaction. Two similar transformations were also studied, one of a cadmium-containing trefoil knot, Cd-TK, into a cadmium-containing catenane, Cd-[2]C, and the other of Cd-TK into Zn-[2]C. The latter transformation could be achieved in one step at high temperature or in two steps via transmetallation to form Zn-TK at room temperature followed by topological conversion of Zn-TK to Zn-[2]C at high temperature.

A State Event Detection Algorithm for Numerically Simulating Hybrid Systems with Model Singularities

DTIC Science & Technology

2007-01-01

the case of non- constant step sizes. Therefore the event dynamics after the predictor and corrector phases are, respectively, gpk +1 = g( xk + hk+1{ m...the Extrapolation Polynomial Using a Taylor series expansion of the predicted event function eq.(6) gpk +1 = gk + hk+1 dgp dt ∣∣∣∣ (x,t)=(xk,tk) + h2k...1 2! d2gp dt2 ∣∣∣∣ (x,t)=(xk,tk) + . . . , (8) we can determine the value of gpk +1 as a function of the, yet undetermined, step size hk+1. Recalling

Identifying populations sensitive to environmental chemicals by simulating toxicokinetic variability.

PubMed

Ring, Caroline L; Pearce, Robert G; Setzer, R Woodrow; Wetmore, Barbara A; Wambaugh, John F

2017-09-01

The thousands of chemicals present in the environment (USGAO, 2013) must be triaged to identify priority chemicals for human health risk research. Most chemicals have little of the toxicokinetic (TK) data that are necessary for relating exposures to tissue concentrations that are believed to be toxic. Ongoing efforts have collected limited, in vitro TK data for a few hundred chemicals. These data have been combined with biomonitoring data to estimate an approximate margin between potential hazard and exposure. The most "at risk" 95th percentile of adults have been identified from simulated populations that are generated either using standard "average" adult human parameters or very specific cohorts such as Northern Europeans. To better reflect the modern U.S. population, we developed a population simulation using physiologies based on distributions of demographic and anthropometric quantities from the most recent U.S. Centers for Disease Control and Prevention National Health and Nutrition Examination Survey (NHANES) data. This allowed incorporation of inter-individual variability, including variability across relevant demographic subgroups. Variability was analyzed with a Monte Carlo approach that accounted for the correlation structure in physiological parameters. To identify portions of the U.S. population that are more at risk for specific chemicals, physiologic variability was incorporated within an open-source high-throughput (HT) TK modeling framework. We prioritized 50 chemicals based on estimates of both potential hazard and exposure. Potential hazard was estimated from in vitro HT screening assays (i.e., the Tox21 and ToxCast programs). Bioactive in vitro concentrations were extrapolated to doses that produce equivalent concentrations in body tissues using a reverse dosimetry approach in which generic TK models are parameterized with: 1) chemical-specific parameters derived from in vitro measurements and predicted from chemical structure; and 2) with physiological parameters for a virtual population. For risk-based prioritization of chemicals, predicted bioactive equivalent doses were compared to demographic-specific inferences of exposure rates that were based on NHANES urinary analyte biomonitoring data. The inclusion of NHANES-derived inter-individual variability decreased predicted bioactive equivalent doses by 12% on average for the total population when compared to previous methods. However, for some combinations of chemical and demographic groups the margin was reduced by as much as three quarters. This TK modeling framework allows targeted risk prioritization of chemicals for demographic groups of interest, including potentially sensitive life stages and subpopulations. Published by Elsevier Ltd.

Comparative investigations of sodium arsenite, arsenic trioxide and cadmium sulphate in combination with gamma-radiation on apoptosis, micronuclei induction and DNA damage in a human lymphoblastoid cell line.

PubMed

Hornhardt, Sabine; Gomolka, Maria; Walsh, Linda; Jung, Thomas

2006-08-30

In the field of radiation protection the combined exposure to radiation and other toxic agents is recognised as an important research area. To elucidate the basic mechanisms of simultaneous exposure, the interaction of the carcinogens and environmental toxicants cadmium and two arsenic compounds, arsenite and arsenic trioxide, in combination with gamma-radiation in human lymphoblastoid cells (TK6) were investigated. Gamma-radiation induced significant genotoxic effects such as micronuclei formation, DNA damage and apoptosis, whereas arsenic and cadmium had no significant effect on these indicators of cellular damage at non-toxic concentrations. However, in combination with gamma-radiation arsenic trioxide induced a more than additive apoptotic rate compared to the sum of the single effects. Here, the level of apoptotic cells was increased, in a dose-dependent way, up to two-fold compared to the irradiated control cells. Arsenite did not induce a significant additive effect at any of the concentrations or radiation doses tested. On the other hand, arsenic trioxide was less effective than arsenite in the induction of DNA protein cross-links. These data indicate that the two arsenic compounds interact through different pathways in the cell. Cadmium sulphate, like arsenite, had no significant effect on apoptosis in combination with gamma-radiation at low concentrations and, at high concentrations, even reduced the radiation-induced apoptosis. An additive effect on micronuclei induction was observed with 1muM cadmium sulphate with an increase of up to 80% compared to the irradiated control cells. Toxic concentrations of cadmium and arsenic trioxide seemed to reduce micronuclei induction. The results presented here indicate that relatively low concentrations of arsenic and cadmium, close to those occuring in nature, may interfere with radiation effects. Differences in action of the two arsenic compounds were identified.

Clinical significance and diagnostic capacity of serum TK1, CEA, CA 19-9 and CA 72-4 levels in gastric and colorectal cancer patients.

PubMed

Ning, Shufang; Wei, Wene; Li, Jilin; Hou, Bingbing; Zhong, Jianhong; Xie, Yuxuan; Liu, Haizhou; Mo, Xianwei; Chen, Jiansi; Zhang, Litu

2018-01-01

Despite extensive progress in treatment for cancer in recent decades, the early diagnosis for gastric cancer (GC) and colorectal cancer (CRC) remains poor. In this study, we explore the diagnostic value of joint detection of thymidine kinase 1 (TK1), carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA 19-9) and carbohydrate antigen 72-4 (CA 72-4) in the diagnosis of GC and CRC, and to evaluated the relationship between TK1 expression and clinical pathological characteristics in the patients. Serum TK1, CA 19-9, CA 72-4 and CEA levels were measured in 169 patients with GC, 344 patients with CRC and 75 healthy controls using electro-chemiluminescence. The TK1 concentration was significantly higher in patients with cancer than in healthy controls and patients with clinical stage Ⅲ+Ⅳ had higher TK1 levels than clinical stage Ⅰ+Ⅱ ( P <0.05). The levels of TK1 is significantly associated with tumor stage, lymph node metastasis, distant metastasis, tumor differentiation and age ( P <0.05). When the tumor markers (TK1, CA 19-9 and CA 72-4) were detected respectively, the area under receiver operating characteristics curve (AUC) of TK1 for three cancers was the highest (0.823-0.895). However, the combination of AUC was higher than that for each tumor marker detected respectively (0.934-0.953), and the Hosmer-Lemeshow test showed an adequate model of calibration (P>0.05). Moreover, the AUCs varied significantly between the combination tests and single biomarker tests (Z test, P <0.01). In conclusion, serum TK1 may be an independent tumor marker for GC and CRC patients, and the combination of TK1, CA 19-9 and CA 72-4 and CEA performed even better. This study suggests that combination detection of four tumor markers may prove to be useful for the diagnosis of GC and CRC.

Co-Targeting Prostate Cancer Epithelium and Bone Stroma by Human Osteonectin-Promoter-Mediated Suicide Gene Therapy Effectively Inhibits Androgen-Independent Prostate Cancer Growth.

PubMed

Sung, Shian-Ying; Chang, Junn-Liang; Chen, Kuan-Chou; Yeh, Shauh-Der; Liu, Yun-Ru; Su, Yen-Hao; Hsueh, Chia-Yen; Chung, Leland W K; Hsieh, Chia-Ling

2016-01-01

Stromal-epithelial interaction has been shown to promote local tumor growth and distant metastasis. We sought to create a promising gene therapy approach that co-targets cancer and its supporting stromal cells for combating castration-resistant prostate tumors. Herein, we demonstrated that human osteonectin is overexpressed in the prostate cancer epithelium and tumor stroma in comparison with their normal counterpart. We designed a novel human osteonectin promoter (hON-522E) containing positive transcriptional regulatory elements identified in both the promoter and exon 1 region of the human osteonectin gene. In vitro reporter assays revealed that the hON-522E promoter is highly active in androgen receptor negative and metastatic prostate cancer and bone stromal cells compared to androgen receptor-positive prostate cancer cells. Moreover, in vivo prostate-tumor-promoting activity of the hON-522E promoter was confirmed by intravenous administration of an adenoviral vector containing the hON-522E promoter-driven luciferase gene (Ad-522E-Luc) into mice bearing orthotopic human prostate tumor xenografts. In addition, an adenoviral vector with the hON-522E-promoter-driven herpes simplex virus thymidine kinase gene (Ad-522E-TK) was highly effective against the growth of androgen-independent human prostate cancer PC3M and bone stromal cell line in vitro and in pre-established PC3M tumors in vivo upon addition of the prodrug ganciclovir. Because of the heterogeneity of human prostate tumors, hON-522E promoter-mediated gene therapy has the potential for the treatment of hormone refractory and bone metastatic prostate cancers.

Loss of thymidine kinase 2 alters neuronal bioenergetics and leads to neurodegeneration

PubMed Central

Bartesaghi, Stefano; Betts-Henderson, Joanne; Cain, Kelvin; Dinsdale, David; Zhou, Xiaoshan; Karlsson, Anna; Salomoni, Paolo; Nicotera, Pierluigi

2010-01-01

Mutations of thymidine kinase 2 (TK2), an essential component of the mitochondrial nucleotide salvage pathway, can give rise to mitochondrial DNA (mtDNA) depletion syndromes (MDS). These clinically heterogeneous disorders are characterized by severe reduction in mtDNA copy number in affected tissues and are associated with progressive myopathy, hepatopathy and/or encephalopathy, depending in part on the underlying nuclear genetic defect. Mutations of TK2 have previously been associated with an isolated myopathic form of MDS (OMIM 609560). However, more recently, neurological phenotypes have been demonstrated in patients carrying TK2 mutations, thus suggesting that loss of TK2 results in neuronal dysfunction. Here, we directly address the role of TK2 in neuronal homeostasis using a knockout mouse model. We demonstrate that in vivo loss of TK2 activity leads to a severe ataxic phenotype, accompanied by reduced mtDNA copy number and decreased steady-state levels of electron transport chain proteins in the brain. In TK2-deficient cerebellar neurons, these abnormalities are associated with impaired mitochondrial bioenergetic function, aberrant mitochondrial ultrastructure and degeneration of selected neuronal types. Overall, our findings demonstrate that TK2 deficiency leads to neuronal dysfunction in vivo, and have important implications for understanding the mechanisms of neurological impairment in MDS. PMID:20123860

Loss of thymidine kinase 2 alters neuronal bioenergetics and leads to neurodegeneration.

PubMed

Bartesaghi, Stefano; Betts-Henderson, Joanne; Cain, Kelvin; Dinsdale, David; Zhou, Xiaoshan; Karlsson, Anna; Salomoni, Paolo; Nicotera, Pierluigi

2010-05-01

Mutations of thymidine kinase 2 (TK2), an essential component of the mitochondrial nucleotide salvage pathway, can give rise to mitochondrial DNA (mtDNA) depletion syndromes (MDS). These clinically heterogeneous disorders are characterized by severe reduction in mtDNA copy number in affected tissues and are associated with progressive myopathy, hepatopathy and/or encephalopathy, depending in part on the underlying nuclear genetic defect. Mutations of TK2 have previously been associated with an isolated myopathic form of MDS (OMIM 609560). However, more recently, neurological phenotypes have been demonstrated in patients carrying TK2 mutations, thus suggesting that loss of TK2 results in neuronal dysfunction. Here, we directly address the role of TK2 in neuronal homeostasis using a knockout mouse model. We demonstrate that in vivo loss of TK2 activity leads to a severe ataxic phenotype, accompanied by reduced mtDNA copy number and decreased steady-state levels of electron transport chain proteins in the brain. In TK2-deficient cerebellar neurons, these abnormalities are associated with impaired mitochondrial bioenergetic function, aberrant mitochondrial ultrastructure and degeneration of selected neuronal types. Overall, our findings demonstrate that TK2 deficiency leads to neuronal dysfunction in vivo, and have important implications for understanding the mechanisms of neurological impairment in MDS.

Safety profile, efficacy, and biodistribution of a bicistronic high-capacity adenovirus vector encoding a combined immunostimulation and cytotoxic gene therapy as a prelude to a phase I clinical trial for glioblastoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Puntel, Mariana; Department of Cell and Developmental Biology, The University of Michigan School of Medicine, MSRB II, RM 4570C, 1150 West Medical Center Drive, Ann Arbor, MI 48109-5689; Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048

2013-05-01

Adenoviral vectors (Ads) are promising gene delivery vehicles due to their high transduction efficiency; however, their clinical usefulness has been hampered by their immunogenicity and the presence of anti-Ad immunity in humans. We reported the efficacy of a gene therapy approach for glioma consisting of intratumoral injection of Ads encoding conditionally cytotoxic herpes simplex type 1 thymidine kinase (Ad-TK) and the immunostimulatory cytokine fms-like tyrosine kinase ligand 3 (Ad-Flt3L). Herein, we report the biodistribution, efficacy, and neurological and systemic effects of a bicistronic high-capacity Ad, i.e., HC-Ad-TK/TetOn-Flt3L. HC-Ads elicit sustained transgene expression, even in the presence of anti-Ad immunity, andmore » can encode large therapeutic cassettes, including regulatory elements to enable turning gene expression “on” or “off” according to clinical need. The inclusion of two therapeutic transgenes within a single vector enables a reduction of the total vector load without adversely impacting efficacy. Because clinically the vectors will be delivered into the surgical cavity, normal regions of the brain parenchyma are likely to be transduced. Thus, we assessed any potential toxicities elicited by escalating doses of HC-Ad-TK/TetOn-Flt3L (1 × 10{sup 8}, 1 × 10{sup 9}, or 1 × 10{sup 10} viral particles [vp]) delivered into the rat brain parenchyma. We assessed neuropathology, biodistribution, transgene expression, systemic toxicity, and behavioral impact at acute and chronic time points. The results indicate that doses up to 1 × 10{sup 9} vp of HC-Ad-TK/TetOn-Flt3L can be safely delivered into the normal rat brain and underpin further developments for its implementation in a phase I clinical trial for glioma. - Highlights: ► High capacity Ad vectors elicit sustained therapeutic gene expression in the brain. ► HC-Ad-TK/TetOn-Flt3L encodes two therapeutic genes and a transcriptional switch. ► We performed a dose escalation study at acute and chronic time points. ► Doses up to 1 × 10{sup 9} vp of HC-Ad-TK/TetOn-Flt3L can be safely delivered in the brain. ► Efficacy and safety of HC-Ad-TK/TetOn-Flt3L merits its use in a GBM Phase I trial.« less

Inter-Individual Variability in High-Throughput Risk Prioritization of Environmental Chemicals (Sot)

EPA Science Inventory

We incorporate realistic human variability into an open-source high-throughput (HT) toxicokinetics (TK) modeling framework for use in a next-generation risk prioritization approach. Risk prioritization involves rapid triage of thousands of environmental chemicals, most which have...

Two novel mutations in thymidine kinase-2 cause early onset fatal encephalomyopathy and severe mtDNA depletion.

PubMed

Lesko, Nicole; Naess, Karin; Wibom, Rolf; Solaroli, Nicola; Nennesmo, Inger; von Döbeln, Ulrika; Karlsson, Anna; Larsson, Nils-Göran

2010-03-01

Deficiency of thymidine kinase-2 (TK2) has been described in children with early onset fatal skeletal myopathy. TK2 is a mitochondrial deoxyribonucleoside kinase required for the phosphorylation of deoxycytidine and deoxythymidine and hence is vital for the maintenance of a balanced mitochondrial dNTP pool in post-mitotic tissues. We describe a patient with two novel TK2 mutations, which caused disease onset shortly after birth and death at the age of three months. One mutation (219insCG) generated an early stop codon, thus preventing the synthesis of a functional protein. The second mutation (R130W) resulted in an amino acid substitution, which caused a severe reduction (<3%) of TK2 enzyme activity. These two novel TK2 mutations cause an extremely severe phenotype with overwhelming central nervous system symptoms not commonly seen in patients with TK2-deficiency. We conclude that the severe clinical presentation in this patient was due to a virtual lack of mitochondrial TK2 activity. Copyright 2009 Elsevier B.V. All rights reserved.

Vitamin K-dependent proteins GAS6 and Protein S and TAM receptors in patients of systemic lupus erythematosus: correlation with common genetic variants and disease activity

PubMed Central

2013-01-01

Introduction Growth arrest-specific gene 6 protein (GAS6) and protein S (ProS) are vitamin K-dependent proteins present in plasma with important regulatory functions in systems of response and repair to damage. They interact with receptor tyrosine kinases of the Tyro3, Axl and MerTK receptor tyrosine kinase (TAM) family, involved in apoptotic cell clearance (efferocytosis) and regulation of the innate immunity. TAM-deficient mice show spontaneous lupus-like symptoms. Here we tested the genetic profile and plasma levels of components of the system in patients with systemic lupus erythematosus (SLE), and compare them with a control healthy population. Methods Fifty SLE patients and 50 healthy controls with matched age, gender and from the same geographic area were compared. Genetic analysis was performed in GAS6 and the TAM receptor genes on SNPs previously identified. The concentrations of GAS6, total and free ProS, and the soluble forms of the three TAM receptors (sAxl, sMerTK and sTyro3) were measured in plasma from these samples. Results Plasma concentrations of GAS6 were higher and, total and free ProS were lower in the SLE patients compared to controls, even when patients on oral anticoagulant treatment were discarded. Those parameters correlated with SLE disease activity index (SLEDAI) score, GAS6 being higher in the most severe cases, while free and total ProS were lower. All 3 soluble receptors increased its concentration in plasma of lupus patients. Conclusions The present study highlights that the GAS6/ProS-TAM system correlates in several ways with disease activity in SLE. We show here that this correlation is affected by common polymorphisms in the genes of the system. These findings underscore the importance of mechanism of regulatory control of innate immunity in the pathology of SLE. PMID:23497733

Vitamin K-dependent proteins GAS6 and Protein S and TAM receptors in patients of systemic lupus erythematosus: correlation with common genetic variants and disease activity.

PubMed

Recarte-Pelz, Pedro; Tàssies, Dolors; Espinosa, Gerard; Hurtado, Begoña; Sala, Núria; Cervera, Ricard; Reverter, Joan Carles; de Frutos, Pablo García

2013-03-12

Growth arrest-specific gene 6 protein (GAS6) and protein S (ProS) are vitamin K-dependent proteins present in plasma with important regulatory functions in systems of response and repair to damage. They interact with receptor tyrosine kinases of the Tyro3, Axl and MerTK receptor tyrosine kinase (TAM) family, involved in apoptotic cell clearance (efferocytosis) and regulation of the innate immunity. TAM-deficient mice show spontaneous lupus-like symptoms. Here we tested the genetic profile and plasma levels of components of the system in patients with systemic lupus erythematosus (SLE), and compare them with a control healthy population. Fifty SLE patients and 50 healthy controls with matched age, gender and from the same geographic area were compared. Genetic analysis was performed in GAS6 and the TAM receptor genes on SNPs previously identified. The concentrations of GAS6, total and free ProS, and the soluble forms of the three TAM receptors (sAxl, sMerTK and sTyro3) were measured in plasma from these samples. Plasma concentrations of GAS6 were higher and, total and free ProS were lower in the SLE patients compared to controls, even when patients on oral anticoagulant treatment were discarded. Those parameters correlated with SLE disease activity index (SLEDAI) score, GAS6 being higher in the most severe cases, while free and total ProS were lower. All 3 soluble receptors increased its concentration in plasma of lupus patients. The present study highlights that the GAS6/ProS-TAM system correlates in several ways with disease activity in SLE. We show here that this correlation is affected by common polymorphisms in the genes of the system. These findings underscore the importance of mechanism of regulatory control of innate immunity in the pathology of SLE.

Influx of kininogens into nasal secretions after antigen challenge of allergic individuals.

PubMed Central

Baumgarten, C R; Togias, A G; Naclerio, R M; Lichtenstein, L M; Norman, P S; Proud, D

1985-01-01

We have recently demonstrated that kinins are generated in vivo after nasal challenge with antigen of allergic, but not nonallergic, individuals. The present study was undertaken as a first step in determining the mechanism(s) of kinin formation during the allergic reaction and was directed towards establishing the availability and origin of kininogens in nasal secretions. Allergic individuals (n = 6) and nonallergic controls (n = 5) were challenged with antigen; and by using specific radioimmunoassays, nasal washes, obtained before and after challenge, were assayed for high molecular weight kininogen (HMWK), total kininogen (TK), albumin, and kinins. Dramatic increases in HMWK (1,730 +/- 510 ng/ml), TK (3,810 +/- 1035 ng/ml), kinin (9.46 +/- 1.75 ng/ml), and albumin (0.85 +/- 0.2 mg/ml) were observed after challenge of allergic individuals which correlated (P less than 0.001) with increases in histamine and N-alpha-tosyl-L-arginine methyl esterase activity and with the onset of clinical symptoms. For nonallergic individuals, levels of kininogens, albumin, and all mediators after antigen challenge were not different from base line. Linear regression analysis revealed excellent correlations (P less than 0.001 in each case) between increases in HMWK, TK, kinin, and albumin during antigen titration experiments and between the time courses of appearance and disappearance of HMWK, TK, kinin, and albumin after antigen challenge. Gel filtration revealed no evidence of degradation products of kininogens in nasal washes. For each allergic individual the ratio of HMWK/TK in postchallenge nasal washes was similar to the ratio of these two proteins in the same individual's plasma. These data suggest that, during the allergic reaction, there is an increase in vascular permeability and a transudation of kininogens from plasma into nasal secretions, where they can provide substrate for kinin-forming enzymes. PMID:4019778

Embedded-Based Graphics Processing Unit Cluster Platform for Multiple Sequence Alignments

PubMed Central

Wei, Jyh-Da; Cheng, Hui-Jun; Lin, Chun-Yuan; Ye, Jin; Yeh, Kuan-Yu

2017-01-01

High-end graphics processing units (GPUs), such as NVIDIA Tesla/Fermi/Kepler series cards with thousands of cores per chip, are widely applied to high-performance computing fields in a decade. These desktop GPU cards should be installed in personal computers/servers with desktop CPUs, and the cost and power consumption of constructing a GPU cluster platform are very high. In recent years, NVIDIA releases an embedded board, called Jetson Tegra K1 (TK1), which contains 4 ARM Cortex-A15 CPUs and 192 Compute Unified Device Architecture cores (belong to Kepler GPUs). Jetson Tegra K1 has several advantages, such as the low cost, low power consumption, and high applicability, and it has been applied into several specific applications. In our previous work, a bioinformatics platform with a single TK1 (STK platform) was constructed, and this previous work is also used to prove that the Web and mobile services can be implemented in the STK platform with a good cost-performance ratio by comparing a STK platform with the desktop CPU and GPU. In this work, an embedded-based GPU cluster platform will be constructed with multiple TK1s (MTK platform). Complex system installation and setup are necessary procedures at first. Then, 2 job assignment modes are designed for the MTK platform to provide services for users. Finally, ClustalW v2.0.11 and ClustalWtk will be ported to the MTK platform. The experimental results showed that the speedup ratios achieved 5.5 and 4.8 times for ClustalW v2.0.11 and ClustalWtk, respectively, by comparing 6 TK1s with a single TK1. The MTK platform is proven to be useful for multiple sequence alignments. PMID:28835734

Male death resulting from hybridization between subspecies of the gypsy moth, Lymantria dispar

PubMed Central

Higashiura, Y; Yamaguchi, H; Ishihara, M; Ono, N; Tsukagoshi, H; Yokobori, S; Tokishita, S; Yamagata, H; Fukatsu, T

2011-01-01

We explored the origin of all-female broods resulting from male death in a Hokkaido population of Lymantria dispar through genetic crosses based on the earlier experiments done by Goldschmidt and by testing for the presence of endosymbionts that are known to cause male killing in some insect species. The mitochondrial DNA haplotypes of the all-female broods in Hokkaido were different from those of normal Hokkaido females and were the same as those widely distributed in Asia, including Tokyo (TK). Goldschmidt obtained all-female broods through backcrossing, that is, F1 females obtained by a cross between TK females (L. dispar japonica) and Hokkaido males (L. dispar praeterea) mated with Hokkaido males. He also obtained all-male broods by mating Hokkaido females with TK males. Goldschmidt inferred that female- and male-determining factors were weakest in the Hokkaido subspecies and stronger in the Honshu (TK) subspecies. According to his theory, the females of all-female broods mated with Honshu males should produce normal sex-ratio broods, whereas weaker Hokkaido sexes would be expected to disappear in F1 or F2 generations after crossing with the Honshu subspecies. We confirmed both of Goldschmidt's results: in the case of all-female broods mated with Honshu males, normal sex-ratio broods were produced, but we obtained only all-female broods in the Goldschmidt backcross and obtained an all-male brood in the F1 generation of a Hokkaido female crossed with a TK male. We found no endosymbionts in all-female broods by 4,′6-diamidino-2-phenylindole (DAPI) staining. Therefore, the all-female broods observed in L. dispar are caused by some incompatibilities between Honshu and Hokkaido subspecies. PMID:20628417

Embedded-Based Graphics Processing Unit Cluster Platform for Multiple Sequence Alignments.

PubMed

Wei, Jyh-Da; Cheng, Hui-Jun; Lin, Chun-Yuan; Ye, Jin; Yeh, Kuan-Yu

2017-01-01

High-end graphics processing units (GPUs), such as NVIDIA Tesla/Fermi/Kepler series cards with thousands of cores per chip, are widely applied to high-performance computing fields in a decade. These desktop GPU cards should be installed in personal computers/servers with desktop CPUs, and the cost and power consumption of constructing a GPU cluster platform are very high. In recent years, NVIDIA releases an embedded board, called Jetson Tegra K1 (TK1), which contains 4 ARM Cortex-A15 CPUs and 192 Compute Unified Device Architecture cores (belong to Kepler GPUs). Jetson Tegra K1 has several advantages, such as the low cost, low power consumption, and high applicability, and it has been applied into several specific applications. In our previous work, a bioinformatics platform with a single TK1 (STK platform) was constructed, and this previous work is also used to prove that the Web and mobile services can be implemented in the STK platform with a good cost-performance ratio by comparing a STK platform with the desktop CPU and GPU. In this work, an embedded-based GPU cluster platform will be constructed with multiple TK1s (MTK platform). Complex system installation and setup are necessary procedures at first. Then, 2 job assignment modes are designed for the MTK platform to provide services for users. Finally, ClustalW v2.0.11 and ClustalWtk will be ported to the MTK platform. The experimental results showed that the speedup ratios achieved 5.5 and 4.8 times for ClustalW v2.0.11 and ClustalWtk, respectively, by comparing 6 TK1s with a single TK1. The MTK platform is proven to be useful for multiple sequence alignments.

Universal and measurable entanglement entropy in the spin-boson model.

PubMed

Kopp, Angela; Le Hur, Karyn

2007-06-01

We study the entanglement between a qubit and its environment from the spin-boson model with Ohmic dissipation. Through a mapping to the anisotropic Kondo model, we derive the entropy of entanglement of the spin E(alpha,Delta,h), where alpha is the dissipation strength, Delta is the tunneling amplitude between qubit states, and h is the level asymmetry. For 1-alpha>Delta/omegac and (Delta,h)TK, E vanishes as (TK/h)2-2alpha, up to a logarithmic correction. For a given h, the maximum entanglement occurs at a value of alpha which lies in the crossover regime h approximately TK. We emphasize the possibility of measuring this entanglement using charge qubits subject to electromagnetic noise.

Discovery of a series of novel phenylpiperazine derivatives as EGFR TK inhibitors

NASA Astrophysics Data System (ADS)

Sun, Juan; Wang, Xin-Yi; Lv, Peng-Cheng; Zhu, Hai-Liang

2015-09-01

Human epidermal growth factor receptor (EGFR) is an important drug target that plays a fundamental role in signal transduction pathways in oncology. We report herein the discovery of a novel class of phenylpiperazine derivatives with improved potency toward EGFR. The biological activity of compound 3p as inhibitor of EGFR was further investigated both in vitro and in vivo. Notably, compound 3p exhibited an IC50 in the nanomolar range in A549 cell cultures and induced a cessation of tumor growth with no toxicity, as determined by loss of body weight and death of treated mice. Compoutational docking studies also showed that compound 3p has interaction with EGFR key residues in the active site.

Prevalence of Intrathecal Acyclovir Resistant Virus in Herpes Simplex Encephalitis Patients.

PubMed

Mitterreiter, Johanna G; Titulaer, Maarten J; van Nierop, Gijsbert P; van Kampen, Jeroen J A; Aron, Georgina I; Osterhaus, Albert D M E; Verjans, Georges M G M; Ouwendijk, Werner J D

2016-01-01

Herpes simplex encephalitis (HSE) is a life-threatening complication of herpes simplex virus (HSV) infection. Acyclovir (ACV) is the antiviral treatment of choice, but may lead to emergence of ACV-resistant (ACVR) HSV due to mutations in the viral UL23 gene encoding for the ACV-targeted thymidine kinase (TK) protein. Here, we determined the prevalence of intrathecal ACVR-associated HSV TK mutations in HSE patients and compared TK genotypes of sequential HSV isolates in paired cerebrospinal fluid (CSF) and blister fluid of mucosal HSV lesions. Clinical samples were obtained from 12 HSE patients, encompassing 4 HSV type 1 (HSV-1) and 8 HSV-2 encephalitis patients. HSV DNA load was determined by real-time PCR and complete HSV TK gene sequences were obtained by nested PCR followed by Sanger sequencing. All HSV-1 HSE patients contained viral TK mutations encompassing 30 unique nucleotide and 13 distinct amino acid mutations. By contrast, a total of 5 unique nucleotide and 4 distinct amino acid changes were detected in 7 of 8 HSV-2 patients. Detected mutations were identified as natural polymorphisms located in non-conserved HSV TK gene regions. ACV therapy did not induce the emergence of ACVR-associated HSV TK mutations in consecutive CSF and mucocutaneous samples of 5 individual patients. Phenotypic susceptibility analysis of these mucocutaneous HSV isolates demonstrated ACV-sensitive virus in 2 HSV-1 HSE patients, whereas in two HSV-2 HSE patients ACVR virus was detected in the absence of known ACVR-associated TK mutations. In conclusion, we did not detect intrathecal ACVR-associated TK mutations in HSV isolates obtained from 12 HSE patients.

mtDNA depletion myopathy: elucidation of the tissue specificity in the mitochondrial thymidine kinase (TK2) deficiency.

PubMed

Saada, Ann; Shaag, Avraham; Elpeleg, Orly

2003-05-01

Decreased mitochondrial thymidine kinase (TK2) activity is associated with mitochondrial DNA (mtDNA) depletion and respiratory chain dysfunction and is manifested by isolated, fatal skeletal myopathy. Other tissues such as liver, brain, heart, and skin remain unaffected throughout the patients' life. In order to elucidate the mechanism of tissue specificity in the disease we have investigated the expression of the mitochondrial deoxynucleotide carrier, the mtDNA content and the activity of TK2 in mitochondria of various tissues. Our results suggest that low basal TK2 activity combined with a high requirement for mitochondrial encoded proteins in muscle predispose this tissue to the devastating effect of TK2 deficiency.

Human variability in high-throughput risk prioritization of environmental chemicals (Texas AM U. webinar)

EPA Science Inventory

We incorporate inter-individual variability into an open-source high-throughput (HT) toxicokinetics (TK) modeling framework for use in a next-generation risk prioritization approach. Risk prioritization involves rapid triage of thousands of environmental chemicals, most which hav...

High Throughput Determination of Critical Human Dosing Parameters (SOT)

EPA Science Inventory

High throughput toxicokinetics (HTTK) is a rapid approach that uses in vitro data to estimate TK for hundreds of environmental chemicals. Reverse dosimetry (i.e., reverse toxicokinetics or RTK) based on HTTK data converts high throughput in vitro toxicity screening (HTS) data int...

Adult cases of mitochondrial DNA depletion due to TK2 defect: an expanding spectrum.

PubMed

Béhin, A; Jardel, C; Claeys, K G; Fagart, J; Louha, M; Romero, N B; Laforêt, P; Eymard, B; Lombès, A

2012-02-28

In this study we aim to demonstrate the occurrence of adult forms of TK2 mutations causing progressive mitochondrial myopathy with significant muscle mitochondrial DNA (mtDNA) depletion. Patients' investigations included serum creatine kinase, blood lactate, electromyographic, echocardiographic, and functional respiratory analyses as well as TK2 gene sequencing and TK2 activity measurement. Mitochondrial activities and mtDNA were analyzed in the patients' muscle biopsy. The 3 adult patients with TK2 mutations presented with slowly progressive myopathy compatible with a fairly normal life during decades. Apart from its much slower progression, these patients' phenotype closely resembled that of pediatric cases including early onset, absence of CNS symptoms, generalized muscle weakness predominating on axial and proximal muscles but affecting facial, ocular, and respiratory muscles, typical mitochondrial myopathy with a mosaic pattern of COX-negative and ragged-red fibers, combined mtDNA-dependent respiratory complexes deficiency and mtDNA depletion. In accordance with the disease's relatively slow progression, the residual mtDNA content was higher than that observed in pediatric cases. That difference was not explained by the type of the TK2 mutations or by the residual TK2 activity. TK2 mutations can cause mitochondrial myopathy with a slow progression. Comparison of patients with similar mutations but different disease progression might address potential mechanisms of mtDNA maintenance modulation.

Thermostable Alcohol Dehydrogenase from Thermococcus kodakarensis KOD1 for Enantioselective Bioconversion of Aromatic Secondary Alcohols

PubMed Central

Wu, Xi; Zhang, Chong; Orita, Izumi; Imanaka, Tadayuki

2013-01-01

A novel thermostable alcohol dehydrogenase (ADH) showing activity toward aromatic secondary alcohols was identified from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (TkADH). The gene, tk0845, which encodes an aldo-keto reductase, was heterologously expressed in Escherichia coli. The enzyme was found to be a monomer with a molecular mass of 31 kDa. It was highly thermostable with an optimal temperature of 90°C and a half-life of 4.5 h at 95°C. The apparent Km values for the cofactors NAD(P)+ and NADPH were similar within a range of 66 to 127 μM. TkADH preferred secondary alcohols and accepted various ketones and aldehydes as substrates. Interestingly, the enzyme could oxidize 1-phenylethanol and its derivatives having substituents at the meta and para positions with high enantioselectivity, yielding the corresponding (R)-alcohols with optical purities of greater than 99.8% enantiomeric excess (ee). TkADH could also reduce 2,2,2-trifluoroacetophenone to (R)-2,2,2-trifluoro-1-phenylethanol with high enantioselectivity (>99.6% ee). Furthermore, the enzyme showed high resistance to organic solvents and was particularly highly active in the presence of H2O–20% 2-propanol and H2O–50% n-hexane or n-octane. This ADH is expected to be a useful tool for the production of aromatic chiral alcohols. PMID:23354700

Insulin Resistance Induced by Hyperinsulinemia Coincides with a Persistent Alteration at the Insulin Receptor Tyrosine Kinase Domain

PubMed Central

Catalano, Karyn J.; Maddux, Betty A.; Szary, Jaroslaw; Youngren, Jack F.; Goldfine, Ira D.; Schaufele, Fred

2014-01-01

Insulin resistance, the diminished response of target tissues to insulin, is associated with the metabolic syndrome and a predisposition towards diabetes in a growing proportion of the worldwide population. Under insulin resistant states, the cellular response of the insulin signaling pathway is diminished and the body typically responds by increasing serum insulin concentrations to maintain insulin signaling. Some evidence indicates that the increased insulin concentration may itself further dampen insulin response. If so, insulin resistance would worsen as the level of circulating insulin increases during compensation, which could contribute to the transition of insulin resistance to more severe disease. Here, we investigated the consequences of excess insulin exposure to insulin receptor (IR) activity. Cells chronically exposed to insulin show a diminished the level of IR tyrosine and serine autophosphorylation below that observed after short-term insulin exposure. The diminished IR response did not originate with IR internalization since IR amounts at the cell membrane were similar after short- and long-term insulin incubation. Förster resonance energy transfer between fluorophores attached to the IR tyrosine kinase (TK) domain showed that a change in the TK domain occurred upon prolonged, but not short-term, insulin exposure. Even though the altered ‘insulin refractory’ IR TK FRET and IR autophosphorylation levels returned to baseline (non-stimulated) levels after wash-out of the original insulin stimulus, subsequent short-term exposure to insulin caused immediate re-establishment of the insulin-refractory levels. This suggests that some cell-based ‘memory’ of chronic hyperinsulinemic exposure acts directly at the IR. An improved understanding of that memory may help define interventions to reset the IR to full insulin responsiveness and impede the progression of insulin resistance to more severe disease states. PMID:25259572

Identification of a Novel Aminopropyltransferase Involved in the Synthesis of Branched-Chain Polyamines in Hyperthermophiles

PubMed Central

Okada, Kazuma; Hidese, Ryota; Fukuda, Wakao; Niitsu, Masaru; Takao, Koichi; Horai, Yuhei; Umezawa, Naoki; Higuchi, Tsunehiko; Oshima, Tairo; Yoshikawa, Yuko; Imanaka, Tadayuki

2014-01-01

Longer- and/or branched-chain polyamines are unique polycations found in thermophiles. N4-aminopropylspermine is considered a major polyamine in Thermococcus kodakarensis. To determine whether a quaternary branched penta-amine, N4-bis(aminopropyl)spermidine, an isomer of N4-aminopropylspermine, was also present, acid-extracted cytoplasmic polyamines were analyzed by high-pressure liquid chromatography, gas chromatography (HPLC), and gas chromatography-mass spectrometry. N4-bis(aminopropyl)spermidine was an abundant cytoplasmic polyamine in this species. To identify the enzyme that catalyzes N4-bis(aminopropyl)spermidine synthesis, the active fraction was concentrated from the cytoplasm and analyzed by linear ion trap–time of flight mass spectrometry with an electrospray ionization instrument after analysis by the MASCOT database. TK0545, TK0548, TK0967, and TK1691 were identified as candidate enzymes, and the corresponding genes were individually cloned and expressed in Escherichia coli. Recombinant forms were purified, and their N4-bis(aminopropyl)spermidine synthesis activity was measured. Of the four candidates, TK1691 (BpsA) was found to synthesize N4-bis(aminopropyl)spermidine from spermidine via N4-aminopropylspermidine. Compared to the wild type, the bpsA-disrupted strain DBP1 grew at 85°C with a slightly longer lag phase but was unable to grow at 93°C. HPLC analysis showed that both N4-aminopropylspermidine and N4-bis(aminopropyl)spermidine were absent from the DBP1 strain grown at 85°C, demonstrating that the branched-chain polyamine synthesized by BpsA is important for cell growth at 93°C. Sequence comparison to orthologs from various microorganisms indicated that BpsA differed from other known aminopropyltransferases that produce spermidine and spermine. BpsA orthologs were found only in thermophiles, both in archaea and bacteria, but were absent from mesophiles. These findings indicate that BpsA is a novel aminopropyltransferase essential for the synthesis of branched-chain polyamines, enabling thermophiles to grow in high-temperature environments. PMID:24610711

The Role of AHR in Breast Cancer Development

DTIC Science & Technology

2005-07-01

Carlsbad, CA) was used according to the manufacturer’s instructions to transfect cells. The renilla luciferase vectorphRL-TK (0.5 [tg/well) was co...the firefly and renilla signals. Briefly, cells were lysed in equal volumes of cell lysis buffer (Promega) and RPMI for 20 min, transferred to a 96...well white wall plate, and analyzed using a Reporter Luminometer (Promega). The renilla signal was read after quenching the firefly output, thus

Deviation from additivity in mixture toxicity: relevance of nonlinear dose-response relationships and cell line differences in genotoxicity assays with combinations of chemical mutagens and gamma-radiation.

PubMed

Lutz, Werner K; Vamvakas, Spyros; Kopp-Schneider, Annette; Schlatter, Josef; Stopper, Helga

2002-12-01

Sublinear dose-response relationships are often seen in toxicity testing, particularly with bioassays for carcinogenicity. This is the result of a superimposition of various effects that modulate and contribute to the process of cancer formation. Examples are saturation of detoxification pathways or DNA repair with increasing dose, or regenerative hyperplasia and indirect DNA damage as a consequence of high-dose cytotoxicity and cell death. The response to a combination treatment can appear to be supra-additive, although it is in fact dose-additive along a sublinear dose-response curve for the single agents. Because environmental exposure of humans is usually in a low-dose range and deviation from linearity is less likely at the low-dose end, combination effects should be tested at the lowest observable effect levels (LOEL) of the components. This principle has been applied to combinations of genotoxic agents in various cellular models. For statistical analysis, all experiments were analyzed for deviation from additivity with an n-factor analysis of variance with an interaction term, n being the number of components tested in combination. Benzo[a]pyrene, benz[a]anthracene, and dibenz[a,c]anthracene were tested at the LOEL, separately and in combination, for the induction of revertants in the Ames test, using Salmonella typhimurium TA100 and rat liver S9 fraction. Combined treatment produced no deviation from additivity. The induction of micronuclei in vitro was investigated with ionizing radiation from a 137Cs source and ethyl methanesulfonate. Mouse lymphoma L5178Y cells revealed a significant 40% supra-additive combination effect in an experiment based on three independent replicates for controls and single and combination treatments. On the other hand, two human lymphoblastoid cell lines (TK6 and WTK1) as well as a pilot study with human primary fibroblasts from fetal lung did not show deviation from additivity. Data derived from one cell line should therefore not be generalized. Regarding the testing of mixtures for deviation from additive toxicity, the suggested experimental protocol is easily followed by toxicologists.

Chloroplast genome resources and molecular markers differentiate rubber dandelion species from weedy relatives.

PubMed

Zhang, Yingxiao; Iaffaldano, Brian J; Zhuang, Xiaofeng; Cardina, John; Cornish, Katrina

2017-02-02

Rubber dandelion (Taraxacum kok-saghyz, TK) is being developed as a domestic source of natural rubber to meet increasing global demand. However, the domestication of TK is complicated by its colocation with two weedy dandelion species, Taraxacum brevicorniculatum (TB) and the common dandelion (Taraxacum officinale, TO). TB is often present as a seed contaminant within TK accessions, while TO is a pandemic weed, which may have the potential to hybridize with TK. To discriminate these species at the molecular level, and facilitate gene flow studies between the potential rubber crop, TK, and its weedy relatives, we generated genomic and marker resources for these three dandelion species. Complete chloroplast genome sequences of TK (151,338 bp), TO (151,299 bp), and TB (151,282 bp) were obtained using the Illumina GAII and MiSeq platforms. Chloroplast sequences were analyzed and annotated for all the three species. Phylogenetic analysis within Asteraceae showed that TK has a closer genetic distance to TB than to TO and Taraxacum species were most closely related to lettuce (Lactuca sativa). By sequencing multiple genotypes for each species and testing variants using gel-based methods, four chloroplast Single Nucleotide Polymorphism (SNP) variants were found to be fixed between TK and TO in large populations, and between TB and TO. Additionally, Expressed Sequence Tag (EST) resources developed for TO and TK permitted the identification of five nuclear species-specific SNP markers. The availability of chloroplast genomes of these three dandelion species, as well as chloroplast and nuclear molecular markers, will provide a powerful genetic resource for germplasm differentiation and purification, and the study of potential gene flow among Taraxacum species.

Studies on the phase diagram of Pb-Fe-O system and standard molar Gibbs energy of formation of 'PbFe5O8.5' and Pb2Fe2O5

NASA Astrophysics Data System (ADS)

Sahu, Sulata Kumari; Ganesan, Rajesh; Gnanasekaran, T.

2012-07-01

Partial phase diagram of Pb-Fe-O system has been established by phase equilibration studies over a wide temperature range coupled with high temperature solid electrolyte based emf cells. Ternary oxides are found to coexist with liquid lead only at temperatures above 900 K. At temperatures below 900 K, iron oxides coexist with liquid lead. Standard molar Gibbs energy of formation of ternary oxides 'PbFe5O8.5' and Pb2Fe2O5 were determined by measuring equilibrium oxygen partial pressures over relevant phase fields using emf cells and are given by the following expressions: ΔfGmo 'PbFeO'±1.0(kJ mol)=-2208.1+0.6677(T/K) (917⩽T/K⩽1117) ΔfGmo PbFeO±0.8(kJ mol)=-1178.4+0.3724(T/K) (1050⩽T/K⩽1131) .

Reciprocal Changes in Sagittal Alignment in Adolescent Idiopathic Scoliosis Patients Following Strategic Pedicle Screw Fixation.

PubMed

Dumpa, Srikanth Reddy; Shetty, Ajoy Prasad; Aiyer, Siddharth N; Kanna, Rishi Mugesh; Rajasekaran, S

2018-04-01

Retrospective observational study. To analyze the effect of low-density (LD) strategic pedicle screw fixation on the correction of coronal and sagittal parameters in adolescent idiopathic scoliosis (AIS) patients. LD screw fixation achieves favorable coronal correction, but its effect on sagittal parameters is not well established. AIS is often associated with decreased thoracic kyphosis (TK), and the use of multi-level pedicle screws may result in further flattening of the sagittal profile. A retrospective analysis was performed on 92 patients with AIS to compare coronal and sagittal parameters preoperatively and at 2-year follow-up. All patients underwent posterior correction via LD strategic pedicle screw fixation. Radiographs were analyzed for primary Cobb angle (PCA), coronal imbalance, cervical sagittal angle (CSA), TK, lumbar lordosis (LL), pelvic incidence, pelvic tilt (PT), sacral slope (SS), C7 plumb line, spino-sacral angle, curve flexibility, and screw density. PCA changed significantly from 57.6°±13.9° to 19°±8.4° ( p <0.0001) with 67% correction, where the mean curve flexibility was 41% and screw density was 68%. Regional sagittal parameters did not change significantly, including CSA (from 10.76° to 10.56°, p =0.893), TK (from 24.4° to 22.8°, p =0.145), and LL (from 50.3° to 51.1°, p =0.415). However, subgroup analysis of the hypokyphosis group (<10°) and the hyperkyphosis group (>40°) showed significant correction of TK ( p <0.0001 in both). Sacro-pelvic parameters showed a significant decrease of PT and increase of SS, suggesting a reduction in pelvic retroversion SS (from 37° to 40°, p =0.0001) and PT (from 15° to 14°, p =0.025). LD strategic pedicle screw fixation provides favorable coronal correction and improves overall sagittal sacro-pelvic parameters. This technique does not cause significant flattening of TK and results in a favorable restoration of TK in patients with hypokyphosis or hyperkyphosis.

Reciprocal Changes in Sagittal Alignment in Adolescent Idiopathic Scoliosis Patients Following Strategic Pedicle Screw Fixation

PubMed Central

Dumpa, Srikanth Reddy; Aiyer, Siddharth N.; Kanna, Rishi Mugesh; Rajasekaran, S

2018-01-01

Study Design Retrospective observational study. Purpose To analyze the effect of low-density (LD) strategic pedicle screw fixation on the correction of coronal and sagittal parameters in adolescent idiopathic scoliosis (AIS) patients. Overview of Literature LD screw fixation achieves favorable coronal correction, but its effect on sagittal parameters is not well established. AIS is often associated with decreased thoracic kyphosis (TK), and the use of multi-level pedicle screws may result in further flattening of the sagittal profile. Methods A retrospective analysis was performed on 92 patients with AIS to compare coronal and sagittal parameters preoperatively and at 2-year follow-up. All patients underwent posterior correction via LD strategic pedicle screw fixation. Radiographs were analyzed for primary Cobb angle (PCA), coronal imbalance, cervical sagittal angle (CSA), TK, lumbar lordosis (LL), pelvic incidence, pelvic tilt (PT), sacral slope (SS), C7 plumb line, spino-sacral angle, curve flexibility, and screw density. Results PCA changed significantly from 57.6°±13.9° to 19°±8.4° (p <0.0001) with 67% correction, where the mean curve flexibility was 41% and screw density was 68%. Regional sagittal parameters did not change significantly, including CSA (from 10.76° to 10.56°, p =0.893), TK (from 24.4° to 22.8°, p =0.145), and LL (from 50.3° to 51.1°, p =0.415). However, subgroup analysis of the hypokyphosis group (<10°) and the hyperkyphosis group (>40°) showed significant correction of TK (p <0.0001 in both). Sacro-pelvic parameters showed a significant decrease of PT and increase of SS, suggesting a reduction in pelvic retroversion SS (from 37° to 40°, p =0.0001) and PT (from 15° to 14°, p =0.025). Conclusions LD strategic pedicle screw fixation provides favorable coronal correction and improves overall sagittal sacro-pelvic parameters. This technique does not cause significant flattening of TK and results in a favorable restoration of TK in patients with hypokyphosis or hyperkyphosis. PMID:29713412

The Aurora kinase inhibitor CCT137690 downregulates MYCN and sensitizes MYCN-amplified neuroblastoma in vivo

PubMed Central

Faisal, Amir; Vaughan, Lynsey; Bavetsias, Vassilios; Sun, Chongbo; Atrash, Butrus; Avery, Sian; Jamin, Yann; Robinson, Simon P.; Workman, Paul; Blagg, Julian; Raynaud, Florence I.; Eccles, Suzanne A.; Chesler, Louis; Linardopoulos, Spiros

2015-01-01

The Aurora kinases regulate key stages of mitosis including centrosome maturation, spindle assembly, chromosome segregation and cytokinesis. Aurora A and B overexpression has also been associated with various human cancers and as such, they have been extensively studied as novel anti-mitotic drug targets. Here we characterise the Aurora kinase inhibitor CCT137690, a highly selective, orally bioavailable imidazo[4,5-b]pyridine derivative that inhibits Aurora A and B kinases with low nanomolar IC50 values in both biochemical and cellular assays and exhibits anti-proliferative activity against a wide range of human solid tumour cell lines. CCT137690 efficiently inhibits histone H3 and TACC3 phosphorylation (Aurora B and Aurora A substrates, respectively) in HCT116 and HeLa cells. Continuous exposure of tumour cells to the inhibitor causes multipolar spindle formation, chromosome misalignment, polyploidy and apoptosis. This is accompanied by p53/p21/BAX induction, thymidine kinase 1 (TK1) downregulation and PARP cleavage. Furthermore, CCT137690 treatment of MYCN-amplified neuroblastoma cell lines inhibits cell proliferation and decreases MYCN protein expression. Importantly, in a transgenic mouse model of neuroblastoma (TH-MYCN) that overexpresses MYCN protein and is predisposed to spontaneous neuroblastoma formation, this compound significantly inhibits tumour growth. The potent preclinical activity of CCT137690 suggests that this inhibitor may benefit patients with MYCN amplified neuroblastoma. PMID:21885865

Acute dermal toxicity and sensitization studies of novel nano-enhanced UV absorbers.

PubMed

Piasecka-Zelga, Joanna; Zelga, Piotr; Górnicz, Magdalena; Strzelczyk, Paweł; Sójka-Ledakowicz, Jadwiga

2015-01-01

Many employees working outside are exposed to the harmful effects of UV radiation. A growing problem is also sensitization to textile materials and allergic reactions to active compounds. Groups of inorganic UV blockers with nanoparticles may provide superior properties over organic UV absorbers with relatively less potential of provoking dermatitis. To assess acute dermal irritation and sensitization of nano UV absorbers. Five UV absorbers with nano-sized particles (Z11, TiO2 - SiO2 [TDPK], TK44, TK11, A8G) and 2 vehicles (paste-based on 10% PEG, and dispersion with 1% HEC) were tested. Acute dermal irritation was tested using group of 3 rabbits for each absorber. The sensitization study was carried out on groups of 15 guinea pigs for each tested textile with a UV absorber showing an Ultraviolet Protection Factor (UPF)>40. This research was designed according to OECD Test Guideline No. 404 and 406, and 21 rabbits and 60 guinea pigs were used in the study. In acute dermal irritation, Z11 and A8G modifiers and the analyzed paste gave results of 0.047 to 0.33 which classifies them as barely perceptible irritants, whereas the other analyzed modifiers and dispersion gave results of 0.00 and were classified as nonirritating. Only the textile with TK 11 did not have UPF>40. The analyzed barrier materials were classified as nonsenitizers (TDPK, A8G) or mild sensitizers (TK44, Z11). None of the analyzed materials or modifiers induced major skin reactions in animals. Therefore, they present low risk of provoking skin reactions in humans.

Amino acid substitutions in the thymidine kinase gene of induced acyclovir-resistant herpes simplex virus type 1

NASA Astrophysics Data System (ADS)

Hussin, Ainulkhir; Nor, Norefrina Shafinaz Md; Ibrahim, Nazlina

2013-11-01

Acyclovir (ACV) is an antiviral drug of choice in healthcare setting to treat infections caused by herpes viruses, including, but not limited to genital herpes, cold sores, shingles and chicken pox. Acyclovir resistance has emerged significantly due to extensive use and misuse of this antiviral in human, especially in immunocompromised patients. However, it remains unclear about the amino acid substitutions in thymidine (TK) gene, which specifically confer the resistance-associated mutation in herpes simplex virus. Hence, acyclovir-resistant HSV-1 was selected at high concentration (2.0 - 4.5 μg/mL), and the TK-gene was subjected to sequencing and genotypic characterization. Genotypic sequences comparison was done using HSV-1 17 (GenBank Accesion no. X14112) for resistance-associated mutation determination whereas HSV-1 KOS, HSV-1 473/08 and HSV clinical isolates sequences were used for polymorphism-associated mutation. The result showed that amino acid substitutions at the non-conserved region (UKM-1: Gln34Lys, UKM-2: Arg32Ser & UKM-5: Arg32Cys) and ATP-binding site (UKM-3: Tyr53End & UKM-4: Ile54Leu) of the TK-gene. These discoveries play an important role to extend another dimension to the evolution of acyclovir-resistant HSV-1 and suggest that selection at high ACV concentration induced ACV-resistant HSV-1 evolution. These findings also expand the knowledge on the type of mutations among acyclovir-resistant HSV-1. In conclusion, HSV-1 showed multiple strategies to exhibit acyclovir resistance, including amino acid substitutions in the TK gene.

High-Throughput Toxicokinetics (HTTK) R package (CompTox CoP presentation)

EPA Science Inventory

Toxicokinetics (TK) provides a bridge between HTS and HTE by predicting tissue concentrations due to exposure, but traditional TK methods are resource intensive. Relatively high throughput TK (HTTK) methods have been used by the pharmaceutical industry to determine range of effic...

Complete mitochondrial genomes of Thai and Lao populations indicate an ancient origin of Austroasiatic groups and demic diffusion in the spread of Tai-Kadai languages.

PubMed

Kutanan, Wibhu; Kampuansai, Jatupol; Srikummool, Metawee; Kangwanpong, Daoroong; Ghirotto, Silvia; Brunelli, Andrea; Stoneking, Mark

2017-01-01

The Tai-Kadai (TK) language family is thought to have originated in southern China and spread to Thailand and Laos, but it is not clear if TK languages spread by demic diffusion (i.e., a migration of people from southern China) or by cultural diffusion, with native Austroasiatic (AA) speakers switching to TK languages. To address this and other questions, we obtained 1234 complete mtDNA genome sequences from 51 TK and AA groups from Thailand and Laos. We find high genetic heterogeneity across the region, with 212 different haplogroups, and significant genetic differentiation among different samples from the same ethnolinguistic group. TK groups are more genetically homogeneous than AA groups, with the latter exhibiting more ancient/basal mtDNA lineages, and showing more drift effects. Modeling of demic diffusion, cultural diffusion, and admixture scenarios consistently supports the spread of TK languages by demic diffusion.

Extending Thymidine Kinase Activity to the Catalytic Repertoire of Human Deoxycytidine Kinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hazra, Saugata; Sabini, Eliszbetta; Ort, Stephan

Salvage of nucleosides in the cytosol of human cells is carried out by deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1). Whereas TK1 is only responsible for thymidine phosphorylation, dCK is capable of converting dC, dA, and dG into their monophosphate forms. Using structural data on dCK, we predicted that select mutations at the active site would, in addition to making the enzyme faster, expand the catalytic repertoire of dCK to include thymidine. Specifically, we hypothesized that steric repulsion between the methyl group of the thymine base and Arg104 is the main factor preventing the phosphorylation of thymidine by wild-typemore » dCK. Here we present kinetic data on several dCK variants where Arg104 has been replaced by select residues, all performed in combination with the mutation of Asp133 to an alanine. We show that several hydrophobic residues at position 104 endow dCK with thymidine kinase activity. Depending on the exact nature of the mutations, the enzyme's substrate preference is modified. The R104M-D133A double mutant is a pyrimidine-specific enzyme due to large K{sub m} values with purines. The crystal structure of the double mutant R104M-D133A in complex with the L-form of thymidine supplies a structural explanation for the ability of this variant to phosphorylate thymidine and thymidine analogs. The replacement of Arg104 by a smaller residue allows L-dT to bind deeper into the active site, making space for the C5-methyl group of the thymine base. The unique catalytic properties of several of the mutants make them good candidates for suicide-gene/protein-therapy applications.« less

Gene targeting and subsequent site-specific transgenesis at the β-actin (ACTB) locus in common marmoset embryonic stem cells.

PubMed

Shiozawa, Seiji; Kawai, Kenji; Okada, Yohei; Tomioka, Ikuo; Maeda, Takuji; Kanda, Akifumi; Shinohara, Haruka; Suemizu, Hiroshi; James Okano, Hirotaka; Sotomaru, Yusuke; Sasaki, Erika; Okano, Hideyuki

2011-09-01

Nonhuman primate embryonic stem (ES) cells have vast promise for preclinical studies. Genetic modification in nonhuman primate ES cells is an essential technique for maximizing the potential of these cells. The common marmoset (Callithrix jacchus), a nonhuman primate, is expected to be a useful transgenic model for preclinical studies. However, genetic modification in common marmoset ES (cmES) cells has not yet been adequately developed. To establish efficient and stable genetic modifications in cmES cells, we inserted the enhanced green fluorescent protein (EGFP) gene with heterotypic lox sites into the β-actin (ACTB) locus of the cmES cells using gene targeting. The resulting knock-in ES cells expressed EGFP ubiquitously under the control of the endogenous ACTB promoter. Using inserted heterotypic lox sites, we demonstrated Cre recombinase-mediated cassette exchange (RMCE) and successfully established a monomeric red fluorescent protein (mRFP) knock-in cmES cell line. Further, a herpes simplex virus-thymidine kinase (HSV-tk) knock-in cmES cell line was established using RMCE. The growth of tumor cells originating from the cell line was significantly suppressed by the administration of ganciclovir. Therefore, the HSV-tk/ganciclovir system is promising as a safeguard for stem cell therapy. The stable and ubiquitous expression of EGFP before RMCE enables cell fate to be tracked when the cells are transplanted into an animal. Moreover, the creation of a transgene acceptor locus for site-specific transgenesis will be a powerful tool, similar to the ROSA26 locus in mice.

20171015 - Capabilities and Evaluation of the US EPA’s HTTK (High Throughput Toxicokinetics) R package (ISES)

EPA Science Inventory

Toxicokinetics (TK) provides a bridge between toxicity and exposure assessment by predicting tissue concentrations due to exposure, however traditional TK methods are resource intensive. Relatively high throughput TK (HTTK) methods have been used by the pharmaceutical industry to...

A novel start codon mutation of the MERTK gene in a patient with retinitis pigmentosa

PubMed Central

Jinda, Worapoj; Poungvarin, Naravat; Taylor, Todd D.; Suzuki, Yutaka; Thongnoppakhun, Wanna; Limwongse, Chanin; Lertrit, Patcharee; Suriyaphol, Prapat

2016-01-01

Purpose Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous group of inherited retinal degenerations characterized by progressive loss of photoreceptor cells and RPE functions. More than 70 causative genes are known to be responsible for RP. This study aimed to identify the causative gene in a patient from a consanguineous family with childhood-onset severe retinal dystrophy. Methods To identify the defective gene, whole exome sequencing was performed. Candidate causative variants were selected and validated using Sanger sequencing. Segregation analysis of the causative gene was performed in additional family members. To verify that the mutation has an effect on protein synthesis, an expression vector containing the first ten amino acids of the mutant protein fused with the DsRed2 fluorescent protein was constructed and transfected into HEK293T cells. Expression of the fusion protein in the transfected cells was measured using fluorescence microscopy. Results By filtering against public variant databases, a novel homozygous missense mutation (c.3G>A) localized in the start codon of the MERTK gene was detected as a potentially pathogenic mutation for autosomal recessive RP. The c.3G>A mutation cosegregated with the disease phenotype in the family. No expression of the first ten amino acids of the MerTK mutant fused with the DsRed2 fluorescent protein was detected in HEK293T cells, indicating that the mutation affects the translation initiation site of the gene that may lead to loss of function of the MerTK signaling pathway. Conclusions We report a novel missense mutation (c.3G>A, p.0?) in the MERTK gene that causes severe vision impairment in a patient. Taken together with previous reports, our results expand the spectrum of MERTK mutations and extend our understanding of the role of the MerTK protein in the pathogenesis of retinitis pigmentosa. PMID:27122965

Immobilised enzyme microreactor for screening of multi-step bioconversions: characterisation of a de novo transketolase-ω-transaminase pathway to synthesise chiral amino alcohols.

PubMed

Matosevic, S; Lye, G J; Baganz, F

2011-09-20

Complex molecules are synthesised via a number of multi-step reactions in living cells. In this work, we describe the development of a continuous flow immobilized enzyme microreactor platform for use in evaluation of multi-step bioconversion pathways demonstrating a de novo transketolase/ω-transaminase-linked asymmetric amino alcohol synthesis. The prototype dual microreactor is based on the reversible attachment of His₆-tagged enzymes via Ni-NTA linkage to two surface derivatised capillaries connected in series. Kinetic parameters established for the model transketolase (TK)-catalysed conversion of lithium-hydroxypyruvate (Li-HPA) and glycolaldehyde (GA) to L-erythrulose using a continuous flow system with online monitoring of reaction output was in good agreement with kinetic parameters determined for TK in stop-flow mode. By coupling the transketolase catalysed chiral ketone forming reaction with the biocatalytic addition of an amine to the TK product using a transaminase (ω-TAm) it is possible to generate chiral amino alcohols from achiral starting compounds. We demonstrated this in a two-step configuration, where the TK reaction was followed by the ω-TAm-catalysed amination of L-erythrulose to synthesise 2-amino-1,3,4-butanetriol (ABT). Synthesis of the ABT product via the dual reaction and the on-line monitoring of each component provided a full profile of the de novo two-step bioconversion and demonstrated the utility of this microreactor system to provide in vitro multi-step pathway evaluation. Copyright © 2011 Elsevier B.V. All rights reserved.

Engineering safer-by-design, transparent, silica-coated ZnO nanorods with reduced DNA damage potential

PubMed Central

Sotiriou, Georgios A.; Watson, Christa; Murdaugh, Kimberly M.; Darrah, Thomas H.; Pyrgiotakis, Georgios; Elder, Alison; Brain, Joseph D.; Demokritou, Philip

2014-01-01

Zinc oxide (ZnO) nanoparticles absorb UV light efficiently while remaining transparent in the visible light spectrum rendering them attractive in cosmetics and polymer films. Their broad use, however, raises concerns regarding potential environmental health risks and it has been shown that ZnO nanoparticles can induce significant DNA damage and cytotoxicity. Even though research on ZnO nanoparticle synthesis has made great progress, efforts on developing safer ZnO nanoparticles that maintain their inherent optoelectronic properties while exhibiting minimal toxicity are limited. Here, a safer-by-design concept was pursued by hermetically encapsulating ZnO nanorods in a biologically inert, nanothin amorphous SiO2 coating during their gas-phase synthesis. It is demonstrated that the SiO2 nanothin layer hermetically encapsulates the core ZnO nanorods without altering their optoelectronic properties. Furthermore, the effect of SiO2 on the toxicological profile of the core ZnO nanorods was assessed using the Nano-Cometchip assay by monitoring DNA damage at a cellular level using human lymphoblastoid cells (TK6). Results indicate significantly lower DNA damage (>3 times) for the SiO2-coated ZnO nanorods compared to uncoated ones. Such an industry-relevant, scalable, safer-by-design formulation of nanostructured materials can liberate their employment in nano-enabled products and minimize risks to the environment and human health. PMID:24955241

Correlation between preoperative spinopelvic alignment and risk of proximal junctional kyphosis after posterior-only surgical correction of Scheuermann kyphosis.

PubMed

Nasto, Luigi A; Perez-Romera, Ana Belen; Shalabi, Saggah Tarek; Quraishi, Nasir A; Mehdian, Hossein

2016-04-01

Surgical correction of Scheuermann kyphosis (SK) is challenging and plagued by relatively high rates of proximal junctional kyphosis and failure (PJK and PJF). Normal sagittal alignment of the spine is determined by pelvic geometric parameters. How these parameters correlate with the risk of developing PJK in SK is not known. The study aimed to investigate the relationship between preoperative and postoperative spinopelvic alignment and occurrence of PJK and PJF. This is a retrospective observational cohort study. The sample included 37 patients who underwent posterior correction of SK from January 2006 to December 2012. The outcome measure was correlation analysis between preoperative and postoperative spinopelvic alignment parameters and the development of PJK over the course of the study period. Whole spine x-rays obtained before surgery, 3 months after surgery, and at the latest follow-up were analyzed. The following parameters were measured: thoracic kyphosis (TK), lumbar lordosis (LL), sagittal vertical axis (SVA), pelvic incidence (PI), pelvic tilt (PT), and sacral slope (SS). The development of PJK was considered the primary end point of the study. Patient population was split into a control and a PJK group; repeated-measures analysis of variance was used to assess group and time differences. Seven patients developed PJK over the study period. Although the severity of the preoperative deformity (TK) did not differ significantly between the two groups, preoperative PI was significantly higher in the PJK group (51.9°C±8.6°C vs. 42.7°C±8.8°C, p=.018). Postoperative correction of TK was similar between the two groups (39.3% and 41.2%, p=.678) and final LL did not differ as well (53.6°C±9.2°C vs. 51.3°C±11.5°C). However, because PJK patients had larger preoperative PI values, a significant deficit of LL was observed at final follow-up in this group compared with the control group (ΔLL -10.5°C±9.8°C vs. 0.6°C±10.5°C, p=.013). Scheuermann kyphosis patients who developed PJK appeared to have a significant postoperative deficit of LL (lumbopelvic mismatch). Lumbar lordosis decreases after surgery following correction of TK; therefore, TK correction should be planned according to preoperative PI values to avoid excessive reduction of LL in patients with higher PI values. Copyright © 2016 Elsevier Inc. All rights reserved.

In vitro and in vivo genotoxicity assessment of HI-6 dimethanesulfonate/oxime.

PubMed

Nakab, Lauren; Bardot, Isabelle; Bardot, Sébastien; Simar, Sophie; Marzin, Daniel; Nesslany, Fabrice

2014-03-01

Organophosphate compounds, which induce organophosphate poisoning, were originally used as pesticides. But this type of product has also been used as warfare nerve agent like sarin, soman, Russian VX, or tabun. HI-6-dimethanesulfonate is a salt of the oxime HI-6 used in the treatment of nerve-agent poisoning. It is known to be the best re-activator component of inactivated acetyl cholinesterase. HI-6-dimethanesulfonate has shown a higher level of solubility with similar potency to reactivate acetyl cholinesterase and a similar pharmacokinetics profile compared with HI-6 dichloride. HI-6 dimethanesulfonate was tested for its mutagenic and genotoxic potential by use of the standard ICH S2R (1) battery for the evaluation of pharmaceuticals. HI-6-dimethanesulfonate was mutagenic in the Ames test only in the presence of metabolic activation. In the mutation assay at the Tk locus in L5178Y mouse-lymphoma cells, HI-6-dimethanesulfonate showed mutagenic activity both with and without metabolic activation, with a significant increase in small colonies. The effects were in favour of a clastogenic activity. It was concluded that the compound was mutagenic and possibly clastogenic in vitro. In contrast, the in vivo micronucleus test in rat bone-marrow did not demonstrate any genotoxic activity and the Comet assay performed in rat liver did not show any statistically or biologically significant increases in DNA strand-breaks. The results of both in vivo studies performed on two different organs with two endpoints are sufficient to conclude the absence of a genotoxic hazard in vivo and to consider that there is no genotoxic concern in humans for HI-6-dimethanesulfonate. Copyright © 2014 Elsevier B.V. All rights reserved.

Synthesis and biological properties of new 5-nitroindazole derivatives.

PubMed

Arán, Vicente J; Ochoa, Carmen; Boiani, Lucía; Buccino, Pablo; Cerecetto, Hugo; Gerpe, Alejandra; González, Mercedes; Montero, David; Nogal, Juan José; Gómez-Barrio, Alicia; Azqueta, Amaya; López de Ceráin, Adela; Piro, Oscar E; Castellano, Eduardo E

2005-05-02

A series of new 3-alkoxy- or 3-hydroxy-1-[omega-(dialkylamino)alkyl]-5-nitroindazoles have been synthesized and their trichomonacidal, antichagasic and antineoplastic properties studied. Five derivatives (5, 6, 8, 9 and 17) showed remarkable trichomonacidal activity against Trichomonas vaginalis at 10 microg/mL concentration. Three compounds (8, 10, 11) exhibited interesting antichagasic activity and these same compounds moderate antineoplastic activity against TK-10 and HT-29 cell lines. Unspecific cytotoxicity against macrophages has also been evaluated and only compounds 9, 10 and 11 resulted cytotoxic at the higher dose evaluated (100 microg/mL), loosing cytotoxicity at lower doses. QSAR studies have been carried out. X-ray crystallographic study of compound 8 has been performed.

Metabolism Dealing with Thermal Degradation of NAD+ in the Hyperthermophilic Archaeon Thermococcus kodakarensis.

PubMed

Hachisuka, Shin-Ichi; Sato, Takaaki; Atomi, Haruyuki

2017-10-01

NAD + is an important cofactor for enzymatic oxidation reactions in all living organisms, including (hyper)thermophiles. However, NAD + is susceptible to thermal degradation at high temperatures. It can thus be expected that (hyper)thermophiles harbor mechanisms that maintain in vivo NAD + concentrations and possibly remove and/or reuse undesirable degradation products of NAD + Here we confirmed that at 85°C, thermal degradation of NAD + results mostly in the generation of nicotinamide and ADP-ribose, the latter known to display toxicity by spontaneously linking to proteins. The hyperthermophilic archaeon Thermococcus kodakarensis possesses a putative ADP-ribose pyrophosphatase (ADPR-PPase) encoded by the TK2284 gene. ADPR-PPase hydrolyzes ADP-ribose to ribose 5-phosphate (R5P) and AMP. The purified recombinant TK2284 protein exhibited activity toward ADP-ribose as well as ADP-glucose. Kinetic analyses revealed a much higher catalytic efficiency toward ADP-ribose, suggesting that ADP-ribose was the physiological substrate. To gain insight into the physiological function of TK2284, a TK2284 gene disruption strain was constructed and examined. Incubation of NAD + in the cell extract of the mutant strain at 85°C resulted in higher ADP-ribose accumulation and lower AMP production compared with those in experiments with the host strain cell extract. The mutant strain also exhibited lower cell yield and specific growth rates in a synthetic amino acid medium compared with those of the host strain. The results obtained here suggest that the ADPR-PPase in T. kodakarensis is responsible for the cleavage of ADP-ribose to R5P and AMP, providing a means to utilize the otherwise dead-end product of NAD + breakdown. IMPORTANCE Hyperthermophilic microorganisms living under high temperature conditions should have mechanisms that deal with the degradation of thermolabile molecules. NAD + is an important cofactor for enzymatic oxidation reactions and is susceptible to thermal degradation to ADP-ribose and nicotinamide. Here we show that an ADP-ribose pyrophosphatase homolog from the hyperthermophilic archaeon Thermococcus kodakarensis converts the detrimental ADP-ribose to ribose 5-phosphate and AMP, compounds that can be directed to central carbon metabolism. This physiological role for ADP-ribose pyrophosphatases might be universal in hyperthermophiles, as their homologs are widely distributed among both hyperthermophilic bacteria and archaea. Copyright © 2017 American Society for Microbiology.

20171024 - Capabilities and Evaluation of the US EPA’s HTTK (High Throughput Toxicokinetics) R package (Webinar Presentation to European Chemical Agency

EPA Science Inventory

Toxicokinetics (TK) provides a bridge between toxicity and exposure assessment by predicting tissue concentrations due to exposure. However traditional TK methods are resource intensive. Relatively high throughput TK (HTTK) methods have been used by the pharmaceutical industry to...

Kondo Effect in Dirac Systems

NASA Astrophysics Data System (ADS)

Yanagisawa, Takashi

2015-07-01

We investigate the Kondo effect in Dirac systems, where Dirac electrons interact with the localized spin via the s-d exchange coupling. The Dirac electron in solid state has the linear dispersion and is described typically by the Hamiltonian such as Hk = vk · σ for the wave number k where σj are Pauli matrices. We derived the formula of the Kondo temperature TK by means of the Green's function theory for small J. The TK is determined from a singularity of Green's functions in the form TK ≃ bar{D}exp ( - const./ρ |J|) when the exchange coupling |J| is small where bar{D} = D/√{1 + D2/(2μ )2} for a cutoff D and ρ is the density of states at the Fermi surface. When |μ| ≪ D, TK is proportional to |μ|: TK ≃ |μ| exp(-const./ρ|J|). The Kondo screening will, however, disappear when the Fermi surface shrinks to a point called the Dirac point, that is, TK vanishes when the chemical potential μ is just at the Dirac point. The resistivity and the specific heat exhibit a log-T singularity in the range TK < T ≪ |μ|/kB. Instead, for T ˜ O(|μ|) or T > |μ|, they never show log-T.

Access and benefits sharing of genetic resources and associated traditional knowledge in northern Canada: understanding the legal environment and creating effective research agreements

PubMed Central

Geary, Janis; Jardine, Cynthia G.; Guebert, Jenilee; Bubela, Tania

2013-01-01

Background Research in northern Canada focused on Aboriginal peoples has historically benefited academia with little consideration for the people being researched or their traditional knowledge (TK). Although this attitude is changing, the complexity of TK makes it difficult to develop mechanisms to preserve and protect it. Protecting TK becomes even more important when outside groups become interested in using TK or materials with associated TK. In the latter category are genetic resources, which may have commercial value and are the focus of this article. Objective This article addresses access to and use of genetic resources and associated TK in the context of the historical power-imbalances in research relationships in Canadian north. Design Review. Results Research involving genetic resources and TK is becoming increasingly relevant in northern Canada. The legal framework related to genetic resources and the cultural shift of universities towards commercial goals in research influence the environment for negotiating research agreements. Current guidelines for research agreements do not offer appropriate guidelines to achieve mutual benefit, reflect unequal bargaining power or take the relationship between parties into account. Conclusions Relational contract theory may be a useful framework to address the social, cultural and legal hurdles inherent in creating research agreements. PMID:23986896

Access and benefits sharing of genetic resources and associated traditional knowledge in northern Canada: understanding the legal environment and creating effective research agreements.

PubMed

Geary, Janis; Jardine, Cynthia G; Guebert, Jenilee; Bubela, Tania

2013-01-01

Research in northern Canada focused on Aboriginal peoples has historically benefited academia with little consideration for the people being researched or their traditional knowledge (TK). Although this attitude is changing, the complexity of TK makes it difficult to develop mechanisms to preserve and protect it. Protecting TK becomes even more important when outside groups become interested in using TK or materials with associated TK. In the latter category are genetic resources, which may have commercial value and are the focus of this article. This article addresses access to and use of genetic resources and associated TK in the context of the historical power-imbalances in research relationships in Canadian north. Review. Research involving genetic resources and TK is becoming increasingly relevant in northern Canada. The legal framework related to genetic resources and the cultural shift of universities towards commercial goals in research influence the environment for negotiating research agreements. Current guidelines for research agreements do not offer appropriate guidelines to achieve mutual benefit, reflect unequal bargaining power or take the relationship between parties into account. Relational contract theory may be a useful framework to address the social, cultural and legal hurdles inherent in creating research agreements.

Imaging Transgene Expression with Radionuclide Imaging Technologies1

PubMed Central

Gambhir, SS; Herschman, HR; Cherry, SR; Barrio, JR; Satyamurthy, N; Toyokuni, T; Phelps, ME; Larson, SM; Balaton, J; Finn, R; Sadelain, M; Tjuvajev, J

2000-01-01

Abstract A variety of imaging technologies are being investigated as tools for studying gene expression in living subjects. Noninvasive, repetitive and quantitative imaging of gene expression will help both to facilitate human gene therapy trials and to allow for the study of animal models of molecular and cellular therapy. Radionuclide approaches using single photon emission computed tomography (SPECT) and positron emission tomography (PET) are the most mature of the current imaging technologies and offer many advantages for imaging gene expression compared to optical and magnetic resonance imaging (MRI)-based approaches. These advantages include relatively high sensitivity, full quantitative capability (for PET), and the ability to extend small animal assays directly into clinical human applications. We describe a PET scanner (micro PET) designed specifically for studies of small animals. We review “marker/reporter gene” imaging approaches using the herpes simplex type 1 virus thymidine kinase (HSV1-tk) and the dopamine type 2 receptor (D2R) genes. We describe and contrast several radiolabeled probes that can be used with the HSV1-tk reporter gene both for SPECT and for PET imaging. We also describe the advantages/disadvantages of each of the assays developed and discuss future animal and human applications. PMID:10933072

Biodegradation of isopropanol and acetone under denitrifying conditions by Thauera sp. TK001 for nitrate-mediated microbially enhanced oil recovery.

PubMed

Fida, Tekle Tafese; Gassara, Fatma; Voordouw, Gerrit

2017-07-15

Amendment of reservoir fluid with injected substrates can enhance the growth and activity of microbes. The present study used isopropyl alcohol (IPA) or acetone to enhance the indigenous anaerobic nitrate-reducing bacterium Thauera sp. TK001. The strain was able to grow on IPA or acetone and nitrate. To monitor effects of strain TK001 on oil recovery, sand-packed columns containing heavy oil were flooded with minimal medium at atmospheric or high (400psi) pressure. Bioreactors were then inoculated with 0.5 pore volume (PV) of minimal medium containing Thauera sp. TK001 with 25mM of acetone or 22.2mM of IPA with or without 80mM nitrate. Incubation without flow for two weeks and subsequent injection with minimal medium gave an additional 17.0±6.7% of residual oil in place (ROIP) from low-pressure bioreactors and an additional 18.3% of ROIP from the high-pressure bioreactors. These results indicate that acetone or IPA, which are commonly used organic solvents, are good substrates for nitrate-mediated microbial enhanced oil recovery (MEOR), comparable to glucose, acetate or molasses, tested previously. This technology may be used for coupling biodegradation of IPA and/or acetone in waste streams to MEOR where these waste streams are generated in close proximity to an oil field. Copyright © 2017 Elsevier B.V. All rights reserved.

Tissue specific distribution of pyrimidine deoxynucleoside salvage enzymes shed light on the mechanism of mitochondrial DNA depletion.

PubMed

Wang, L; Eriksson, S

2010-06-01

Deficiency in thymidine kinase 2 (TK2) activity due to genetic alterations caused tissue specific mitochondrial DNA (mtDNA) depletion syndrome with symptoms resembling these of AIDS patients treated with nucleoside analogues. Mechanisms behind this mitochondrial effects is still not well understood. With rat as a model we isolated mitochondrial and cytosolic fractions from major organs and studied enzymes involved in thymidine (dT) and deoxycytidine (dC) phosphorylation by using ionic exchange column chromatography. A cytosolic form of TK2 was identified in all tested tissues in addition to mitochondrial TK2. TK1 was detected in liver and spleen cytosolic extracts while dCK was found in liver, spleen and lung cytosolic extracts. Thus, the nature of dT and dC salvage enzymes in each tissue type was determined. In most tissues TK2 is the only salvage enzyme present except liver and spleen. These results may help to explain the mechanisms of mitochondrial toxicity of antiviral nucleoside analogues and mtDNA depletion caused by TK2 deficiency.

Applications of temporal kernel canonical correlation analysis in adherence studies.

PubMed

John, Majnu; Lencz, Todd; Ferbinteanu, Janina; Gallego, Juan A; Robinson, Delbert G

2017-10-01

Adherence to medication is often measured as a continuous outcome but analyzed as a dichotomous outcome due to lack of appropriate tools. In this paper, we illustrate the use of the temporal kernel canonical correlation analysis (tkCCA) as a method to analyze adherence measurements and symptom levels on a continuous scale. The tkCCA is a novel method developed for studying the relationship between neural signals and hemodynamic response detected by functional MRI during spontaneous activity. Although the tkCCA is a powerful tool, it has not been utilized outside the application that it was originally developed for. In this paper, we simulate time series of symptoms and adherence levels for patients with a hypothetical brain disorder and show how the tkCCA can be used to understand the relationship between them. We also examine, via simulations, the behavior of the tkCCA under various missing value mechanisms and imputation methods. Finally, we apply the tkCCA to a real data example of psychotic symptoms and adherence levels obtained from a study based on subjects with a first episode of schizophrenia, schizophreniform or schizoaffective disorder.

Rb silencing mediated by the down-regulation of MeCP2 is involved in cell transformation induced by long-term exposure to hydroquinone.

PubMed

Liu, Linhua; Ling, Xiaoxuan; Wu, Minhua; Chen, Jialong; Chen, Shaoqiao; Tan, Qiang; Chen, Jiansong; Liu, Jiaxian; Zou, Fei

2017-02-01

Hydroquinone (HQ), a metabolite of benzene, is a well-known human carcinogen; however, its molecular mechanisms of action remain unclear. MeCP2 has been traditionally described as a transcriptional repressor, though growing evidence indicates that it also activates gene expression. Here, we investigated whether some epigenetic machinery genes are aberrantly expressed as target tumor suppressor genes in HQ-transformed TK6 lymphoblastoid cells. Our results showed that treatment with 5-Aza-2'-deoxycytidine or trichostatin A enhanced the expression of Rb, resulting in cell arrest in G1-phase, and subsequently, an increase in apoptosis and a decrease in cell growth. Moreover, we hypothesised that Rb was silenced by the down-regulation of MeCP2 in HQ-transformed cells, resulting in the dynamic expression of Rb and epigenetic machinery proteins in HQ-transformed cells at different time points. The expression of Rb and MeCP2 in patients with B-cell non-Hodgkin's lymphoma (B-NHL) showed that positive staining for MeCP2 or Rb was significantly lower in B-NHL tumor tissues, and these changes were significantly and negatively correlated with the grade of B-NHL. The restoration of MeCP2 in HQ-transformed cells enhanced the expression of Rb, promoted cell apoptosis, and inhibited cell growth. The changes in the expression patterns of MeCP2 and Rb were inversely correlated with the degree of DNA methylation. A ChiP assay revealed that MeCP2 proteins were recruited to the Rb promoter with lower 5'-methylcytosine levels. In conclusion, we demonstrated that the down-regulation of MeCP2 silences Rb, a process involved in cell transformation resulting from long-term exposure to HQ. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Phase I Study of Cellular Immunotherapy for Recurrent/Refractory Malignant Glioma Using Intratumoral Infusions of GRm13Z40-2, An Allogeneic CD8+ Cytolitic T-Cell Line Genetically Modified to Express the IL 13-Zetakine and HyTK and to be Resistant to Glucocorticoids, in Combination With Interleukin-2

ClinicalTrials.gov

2015-06-03

Anaplastic Astrocytoma; Anaplastic Ependymoma; Anaplastic Meningioma; Anaplastic Oligodendroglioma; Brain Stem Glioma; Ependymoblastoma; Giant Cell Glioblastoma; Glioblastoma; Gliosarcoma; Grade III Meningioma; Meningeal Hemangiopericytoma; Mixed Glioma; Pineal Gland Astrocytoma; Brain Tumor

Spiny Nanorod and Upconversion Nanoparticle Satellite Assemblies for Ultrasensitive Detection of Messenger RNA in Living Cells.

PubMed

Gao, Rui; Hao, Changlong; Xu, Liguang; Xu, Chuanlai; Kuang, Hua

2018-04-17

Quantitation and in situ monitoring of target mRNA (mRNA) in living cells remains a significant challenge for the chemical and biomedical communities. To quantitatively detect mRNA expression levels in living cells, we have developed DNA-driven gold nanorod coated platinum-upconversion nanoparticle satellite assemblies (termed Au NR@Pt-UCNP satellites) for intracellular thymidine kinase 1 (TK1) mRNA analysis. The nanostructures were capable of recognizing target mRNA in a sequence-specific manner as luminescence of UCNPs was effectively quenched by Au NR@Pt within the assemblies. Following recognition, UCNPs detached from Au NR@Pt, resulting in luminescence restoration to achieve effective in situ imaging and quantifiable detection of target mRNA. The upconversional luminescence intensity of confocal images showed a good linear relationship with intracellular TK1 mRNA ranging from 1.17 to 65.21 fmol/10 μg RNA and a limit of detection (LOD) of 0.67 fmol/10 μg RNA. We believe that our present assay can be broadly applied for detection of endogenous biomolecules at the cellular and tissue levels and restoration of tissue homeostasis in vivo.

Post-synthetic modifications of cadmium-based knots and links.

PubMed

Prakasam, Thirumurugan; Bilbeisi, Rana A; Lusi, Matteo; Olsen, John-Carl; Platas-Iglesias, Carlos; Trabolsi, Ali

2016-05-31

Three topologically non-trivial cadmium(ii)-based complexes-Cd-[2]C, Cd-TK and Cd-SL-were simultaneously self-assembled in a dynamic library, individually isolated and fully characterized using solid-state, gas-phase and solution-phase techniques. Post-synthetic modifications, including reduction and transmetalation, were subsequently achieved. Imine bond reduction followed by demetallation led to the isolation of the corresponding organic molecules [2]C, TK and SL. Transmetalation of Cd-TK and Cd-SL with the zinc(ii) cation resulted in isolation of the corresponding zinc(ii)-containing complexes Zn-TK and Zn-SL.

TK Modeler version 1.0, a Microsoft® Excel®-based modeling software for the prediction of diurnal blood/plasma concentration for toxicokinetic use.

PubMed

McCoy, Alene T; Bartels, Michael J; Rick, David L; Saghir, Shakil A

2012-07-01

TK Modeler 1.0 is a Microsoft® Excel®-based pharmacokinetic (PK) modeling program created to aid in the design of toxicokinetic (TK) studies. TK Modeler 1.0 predicts the diurnal blood/plasma concentrations of a test material after single, multiple bolus or dietary dosing using known PK information. Fluctuations in blood/plasma concentrations based on test material kinetics are calculated using one- or two-compartment PK model equations and the principle of superposition. This information can be utilized for the determination of appropriate dosing regimens based on reaching a specific desired C(max), maintaining steady-state blood/plasma concentrations, or other exposure target. This program can also aid in the selection of sampling times for accurate calculation of AUC(24h) (diurnal area under the blood concentration time curve) using sparse-sampling methodologies (one, two or three samples). This paper describes the construction, use and validation of TK Modeler. TK Modeler accurately predicted blood/plasma concentrations of test materials and provided optimal sampling times for the calculation of AUC(24h) with improved accuracy using sparse-sampling methods. TK Modeler is therefore a validated, unique and simple modeling program that can aid in the design of toxicokinetic studies. Copyright © 2012 Elsevier Inc. All rights reserved.

Thymidine kinase 2 enzyme kinetics elucidate the mechanism of thymidine-induced mitochondrial DNA depletion.

PubMed

Sun, Ren; Wang, Liya

2014-10-07

Mitochondrial thymidine kinase 2 (TK2) is a nuclear gene-encoded protein, synthesized in the cytosol and subsequently translocated into the mitochondrial matrix, where it catalyzes the phosphorylation of thymidine (dT) and deoxycytidine (dC). The kinetics of dT phosphorylation exhibits negative cooperativity, but dC phosphorylation follows hyperbolic Michaelis-Menten kinetics. The two substrates compete with each other in that dT is a competitive inhibitor of dC phosphorylation, while dC acts as a noncompetitive inhibitor of dT phosphorylation. In addition, TK2 is feedback inhibited by dTTP and dCTP. TK2 also phosphorylates a number of pyrimidine nucleoside analogues used in antiviral and anticancer therapy and thus plays an important role in mitochondrial toxicities caused by nucleoside analogues. Deficiency in TK2 activity due to genetic alterations causes devastating mitochondrial diseases, which are characterized by mitochondrial DNA (mtDNA) depletion or multiple deletions in the affected tissues. Severe TK2 deficiency is associated with early-onset fatal mitochondrial DNA depletion syndrome, while less severe deficiencies result in late-onset phenotypes. In this review, studies of the enzyme kinetic behavior of TK2 enzyme variants are used to explain the mechanism of mtDNA depletion caused by TK2 mutations, thymidine overload due to thymidine phosphorylase deficiency, and mitochondrial toxicity caused by antiviral thymidine analogues.

Structural stability of E. coli transketolase to temperature and pH denaturation.

PubMed

Jahromi, Raha R F; Morris, Phattaraporn; Martinez-Torres, Ruben J; Dalby, Paul A

2011-09-10

We have previously shown that the denaturation of TK with urea follows a non-aggregating though irreversible denaturation pathway in which the cofactor binding appears to become altered but without dissociating, then followed at higher urea by partial denaturation of the homodimer prior to any further unfolding or dissociation of the two monomers. Urea is not typically present during biocatalysis, whereas access to TK enzymes that retain activity at increased temperature and extreme pH would be useful for operation under conditions that increase substrate and product stability or solubility. To provide further insight into the underlying causes of its deactivation in process conditions, we have characterised the effects of temperature and pH on the structure, stability, aggregation and activity of Escherichia coli transketolase. The activity of TK was initially found to progressively improve after pre-incubation at increasing temperatures. Loss of activity at higher temperature and low pH resulted primarily from protein denaturation and subsequent irreversible aggregation. By contrast, high pH resulted in the formation of a native-like state that was only partially inactive. The apo-TK enzyme structure content also increased at pH 9 to converge on that of the holo-TK. While cofactor dissociation was previously proposed for high pH deactivation, the observed structural changes in apo-TK but not holo-TK indicate a more complex mechanism. Copyright © 2011 Elsevier B.V. All rights reserved.

Nuclear Imaging for Assessment of Prostate Cancer Gene Therapy

DTIC Science & Technology

2007-03-01

thymidine kinase transfected EL4 cells . Further exploration of Tc-99m conjugated potential HSV1-TK substrates is still undergoing in our laboratory...prostate cancer cells , has been demonstrated the utility for tissue-specific toxic gene therapy for prostate cancer[10, 11]. Therefore, an adenovirus...BJ5183 together with pAdeasy-1, the viral DNA plasmid. The pAdeasy-1 is E1 and E3 deleted, its E1 function can be complemented in 293A cells . The

The NifTK software platform for image-guided interventions: platform overview and NiftyLink messaging.

PubMed

Clarkson, Matthew J; Zombori, Gergely; Thompson, Steve; Totz, Johannes; Song, Yi; Espak, Miklos; Johnsen, Stian; Hawkes, David; Ourselin, Sébastien

2015-03-01

To perform research in image-guided interventions, researchers need a wide variety of software components, and assembling these components into a flexible and reliable system can be a challenging task. In this paper, the NifTK software platform is presented. A key focus has been high-performance streaming of stereo laparoscopic video data, ultrasound data and tracking data simultaneously. A new messaging library called NiftyLink is introduced that uses the OpenIGTLink protocol and provides the user with easy-to-use asynchronous two-way messaging, high reliability and comprehensive error reporting. A small suite of applications called NiftyGuide has been developed, containing lightweight applications for grabbing data, currently from position trackers and ultrasound scanners. These applications use NiftyLink to stream data into NiftyIGI, which is a workstation-based application, built on top of MITK, for visualisation and user interaction. Design decisions, performance characteristics and initial applications are described in detail. NiftyLink was tested for latency when transmitting images, tracking data, and interleaved imaging and tracking data. NiftyLink can transmit tracking data at 1,024 frames per second (fps) with latency of 0.31 milliseconds, and 512 KB images with latency of 6.06 milliseconds at 32 fps. NiftyIGI was tested, receiving stereo high-definition laparoscopic video at 30 fps, tracking data from 4 rigid bodies at 20-30 fps and ultrasound data at 20 fps with rendering refresh rates between 2 and 20 Hz with no loss of user interaction. These packages form part of the NifTK platform and have proven to be successful in a variety of image-guided surgery projects. Code and documentation for the NifTK platform are available from http://www.niftk.org . NiftyLink is provided open-source under a BSD license and available from http://github.com/NifTK/NiftyLink . The code for this paper is tagged IJCARS-2014.

Tumor cell apoptosis induces tumor-specific immunity in a CC chemokine receptor 1- and 5-dependent manner in mice.

PubMed

Iida, Noriho; Nakamoto, Yasunari; Baba, Tomohisa; Kakinoki, Kaheita; Li, Ying-Yi; Wu, Yu; Matsushima, Kouji; Kaneko, Shuichi; Mukaida, Naofumi

2008-10-01

The first step in the generation of tumor immunity is the migration of dendritic cells (DCs) to the apoptotic tumor, which is presumed to be mediated by various chemokines. To clarify the roles of chemokines, we induced apoptosis using suicide gene therapy and investigated the immune responses following tumor apoptosis. We injected mice with a murine hepatoma cell line, BNL 1ME A.7R.1 (BNL), transfected with HSV-thymidine kinase (tk) gene and then treated the animals with ganciclovir (GCV). GCV treatment induced massive tumor cell apoptosis accompanied with intratumoral DC infiltration. Tumor-infiltrating DCs expressed chemokine receptors CCR1 and CCR5, and T cells and macrophages expressed CCL3, a ligand for CCR1 and CCR5. Moreover, tumor apoptosis increased the numbers of DCs migrating into the draining lymph nodes and eventually generated a specific cytotoxic cell population against BNL cells. Although GCV completely eradicated HSV-tk-transfected BNL cells in CCR1-, CCR5-, or CCL3-deficient mice, intratumoral and intranodal DC infiltration and the subsequent cytotoxicity generation were attenuated in these mice. When parental cells were injected again after complete eradication of primary tumors by GCV treatment, the wild-type mice completely rejected the rechallenged cells, but the deficient mice exhibited impairment in rejection. Thus, we provide definitive evidence indicating that CCR1 and CCR5 and their ligand CCL3 play a crucial role in the regulation of intratumoral DC accumulation and the subsequent establishment of tumor immunity following induction of tumor apoptosis by suicide genes.

Characterization and multi-step transketolase-ω-transaminase bioconversions in an immobilized enzyme microreactor (IEMR) with packed tube.

PubMed

Halim, Amanatuzzakiah Abdul; Szita, Nicolas; Baganz, Frank

2013-12-01

The concept of de novo metabolic engineering through novel synthetic pathways offers new directions for multi-step enzymatic synthesis of complex molecules. This has been complemented by recent progress in performing enzymatic reactions using immobilized enzyme microreactors (IEMR). This work is concerned with the construction of de novo designed enzyme pathways in a microreactor synthesizing chiral molecules. An interesting compound, commonly used as the building block in several pharmaceutical syntheses, is a single diastereoisomer of 2-amino-1,3,4-butanetriol (ABT). This chiral amino alcohol can be synthesized from simple achiral substrates using two enzymes, transketolase (TK) and transaminase (TAm). Here we describe the development of an IEMR using His6-tagged TK and TAm immobilized onto Ni-NTA agarose beads and packed into tubes to enable multi-step enzyme reactions. The kinetic parameters of both enzymes were first determined using single IEMRs evaluated by a kinetic model developed for packed bed reactors. The Km(app) for both enzymes appeared to be flow rate dependent, while the turnover number kcat was reduced 3 fold compared to solution-phase TK and TAm reactions. For the multi-step enzyme reaction, single IEMRs were cascaded in series, whereby the first enzyme, TK, catalyzed a model reaction of lithium-hydroxypyruvate (HPA) and glycolaldehyde (GA) to L-erythrulose (ERY), and the second unit of the IEMR with immobilized TAm converted ERY into ABT using (S)-α-methylbenzylamine (MBA) as amine donor. With initial 60mM (HPA and GA each) and 6mM (MBA) substrate concentration mixture, the coupled reaction reached approximately 83% conversion in 20 min at the lowest flow rate. The ability to synthesize a chiral pharmaceutical intermediate, ABT in relatively short time proves this IEMR system as a powerful tool for construction and evaluation of de novo pathways as well as for determination of enzyme kinetics. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Characterization of Stearoyl-CoA Desaturases from a Psychrophilic Antarctic Copepod, Tigriopus kingsejongensis.

PubMed

Jung, Woongsic; Kim, Eun Jae; Han, Se Jong; Choi, Han-Gu; Kim, Sanghee

2016-10-01

Stearoyl-CoA desaturase is a key regulator in fatty acid metabolism that catalyzes the desaturation of stearic acid to oleic acid and controls the intracellular levels of monounsaturated fatty acids (MUFAs). Two stearoyl-CoA desaturases (SCD, Δ9 desaturases) genes were identified in an Antarctic copepod, Tigriopus kingsejongensis, that was collected in a tidal pool near the King Sejong Station, King George Island, Antarctica. Full-length complementary DNA (cDNA) sequences of two T. kingsejongensis SCDs (TkSCDs) were obtained from next-generation sequencing and isolated by reverse transcription PCR. DNA sequence lengths of the open reading frames of TkSCD-1 and TkSCD-2 were determined to be 1110 and 681 bp, respectively. The molecular weights deduced from the corresponding genes were estimated to be 43.1 kDa (TkSCD-1) and 26.1 kDa (TkSCD-2). The amino acid sequences were compared with those of fatty acid desaturases and sterol desaturases from various organisms and used to analyze the relationships among TkSCDs. As assessed by heterologous expression of recombinant proteins in Escherichia coli, the enzymatic functions of both stearoyl-CoA desaturases revealed that the amount of C16:1 and C18:1 fatty acids increased by greater than 3-fold after induction with isopropyl β-D-thiogalactopyranoside. In particular, C18:1 fatty acid production increased greater than 10-fold in E. coli expressing TkSCD-1 and TkSCD-2. The results of this study suggest that both SCD genes from an Antarctic marine copepod encode a functional desaturase that is capable of increasing the amounts of palmitoleic acid and oleic acid in a prokaryotic expression system.

Pseudorabies virus glycoprotein gIII is a major target antigen for murine and swine virus-specific cytotoxic T lymphocytes.

PubMed Central

Zuckermann, F A; Zsak, L; Mettenleiter, T C; Ben-Porat, T

1990-01-01

Pseudorabies virus (PrV) is the etiological agent of Aujeszky's disease, a disease that causes heavy economic losses in the swine industry. A rational approach to the generation of an effective vaccine against this virus requires an understanding of the immune response induced by it and of the role of the various viral antigens in inducing such a response. We have constructed mutants of PrV [strain PrV (Ka)] that differ from each other only in expression of the viral nonessential glycoproteins gI, gp63, gX, and gIII (i.e., are otherwise isogenic). These mutants were used to ascertain the importance of each of the nonessential glycoproteins in eliciting a PrV-specific cytotoxic T-lymphocyte (CTL) response in mice and pigs. Immunization of DBA/2 mice and pigs with a thymidine kinase-deficient (TK-) mutant of PrV elicits the formation of cytotoxic cells that specifically lyse syngeneic infected target cells. These PrV-specific cytolytic cells have the phenotype of major histocompatibility complex class I antigen-restricted CTLs. The relative number of CTLs specific for glycoproteins gI, gp63, gX, and gIII induced in mice vaccinated with a TK- mutant of PrV was ascertained by comparing their levels of cytotoxicity against syngeneic cells infected with either wild-type virus or gI-/gp63-, gX-, or gIII- virus deletion mutants. The PrV-specific CLTs were significantly less effective in lysing gIII(-)-infected targets than in lysing gI-/gp63-, gX-, or wild-type-infected targets. The in vitro secondary CTL response of lymphocytes obtained from either mice or pigs 6 or more weeks after immunization with a TK- mutant of PrV was also tested. Lymphocytes obtained from these animals were cultured with different glycoprotein-deficient mutants of PrV, and their cytolytic activities against wild-type-infected targets were ascertained. The importance of each of the nonessential viral glycoproteins in eliciting CTLs was assessed from the effectiveness of each of the virus mutants to stimulate the secondary anti-PrV CTL response. Cultures of both murine or swine lymphocytes that had been stimulated with gIII- virus contained only approximately half as many lytic units as did those stimulated with either wild-type virus, a gX- virus mutant, or a gI-/gp63- virus mutant. Thus, a large proportion of the PrV-specific CTLs that are induced by immunization with PrV of both mice and pigs are directed against gIII. Furthermore, glycoproteins gI, gp63, and gX play at most a minor role in the CTL response of these animals to PrV. PMID:2153244

SH003 represses tumor angiogenesis by blocking VEGF binding to VEGFR2

PubMed Central

Choi, Hyeong Sim; Kim, Min Kyoung; Lee, Kangwook; Lee, Kang Min; Choi, Youn Kyung; Shin, Yong Cheol; Cho, Sung-Gook; Ko, Seong-Gyu

2016-01-01

Tumor angiogenesis is a key feature of cancer progression, because a tumor requires abundant oxygen and nutrition to grow. Here, we demonstrate that SH003, a mixed herbal extract containing Astragalus membranaceus (Am), Angelica gigas (Ag) and Trichosanthes Kirilowii Maximowicz (Tk), represses VEGF-induced tumor angiogenesis both in vitro and in vivo. SH003 inhibited VEGF-induced migration, invasion and tube formation in human umbilical vein endothelial cells (HUVEC) with no effect on the proliferation. SH003 reduced CD31-positive vessel numbers in tumor tissues and retarded tumor growth in our xenograft mouse tumor model, while SH003 did not affect pancreatic tumor cell viability. Consistently, SH003 inhibited VEGF-stimulated vascular permeability in ears and back skins. Moreover, SH003 inhibited VEGF-induced VEGFR2-dependent signaling by blocking VEGF binding to VEGFR2. Therefore, our data conclude that SH003 represses tumor angiogenesis by inhibiting VEGF-induced VEGFR2 activation, and suggest that SH003 may be useful for treating cancer. PMID:27105528

(1→3)-β-d-Glucan and Galactomannan for Differentiating Chemical "Black Particles" and Fungal Particles Inside Peritoneal Dialysis Tubing.

PubMed

Leelahavanichkul, Asada; Pongpirul, Krit; Thongbor, Nisa; Worasilchai, Navaporn; Petphuak, Kwanta; Thongsawang, Bussakorn; Towannang, Piyaporn; Lorvinitnun, Pichet; Sukhontasing, Kanya; Katavetin, Pisut; Praditpornsilpa, Kearkiat; Eiam-Ong, Somchai; Chindamporn, Ariya; Kanjanabuch, Talerngsak

2016-01-01

♦ Aseptic, sheet-like foreign bodies observed inside Tenckhoff (TK) catheter lumens (referred to as "black particles") are, on gross morphology, hardly distinguishable from fungal colonization because these contaminants adhere tightly to the catheter. Detection of fungal cell wall components using (1→3)-β-d-glucan (BG) and galactomannan index (GMI) might be an alternative method for differentiating the particles. ♦ Foreign particles retrieved from TK catheters in 19 peritoneal dialysis patients were examined microscopically and cultured for fungi and bacteria. Simultaneously, a Fungitell test (Associates of Cape Cod, Falmouth, MA, USA) and a Platelia Aspergillus ELISA assay (Bio-Rad Laboratories, Marnes-La-Coquette, France) were used to test the spent dialysate for BG and GMI respectively. ♦ Of the 19 patients, 9 had aseptic black particles and 10 had fungal particles in their tubing. The fungal particles looked grainy, were tightly bound to the catheter, and appeared more "colorful" than the black particles, which looked sheet-like and could easily be removed by milking the tubing. Compared with effluent from patients having aseptic particles, effluent from patients with fungal particles had significantly higher levels of BG (501 ± 70 pg/mL vs. 46 ± 10 pg/mL) and GMI (10.98 ± 2.17 vs. 0.25 ± 0.05). Most of the fungi that formed colonies inside the catheter lumen were molds not usually found in clinical practice, but likely from water or soil, suggesting environmental contamination. Interestingly, in all 10 patients with fungal colonization, visualization of black particles preceded a peritonitis episode and TK catheter removal by approximately 1-3 weeks; in patients with aseptic particles, a 17-week onset to peritonitis was observed. ♦ In all patients with particle-coated peritoneal dialysis tubing, spent dialysate should be screened for BG and GMI. Manipulation of the TK catheter by squeezing, hard flushing, or even brushing to dislodge black particles should be avoided. Replacement of the TK catheter should be suspended until a cause for the particles is determined. Copyright © 2016 International Society for Peritoneal Dialysis.

Hematopoiesis is severely altered in mice with an induced osteoblast deficiency.

PubMed

Visnjic, Dora; Kalajzic, Zana; Rowe, David W; Katavic, Vedran; Lorenzo, Joseph; Aguila, Hector L

2004-05-01

We previously reported a transgenic mouse model expressing herpesvirus thymidine kinase (TK) gene under the control of a 2.3-kilobase fragment of the rat collagen alpha1 type I promoter (Col2.3 Delta TK). This construct confers lineage-specific expression in developing osteoblasts, allowing the conditional ablation of osteoblast lineage after treatment with ganciclovir (GCV). After GCV treatment these mice have profound alterations on bone formation leading to a progressive bone loss. In addition, treated animals also lose bone marrow cellularity. In this report we characterized hematopoietic parameters in GCV-treated Col2.3 Delta TK mice, and we show that after treatment transgenic animals lose lymphoid, erythroid, and myeloid progenitors in the bone marrow, followed by decreases in the number of hematopoietic stem cells (HSCs). Together with the decrease in bone marrow hematopoiesis, active extramedullary hematopoiesis was observed in the spleen and liver, as measured by an increase in peripheral HSCs and active primary in vitro hematopoiesis. After withdrawal of GCV, osteoblasts reappeared in the bone compartment together with a recovery of medullary and decrease in extramedullary hematopoiesis. These observations directly demonstrate the role of osteoblasts in hematopoiesis and provide a model to study the interactions between the mesenchymal and hematopoietic compartments in the marrow.

Noninvasive imaging of cationic lipid-mediated delivery of optical and PET reporter genes in living mice.

PubMed

Iyer, Meera; Berenji, Manijeh; Templeton, Nancy S; Gambhir, Sanjiv S

2002-10-01

Gene therapy involves the safe and effective delivery of one or more genes of interest to target cells in vivo. The advantages of using nonviral delivery systems include ease of preparation, low toxicity, and weak immunogenicity. Nonviral delivery methods, when combined with a noninvasive, clinically applicable imaging assay, will greatly aid in the optimization of gene therapy approaches for cancer. We demonstrate cationic lipid-mediated noninvasive monitoring of reporter gene expression of firefly (Photinus pyralis) luciferase (fl) and a mutant herpes simplex virus type I thymidine kinase (HSV1-sr39tk, tk) in living mice using a cooled charge coupled device (CCD) camera and positron emission tomography (PET), respectively. We observe a high level of fl and tk reporter gene expression predominantly in the lungs after a single injection of the extruded DOTAP:cholesterol DNA liposome complexes by way of the tail vein, seen to be time- and dose-dependent. We observe a good correlation between the in vivo bioluminescent signal and the ex vivo firefly luciferase enzyme (FL) activity in different organs. We further demonstrate the feasibility of noninvasively imaging both optical and PET reporter gene expression in the same animal using the CCD camera and microPET, respectively.

Environmental Xenobiotics and the Antihormones Cyproterone Acetate and Spironolactone Use the Nuclear Hormone Pregnenolone X Receptor to Activate the CYP3A23 Hormone Response Element

PubMed Central

SCHUETZ, ERIN G.; BRIMER, CYNTHIA; SCHUETZ, JOHN D.

2013-01-01

The pregnenolone X receptor (PXR), a new member of the nuclear hormone receptor superfamily, was recently demonstrated to mediate glucocorticoid agonist and antagonist activation of a hormone response element spaced by three nucleotides (DR-3) within the rat CYP3A23 promoter. Because many other steroids and xenobiotics can up-regulate CYP3A23 expression, we determined whether some of these other regulators used PXR to activate the CYP3A23 DR-3. Transient cotransfection of LLC-PK1 cells with (CYP3A23)2-tk-CAT and mouse PXR demonstrated that the organochlorine pesticides transnonachlor and chlordane and the nonplanar polychlorinated biphenyls (PCBs) each induced the CYP3A23 DR-3 element, and this activation required PXR. Additionally, this study found that PXR is activated to induce (CYP3A23)2-tk-CAT by antihormones of several steroid classes including the antimineralocorticoid spironolactone and the antiandrogen cyproterone acetate. These studies reveal that PXR is involved in the induction of CYP3A23 by pharmacologically and structurally distinct steroids and xenobiotics. Moreover, PXR-mediated PCB activation of the (CYP3A23)2-tk-CAT may serve as a rapid assay for effects of nonplanar PCBs. PMID:9855641

Synergy of 2-deoxy-D-glucose combined with berberine in inducing the lysosome/autophagy and transglutaminase activation-facilitated apoptosis.

PubMed

Halicka, H Dorota; Garcia, Jorge; Li, Jiangwei; Zhao, Hong; Darzynkiewicz, Zbigniew

2017-02-01

Utilizing a variety of flow cytometric methods evidence was obtained indicating that a combination of the glucose analog 2-deoxy-D-glucose (2-dG) and the plant alkaloid berberine (BRB) produces synergistic effect in the induction of apoptosis in human lymphoblastoid TK6 cells. The synergistic effect is seen at concentrations of the drugs at which each of them alone shows no cytotoxicity at all. The data suggest that the combination of these drugs, which are known in terms of their overall toxicity, side effects and pharmacokinetics may be considered for further studies as chemopreventive and cancer treatment modalities. Of interest are results indicating that rapamycin, which similarly to BRB, suppresses mTOR signaling, when combined with 2-dG shows no synergistic properties. Metformin, on other hand, requires much higher concentration to show the synergy with 2-dG. Also of interest are the findings pertaining to the methodology of the present study. Specifically, dynamic assessment of cellular viability was performed by using the DRAQ7 cell exclusion fluorochrome present in cultures from 0 to 72 h. Concurrent measurement of lysosomal proton pump using acridine orange as the probe shows activation of lysosomes in the cells treated with 2-dG or BRB alone as well as with the drugs combined. Apoptosis was assessed by measuring DNA fragmentation, cell cycle, activation of caspase-3 and tissue transglutaminase (Tgase). A novel cytometric method was developed based on analysis of lysosomal (acidic vesicles) proton pump in live cells followed by cell lysis with detergent and fluorochrome labeling of proteins and DNA to analyze Tgase activation concurrently with cell cycle, in same population of cells. The data show that the cell subpopulation undergoing apoptosis has increased side (right-angle) light scatter likely due to the presence of the crosslinked (solid state) proteins, the consequence Tgase activation.

The mechanical properties of polyimide films after exposure to high pH

NASA Technical Reports Server (NTRS)

Croall, Catharine I.; St.clair, Terry L.

1992-01-01

Wiring failures linked to insulation damage have drawn much attention in the aerospace industry and concerns have developed regarding the stability and safety of polyimide insulated electrical wire. Several polyimides were selected for evaluation for resistance to degradation by various aqueous alkaline solutions. The polyimides under evaluation include commercially available films such as Kapton (tk), Apical (tk), LaRC(tk)-TPI, and Upilex(tk)R and S, as well as a number of experimental films prepared by NASA Langley. Thermally imidized films were studied for their retention of mechanical properties after exposure to high pH solutions under stressed conditions.

Deoxyribonucleoside kinases in mitochondrial DNA depletion.

PubMed

Saada-Reisch, Ann

2004-10-01

Mitochondrial DNA (mtDNA) depletion syndromes (MDS) are a heterogeneous group of mitochondrial disorders, manifested by a decreased mtDNA copy number and respiratory chain dysfunction. Primary MDS are inherited autosomally and may affect a single organ or multiple tissues. Mutated mitochondrial deoxyribonucleoside kinases; deoxyguanosine kinase (dGK) and thymidine kinase 2 (TK2), were associated with the hepatocerebral and myopathic forms of MDS respectively. dGK and TK2 are key enzymes in the mitochondrial nucleotide salvage pathway, providing the mitochondria with deoxyribonucleotides (dNP) essential for mtDNA synthesis. Although the mitochondrial dNP pool is physically separated from the cytosolic one, dNP's may still be imported through specific transport. Non-replicating tissues, where cytosolic dNP supply is down regulated, are thus particularly vulnerable to dGK and TK2 deficiency. The overlapping substrate specificity of deoxycytidine kinase (dCK) may explain the relative sparing of muscle in dGK deficiency, while low basal TK2 activity render this tissue susceptible to TK2 deficiency. The precise pathophysiological mechanisms of mtDNA depletion due to dGK and TK2 deficiencies remain to be determined, though recent findings confirm that it is attributed to imbalanced dNTP pools.

The primary structure of the thymidine kinase gene of fish lymphocystis disease virus.

PubMed

Schnitzler, P; Handermann, M; Szépe, O; Darai, G

1991-06-01

The DNA nucleotide sequence of the thymidine kinase (TK) gene of fish lymphocystis disease virus (FLDV) which has been localized between the coordinates 0.678 to 0.688 of the viral genome was determined. The analysis of the DNA nucleotide sequence located between the recognition sites of HindIII (0.669 map unit; nucleotide position 1) and AccI (nucleotide position 2032) revealed the presence of an open reading frame of 954 bp on the lower strand of this region between nucleotide positions 1868 (ATG) and 915 (TAA). It encodes for a protein of 318 amino acid residues. The evolutionary relationships of the TK gene of FLDV to the other known TK genes was investigated using the method of progressive sequence alignment. These analyses revealed a high degree of diversity between the protein sequence of FLDV TK gene and the amino acid composition of other TKs tested. However, significant conservations were detected at several regions of amino acid residues of the FLDV TK protein when compared to the amino acid sequence of TKs of African swine fever virus, fowlpox virus, shope fibroma virus, and vaccinia virus and to the amino acid sequences of the cellular cytoplasmic TK of chicken, mouse, and man.

Kondo temperature and Heavy Fermion behavior in Yb1-xYxCuAl series of alloys

NASA Astrophysics Data System (ADS)

Rojas, D. P.; Gandra, F. G.; Medina, A. N.; Fernández Barquín, L.; Gómez Sal, J. C.

2018-05-01

Results on x-ray diffraction, electrical resistivity, specific heat and magnetization on the Yb1-xYxCuAl series of compounds are reported. The analysis of the x-ray data shows the increase of the unit cell volume with the Y dilution. The electrical resistivity shows an evolution from Kondo lattice regime for x ≤ 0.6 to single impurity behavior for x = 0.8 and 0.94. The electronic coefficient γ shows values of Heavy Fermion systems along the series for 0 ≤ x < 1 . On the other hand, dc magnetic susceptibility measurements show typical curves of intermediate valence systems with a maximum around 25 K. Below this maximum, the values of low temperature susceptibility (χ (0)) decrease with the increase of Y content. From the dependence of χ (0) and γ upon Y substitution, an increase of 12% of the Kondo temperature (TK) for x = 0.8 alloy respect to the reference YbCuAl (x = 0) is estimated. This is further supported by the evolution of the temperature of the maximum in the magnetic contribution of the specific heat. The overall results can be explained by the increase of the hybridization as consequence of negative pressure effects obtained by the chemical substitution of Yb by Y, thus leading to the increase of TK, in agreement with the Doniach's diagram.

The Role of AhR in Breast Cancer Development

DTIC Science & Technology

2006-07-01

was used according to the manufacturer’s instructions to transfect cells. The Renilla luciferase vector phRL-TK (0.05 g) was cotransfected with...sequential reading of the Firefly and Renilla signals. Cells were lysed according to the manufacturer’s directions (Promega, Madison, WI, USA...transferred to 96-well white wall plates, and analysed using a Reporter Luminometer (Promega, Madison, WI, USA). The Renilla signal was 7 read after

DEVELOPMENT OF AN INTACT HEPATOCYTE ACTIVATION SYSTEM FOR ROUTINE USE WITH THE MOUSE LYMPHOMA ASSAY

EPA Science Inventory

The authors have developed a method for cocultivating primary rat hepatocytes with L5178Y/TK+/- 3.7.2C mouse lymphoma cells. The system should provide a means to simulate more closely in vivo metabolism compared to metabolism by liver homogenates, while still being useful for rou...

Isolation and identification of feline herpesvirus type 1 from a South China tiger in China.

PubMed

Sun, Heting; Li, Yuanguo; Jiao, Weiyi; Liu, Cunfa; Liu, Xiujuan; Wang, Haijun; Hua, Fuyou; Dong, Jianxiu; Fan, Shengtao; Yu, Zhijun; Gao, Yuwei; Xia, Xianzhu

2014-02-28

In 2012, an FHV-1-like virus was isolated from a tiger that presented with clinical signs of sialorrhea, sneezing and purulent rhinorrhea. Isolation was performed with the FK81 cell line, and the virus was identified by PCR, transmission electron microscopy (TEM), and the phylogenetic analysis of the partial thymidine kinase (TK) and glycoprotein B (gB) genes. A total of 253 bp of the TK gene and 566 bp of the gB gene were amplified from the trachea of the tiger by PCR/RT-PCR method. Phylogenetic analysis showed that the isolate belonged to the same cluster with other FHV-1 strains obtained from GenBank. Herpes-like viruses with an envelope and diameters of approximately 200 nm were observed in the culture supernatants of FK81 cells inoculated with samples from the tiger. The FHV-1 infection was confirmed by an animal challenge experiment in a cat model. Our finding extends the host range of FHV-1 and has implications for FHV-1 infection and South China tiger conservation.

Altered levels of the Taraxacum kok-saghyz (Russian dandelion) small rubber particle protein, TkSRPP3, result in qualitative and quantitative changes in rubber metabolism.

PubMed

Collins-Silva, Jillian; Nural, Aise Taban; Skaggs, Amanda; Scott, Deborah; Hathwaik, Upul; Woolsey, Rebekah; Schegg, Kathleen; McMahan, Colleen; Whalen, Maureen; Cornish, Katrina; Shintani, David

2012-07-01

Several proteins have been identified and implicated in natural rubber biosynthesis, one of which, the small rubber particle protein (SRPP), was originally identified in Hevea brasiliensis as an abundant protein associated with cytosolic vesicles known as rubber particles. While previous in vitro studies suggest that SRPP plays a role in rubber biosynthesis, in vivo evidence is lacking to support this hypothesis. To address this issue, a transgene approach was taken in Taraxacum kok-saghyz (Russian dandelion or Tk) to determine if altered SRPP levels would influence rubber biosynthesis. Three dandelion SRPPs were found to be highly abundant on dandelion rubber particles. The most abundant particle associated SRPP, TkSRPP3, showed temporal and spatial patterns of expression consistent with patterns of natural rubber accumulation in dandelion. To confirm its role in rubber biosynthesis, TkSRPP3 expression was altered in Russian dandelion using over-expression and RNAi methods. While TkSRPP3 over-expressing lines had slightly higher levels of rubber in their roots, relative to the control, TkSRPP3 RNAi lines showed significant decreases in root rubber content and produced dramatically lower molecular weight rubber than the control line. Not only do results here provide in vivo evidence of TkSRPP proteins affecting the amount of rubber in dandelion root, but they also suggest a function in regulating the molecular weight of the cis-1, 4-polyisoprene polymer. Copyright © 2012 Elsevier Ltd. All rights reserved.

[131I]FIAU labeling of genetically transduced, tumor-reactive lymphocytes: cell-level dosimetry and dose-dependent toxicity.

PubMed

Zanzonico, Pat; Koehne, Guenther; Gallardo, Humilidad F; Doubrovin, Mikhail; Doubrovina, Ekaterina; Finn, Ronald; Blasberg, Ronald G; Riviere, Isabelle; O'Reilly, Richard J; Sadelain, Michel; Larson, Steven M

2006-09-01

Donor T cells have been shown to be reactive against and effective in adoptive immunotherapy of Epstein-Barr virus (EBV) lymphomas which develop in some leukemia patients post marrow transplantation. These T cells may be genetically modified by incorporation of a replication-incompetent viral vector (NIT) encoding both an inactive mutant nerve growth factor receptor (LNGFR), as an immunoselectable surface marker, and a herpes simplex virus thymidine kinase (HSV-TK), rendering the cells sensitive to ganciclovir. The current studies are based on the selective HSV-TK-catalyzed trapping (phosphorylation) of the thymidine analog [(131)I]-2'-fluoro-2'-deoxy-1-beta-D-arabinofuransyl-5-iodo-uracil (FIAU) as a means of stably labeling such T cells for in vivo trafficking (including tumor targeting) studies. Because of the radiosensitivity of lymphocytes and the potentially high absorbed dose to the nucleus from intracellular (131)I (even at tracer levels), the nucleus absorbed dose (D ( n )) and dose-dependent immune functionality were evaluated for NIT(+) T cells labeled ex vivo in [(131)I]FIAU-containing medium. Based on in vitro kinetic studies of [(131)I]FIAU uptake by NIT(+) T cells, D ( n ) was calculated using an adaptation of the MIRD formalism and the recently published MIRD cellular S factors. Immune cytotoxicity of [(131)I]FIAU-labeled cells was assayed against (51)Cr-labeled target cells [B-lymphoblastoid cells (BLCLs)] in a standard 4-h release assay. At median nuclear absorbed doses up to 830 cGy, a (51)Cr-release assay against BLCLs showed no loss of immune cytotoxicity, thus demonstrating the functional integrity of genetically transduced, tumor-reactive T cells labeled at this dose level for in vivo cell trafficking and tumor targeting studies.

Integrated evaluation of the vulnerability to thermokarst disturbance and its implications for the regional carbon balance in boreal Alaska

NASA Astrophysics Data System (ADS)

Helene, G.; Lara, M. J.; McGuire, A. D.; Euskirchen, E. S.; Bolton, W. R.; Romanovsky, V. E.

2017-12-01

Our capacity to project future ecosystem trajectories in northern permafrost regions depends on our ability to characterize complex interactions between climatic and ecological processes at play in the soil, the vegetation, and the atmosphere. We present a study that uses remote sensing analyses, field observations, and data synthesis to inform models for the prediction of ecosystem responses to climate change in the boreal zone of Alaska. Recent warming, altered precipitation and fire regimes are driving permafrost degradation, threatening to mobilize vast reservoirs of ancient carbon previously protected from decomposition. Although large scale, progressive, top-down permafrost thaw have been well studied and represented in high-latitude ecosystem models, the consequences of abrupt and local thermokarst disturbances (TK) are less well understood. To fill this gap, we conducted a detection analysis characterizing 60 years of land cover change in the Tanana Flats, a wetland complex subjected to TK disturbance in Interior Alaska, using aerial and satellite images. We observed a nonlinear loss of permafrost plateau forest associated with TK and driven by precipitation and forest fragmentation. The results of this analysis were integrated into the Alaska Thermokarst Model (ATM), a state-and-transition model that simulates land cover change associated with TK disturbance. Thermokarst-related land cover change was simulated from 2000 to 2100 across the Tanana Flats. By 2100, the model predicts a mean decrease of 7.4% (sd 1.8%) in permafrost plateau forests associated with an increase in TK fens and bogs. Transitions from permafrost plateau forests to TK wetlands are accompanied with changes in physical and biogeochemical processes affecting ecosystem carbon balance. We evaluated the consequences of TK disturbances on the regional carbon balance by coupling outputs from the ATM and from a process-based biogeochemical model. We used long-term field observations of vegetation and soil physical and biogeochemical attributes to develop new parameterizations for TK wetlands and permafrost plateau forest land cover types. Preliminary simulations from 2000 to 2100 estimate that the conversion of permafrost plateau forest to young TK wetlands would result in a 7.5% (sd 3.5%) decrease in Net Ecosystem Exchange.

FSP1+ fibroblast subpopulation is essential for the maintenance and regeneration of medullary thymic epithelial cells

PubMed Central

Sun, Lina; Sun, Chenming; Liang, Zhanfeng; Li, Hongran; Chen, Lin; Luo, Haiying; Zhang, Hongmei; Ding, Pengbo; Sun, Xiaoning; Qin, Zhihai; Zhao, Yong

2015-01-01

Thymic epithelial cells (TECs) form a 3-dimentional network supporting thymocyte development and maturation. Besides epithelium and thymocytes, heterogeneous fibroblasts are essential components in maintaining thymic microenvironments. However, thymic fibroblast characteristics, development and function remain to be determined. We herein found that thymic non-hematopoietic CD45-FSP1+ cells represent a unique Fibroblast specific protein 1 (FSP1)—fibroblast-derived cell subset. Deletion of these cells in FSP1-TK transgenic mice caused thymus atrophy due to the loss of TECs, especially mature medullary TECs (MHCIIhigh, CD80+ and Aire+). In a cyclophosphamide-induced thymus injury and regeneration model, lack of non-hematopoietic CD45-FSP1+ fibroblast subpopulation significantly delayed thymus regeneration. In fact, thymic FSP1+ fibroblasts released more IL-6, FGF7 and FSP1 in the culture medium than their FSP1- counterparts. Further experiments showed that the FSP1 protein could directly enhance the proliferation and maturation of TECs in the in vitro culture systems. FSP1 knockout mice had significantly smaller thymus size and less TECs than their control. Collectively, our studies reveal that thymic CD45-FSP1+ cells are a subpopulation of fibroblasts, which is crucial for the maintenance and regeneration of TECs especially medullary TECs through providing IL-6, FGF7 and FSP1. PMID:26445893

Simulation of human plasma concentration-time profiles of the partial glucokinase activator PF-04937319 and its disproportionate N-demethylated metabolite using humanized chimeric mice and semi-physiological pharmacokinetic modeling.

PubMed

Kamimura, Hidetaka; Ito, Satoshi; Chijiwa, Hiroyuki; Okuzono, Takeshi; Ishiguro, Tomohiro; Yamamoto, Yosuke; Nishinoaki, Sho; Ninomiya, Shin-Ichi; Mitsui, Marina; Kalgutkar, Amit S; Yamazaki, Hiroshi; Suemizu, Hiroshi

2017-05-01

1. The partial glucokinase activator N,N-dimethyl-5-((2-methyl-6-((5-methylpyrazin-2-yl)carbamoyl)benzofuran-4-yl)oxy)pyrimidine-2-carboxamide (PF-04937319) is biotransformed in humans to N-methyl-5-((2-methyl-6-((5-methylpyrazin-2-yl)carbamoyl)benzofuran-4-yl)oxy)pyrimidine-2-carboxamide (M1), accounting for ∼65% of total exposure at steady state. 2. As the disproportionately abundant nature of M1 could not be reliably predicted from in vitro metabolism studies, we evaluated a chimeric mouse model with humanized liver on TK-NOG background for its ability to retrospectively predict human disposition of PF-04937319. Since livers of chimeric mice were enlarged by hyperplasia and contained remnant mouse hepatocytes, hepatic intrinsic clearances normalized for liver weight, metabolite formation and liver to plasma concentration ratios were plotted against the replacement index by human hepatocytes and extrapolated to those in the virtual chimeric mouse with 100% humanized liver. 3. Semi-physiological pharmacokinetic analyses using the above parameters revealed that simulated concentration curves of PF-04937319 and M1 were approximately superimposed with the observed clinical data in humans. 4. Finally, qualitative profiling of circulating metabolites in humanized chimeric mice dosed with PF-04937319 or M1 also revealed the presence of a carbinolamide metabolite, identified in the clinical study as a human-specific metabolite. The case study demonstrates that humanized chimeric mice may be potentially useful in preclinical discovery towards studying disproportionate or human-specific metabolism of drug candidates.

Myopathic mtDNA Depletion Syndrome Due to Mutation in TK2 Gene.

PubMed

Martín-Hernández, Elena; García-Silva, María Teresa; Quijada-Fraile, Pilar; Rodríguez-García, María Elena; Hernández-Laín, Aurelio; Coca-Robinot, David; Rivera, Henry; Fernández-Toral, Joaquín; Arenas, Joaquín; Martín, MiguelÁngel; Martínez-Azorín, Francisco

2016-02-29

Whole-exome sequencing (WES) was used to identify the disease gene(s) in a Spanish girl with failure to thrive, muscle weakness, mild facial weakness, elevated creatine kinase (CK), deficiency of mitochondrial complex III and depletion of mtDNA. With WES data, it was possible to get the whole mtDNA sequencing and discard any pathogenic variant in this genome. The analysis of whole exome uncovered a homozygous pathogenic mutation in Thymidine kinase 2 gene (TK2; NM_004614.4:c.323C>T, p.T108M). TK2 mutations have been identified mainly in patients with the myopathic form of mtDNA depletion syndromes (MDS). This patient presents an atypical TK2 related-myopathic form of MDS, because despite having a very low content of mtDNA (<20%), she presents a slower and less severe evolution of the disease. In conclusion, our data confirm the role of TK2 gene in MDS and expanded the phenotypic spectrum.

Myopathic mtDNA Depletion Syndrome Due to Mutation in TK2 Gene.

PubMed

Martín-Hernández, Elena; García-Silva, María Teresa; Quijada-Fraile, Pilar; Rodríguez-García, María Elena; Rivera, Henry; Hernández-Laín, Aurelio; Coca-Robinot, David; Fernández-Toral, Joaquín; Arenas, Joaquín; Martín, Miguel A; Martínez-Azorín, Francisco

2017-01-01

Whole-exome sequencing was used to identify the disease gene(s) in a Spanish girl with failure to thrive, muscle weakness, mild facial weakness, elevated creatine kinase, deficiency of mitochondrial complex III and depletion of mtDNA. With whole-exome sequencing data, it was possible to get the whole mtDNA sequencing and discard any pathogenic variant in this genome. The analysis of whole exome uncovered a homozygous pathogenic mutation in thymidine kinase 2 gene ( TK2; NM_004614.4:c.323 C>T, p.T108M). TK2 mutations have been identified mainly in patients with the myopathic form of mtDNA depletion syndromes. This patient presents an atypical TK2-related myopathic form of mtDNA depletion syndromes, because despite having a very low content of mtDNA (<20%), she presents a slower and less severe evolution of the disease. In conclusion, our data confirm the role of TK2 gene in mtDNA depletion syndromes and expanded the phenotypic spectrum.

New insights from Thailand into the maternal genetic history of Mainland Southeast Asia.

PubMed

Kutanan, Wibhu; Kampuansai, Jatupol; Brunelli, Andrea; Ghirotto, Silvia; Pittayaporn, Pittayawat; Ruangchai, Sukhum; Schröder, Roland; Macholdt, Enrico; Srikummool, Metawee; Kangwanpong, Daoroong; Hübner, Alexander; Arias, Leonardo; Stoneking, Mark

2018-02-26

Tai-Kadai (TK) is one of the major language families in Mainland Southeast Asia (MSEA), with a concentration in the area of Thailand and Laos. Our previous study of 1234 mtDNA genome sequences supported a demic diffusion scenario in the spread of TK languages from southern China to Laos as well as northern and northeastern Thailand. Here we add an additional 560 mtDNA genomes from 22 groups, with a focus on the TK-speaking central Thai people and the Sino-Tibetan speaking Karen. We find extensive diversity, including 62 haplogroups not reported previously from this region. Demic diffusion is still a preferable scenario for central Thais, emphasizing the expansion of TK people through MSEA, although there is also some support for gene flow between central Thai and native Austroasiatic speaking Mon and Khmer. We also tested competing models concerning the genetic relationships of groups from the major MSEA languages, and found support for an ancestral relationship of TK and Austronesian-speaking groups.

The success story of Fatima Begum.

PubMed

1999-12-01

This article tells the success story of Fatima Begum from Jhenaidaha, Bangladesh. Having learned of the Integrated Rural Women Development Program of the Bangladesh Rural Development Board, Fatima started a Mohila Samobaya Samity or Women's Cooperative Society on August 21, 1993, with a deposit of Tk. 15 as savings and Tk. 10 as share. In 1994 and 1997 she received loans which she used to purchase a cow, for small trade, and for starting a grocery shop. She had repaid all her loans and had increased her savings deposit to Tk. 1530 and share to Tk. 710. She also had purchased 5 decimals of land and constructed a small house. Meanwhile, Fatima had adopted family planning methods and had two children. Fatima is now the elected Director of Jhenaidaha Sadar Thana Cooperative Societies Federation. Her own society is considered an ideal organization under her leadership. Her society now has 28 members and the amount of thrift-deposit has become Tk. 25,590.

The use of Tcl and Tk to improve design and code reutilization

NASA Technical Reports Server (NTRS)

Rodriguez, Lisbet; Reinholtz, Kirk

1995-01-01

Tcl and Tk facilitate design and code reuse in the ZIPSIM series of high-performance, high-fidelity spacecraft simulators. Tcl and Tk provide a framework for the construction of the Graphical User Interfaces for the simulators. The interfaces are architected such that a large proportion of the design and code is used for several applications, which has reduced design time and life-cycle costs.

Near-infrared indocyanine dye permits real-time characterization of both venous and lymphatic circulation

NASA Astrophysics Data System (ADS)

Kurahashi, Toshikazu; Iwatsuki, Katsuyuki; Onishi, Tetsuro; Arai, Tetsuya; Teranishi, Katsunori; Hirata, Hitoshi

2016-08-01

We investigated the optical properties of a near-infrared (NIR) fluorochrome, di-β-cyclodextrin-binding indocyanine derivative (TK-1), and its pharmacokinetic differences with indocyanine green (ICG). TK-1 was designed to have hydrophilic cyclodextrin molecules and, thus, for higher water solubility and smaller particle sizes than the plasma protein-bound ICG. We compared optical properties such as the absorption and fluorescence spectra, quantum yield, and photostability between both dyes in vitro. In addition, we subcutaneously injected a 1 mM solution of TK-1 or ICG into the hind footpad of rats and observed real-time NIR fluorescence intensities in their femoral veins and accompanying lymphatics at the exposed groin site to analyze the dye pharmacokinetics. These optical experiments demonstrated that TK-1 has high water solubility, a low self-aggregation tendency, and high optical and chemical stabilities. Our in vivo imaging showed that TK-1 was transported via peripheral venous flow and lymphatic flow, whereas ICG was drained only through lymphatics. The results of this study showed that lymphatic and venous transport can be differentially regulated and is most likely influenced primarily by particle size, and that TK-1 can enable real-time NIR fluorescence imaging of whole fluids and solute movement via both microvessels and lymphatics, which conventional ICG cannot achieve.

The structures of thymidine kinase from herpes simplex virus type 1 in complex with substrates and a substrate analogue.

PubMed Central

Wild, K.; Bohner, T.; Folkers, G.; Schulz, G. E.

1997-01-01

Thymidine kinase from Herpes simplex virus type 1 (TK) was crystallized in an N-terminally truncated but fully active form. The structures of TK complexed with ADP at the ATP-site and deoxythymidine-5'-monophosphate (dTMP), deoxythymidine (dT), or idoxuridine-5'-phosphate (5-iodo-dUMP) at the substrate-site were refined to 2.75 A, 2.8 A, and 3.0 A resolution, respectively. TK catalyzes the phosphorylation of dT resulting in an ester, and the phosphorylation of dTMP giving rise to an anhydride. The presented TK structures indicate that there are only small differences between these two modes of action. Glu83 serves as a general base in the ester reaction. Arg163 parks at an internal aspartate during ester formation and binds the alpha-phosphate of dTMP during anhydride formation. The bound deoxythymidine leaves a 35 A3 cavity at position 5 of the base and two sequestered water molecules at position 2. Cavity and water molecules reduce the substrate specificity to such an extent that TK can phosphorylate various substrate analogues useful in pharmaceutical applications. TK is structurally homologous to the well-known nucleoside monophosphate kinases but contains large additional peptide segments. PMID:9336833

A Japanese Stretching Intervention Can Modify Lumbar Lordosis Curvature.

PubMed

Kadono, Norio; Tsuchiya, Kazushi; Uematsu, Azusa; Kamoshita, Hiroshi; Kiryu, Kazunori; Hortobágyi, Tibor; Suzuki, Shuji

2017-08-01

Eighteen healthy male adults were assigned to either an intervention or control group. Isogai dynamic therapy (IDT) is one of Japanese stretching interventions and has been practiced for over 70 years. However, its scientific quantitative evidence remains unestablished. The objective of this study was to determine whether IDT could modify lumbar curvature in healthy young adults compared with stretching exercises used currently in clinical practice. None of previous studies have provided data that conventional stretching interventions could modify spinal curvatures. However, this study provides the first evidence that a specific form of a Japanese stretching intervention can acutely modify the spinal curvatures. We compared the effects of IDT, a Japanese stretching intervention (n=9 males), with a conventional stretching routine (n=9 males) used widely in clinics to modify pelvic tilt and lumbar lordosis (LL) angle. We measured thoracic kyphosis (TK) and LL angles 3 times during erect standing using the Spinal Mouse before and after each intervention. IDT consisted of: (1) hip joint correction, (2) pelvic tilt correction, (3) lumbar alignment correction, and (4) squat exercise stretch. The control group performed hamstring stretches while (1) standing and (2) sitting. IDT increased LL angle to 25.1 degrees (±5.9) from 21.2 degrees (±6.9) (P=0.047) without changing TK angle (pretest: 36.8 degrees [±6.9]; posttest: 36.1 degrees [±6.5]) (P=0.572). The control group showed no changes in TK (P=0.819) and LL angles (P=0.744). IDT can thus be effective for increasing LL angle, hence anterior pelvic tilt. Such modifications could ameliorate low back pain and improve mobility in old adults with an unfavorable pelvic position.

Valorizing the 'Irulas' traditional knowledge of medicinal plants in the Kodiakkarai Reserve Forest, India

PubMed Central

Ragupathy, Subramanyam; Newmaster, Steven G

2009-01-01

A mounting body of critical research is raising the credibility of Traditional Knowledge (TK) in scientific studies. These studies have gained credibility because their claims are supported by methods that are repeatable and provide data for quantitative analyses that can be used to assess confidence in the results. The theoretical importance of our study is to test consensus (reliability/replicable) of TK within one ancient culture; the Irulas of the Kodiakkarai Reserve Forest (KRF), India. We calculated relative frequency (RF) and consensus factor (Fic) of TK from 120 Irulas informants knowledgeable of medicinal plants. Our research indicates a high consensus of the Irulas TK concerning medicinal plants. The Irulas revealed a diversity of plants that have medicinal and nutritional utility in their culture and specific ethnotaxa used to treat a variety of illnesses and promote general good health in their communities. Throughout history aboriginal people have been the custodians of bio-diversity and have sustained healthy life-styles in an environmentally sustainable manner. However this knowledge has not been transferred to modern society. We suggest this may be due to the asymmetry between scientific and TK, which demands a new approach that considers the assemblage of TK and scientific knowledge. A greater understanding of TK is beginning to emerge based on our research with both the Irulas and Malasars; they believe that a healthy lifestyle is founded on a healthy environment. These aboriginal groups chose to share this knowledge with society-at-large in order to promote a global lifestyle of health and environmental sustainability. PMID:19366462

Impact of kitchen organization on oral intake of malnourished inpatients: A two-center study.

PubMed

Calleja-Fernández, Alicia; Velasco-Gimeno, Cristina; Vidal-Casariego, Alfonso; Pintor-de-la-Maza, Begoña; Frías-Soriano, Laura; Villar-Taibo, Rocío; García-Peris, Pilar; Cano-Rodríguez, Isidoro; García-Fernández, Camino; Ballesteros-Pomar, María D

2017-10-01

To determine the impact of the type of hospital kitchen on the dietary intake of patients. A cross-sectional, two-centre study, of cooking in a traditional kitchen (TK) and in a chilled kitchen (CK). Subjective global assessment (SGA) was used for nutritional diagnosis. Before study start, a dietician performed a nutritional assessment of the menus of each hospital. All dishes were weighed upon arrival to the ward and at the end of the meal. 201 and 41 patients from the centres with TK and CK respectively were evaluated. Prevalence of malnutrition risk was 50.2% at the hospital with TK and 48.8% at the hospital with CK (p=0.328). Forty-eight and 56 dishes were nutritionally evaluated at the hospitals with TK and CK respectively. Intake analysis consisted of 1993 and 846 evaluations in the hospitals with TK and CK respectively. Median food consumption was 76.83% at the hospital with TK (IQR 45.76%) and 83.43% (IQR 40.49%) at the hospital with CK (p<0.001). Based on the prevalence of malnutrition, a higher protein and energy intake was seen in malnourished patients from the CK as compared to the TK hospital, but differences were not significant after adjustment for other factors. Cooking in a chilled kitchen, as compared to a traditional kitchen, may increase energy and protein intake in hospitalized patients, which is particularly beneficial for malnourished patients. Copyright © 2017 SEEN. Publicado por Elsevier España, S.L.U. All rights reserved.

Influence of screw density on thoracic kyphosis restoration in hypokyphotic adolescent idiopathic scoliosis.

PubMed

Luo, Ming; Jiang, Honghui; Wang, Wengang; Li, Ning; Shen, Mingkui; Li, Peng; Xu, Genzhong; Xia, Lei

2017-12-13

Previous studies have reported that rod composition and diameter, as well as the correction technique are key factors associated with thoracic kyphosis (TK) restoration. However, few study has analyzed the correlation between screw density and TK restoration in hypokyphotic adolescent idiopathic scoliosis (AIS). Fifty-seven thoracic AIS patients with preoperative TK < 10° treated with all pedicle screw fixation with a minimum 2-year follow-up were recruited. Preoperative and postoperative radiographic measurements, and information of posterior instrumentation were reviewed. Pearson and Spearman correlation coefficient analysis were used to assess relationships between change in TK and number of variables. Then, the included patients were classified into two groups (Group 1: postoperative TK ≥ 20°; Group 2: postoperative TK < 20°) to evaluate the influence factors of TK restoration. The average preoperative TK was 4.75°, which was significantly restored to 17.30° (P < 0.001). Significant correlations were found between change in TK and flexibility of major thoracic curve (r = 0.357, P = 0.006), preoperative TK (r = -0.408, P = 0.002), and screw density of concave side (r = 0.306, P = 0.021), respectively. In the subgroup comparison, 17 patients (29.8%) maintain the postoperative TK ≥ 20°, increased flexibility of major thoracic curve (P < 0.001), screw number of concave side (P = 0. 029), and cobalt chromium rods (P = 0.041) were found in the group of postoperative TK ≥ 20°. TK restoration remains a challenge for AIS patients with hypokyphosis, especially for the poor flexibility ones. Except for thicker and cobalt chromium rods, screw density of concave side might be another positive predictor of restoring normal kyphosis, which provides a stronger corrective force on the sagittal plane with more pedicle screws.