Characterization of a silencer regulatory element in the human interferon-gamma promoter.

Previous analysis of the human interferon-gamma (IFN-gamma promoter indicated that the region of DNA from -251 to -215 (designated here as BE (binding element)) possessed silencer activity, as deletion of this region caused an increase in promoter activity. Based on this finding, we have conducted a series of experiments to characterize BE function and analyze the binding proteins which interact with this region. Transient transfection assays in the Jurkat T cell line revealed that the BE region possesses silencer activity, which is orientation-dependent when reinserted 5' to the IFN-gamma core promoter.

Interferon gamma-induced reduction in erbB-2 tyrosyl phosphorylation in human ovarian carcinoma cells.

Interferon, which inhibits growth of ovarian cancer cells in vivo and in vitro, decreases expression of erbB-2 protein in ovarian carcinoma cell lines. We now show that interferon-gamma (IFN-gamma) also decreases constitutive tyrosine phosphorylation of erbB-2 and inhibits erbB-2 kinase activity in an ovarian cancer cell line. SK-OV3 ovarian cancer cells, which over-express erbB-2, were treated with IFN-gamma for 0-72 hr.

Modulation of the cytotoxic activity of tumor necrosis factor by protein tyrosine kinase and protein tyrosine phosphatase inhibitors.

The mechanism by which tumor necrosis factor (TNF) inhibits cell growth is not known. The importance of tyrosine phosphorylation in mediating the cytotoxicity of TNF has been suggested from previous studies. In this report, we investigated the effects of both tyrosine kinase (TK) and protein tyrosine phosphatase (PTP) inhibitors on the cytotoxic effects of TNF.

Analysis of Eimeria acervulina-induced changes in the intestinal T lymphocyte subpopulations in two chicken strains showing different levels of susceptibility to coccidiosis.

The major T lymphocyte subpopulations expressing CD8, CD4, or antigen-specific T cell receptor (TCR) heterodimer alpha beta (TCR2) or gamma delta (TCR1) were assessed in duodenum intraepithelium of two inbred chicken strains, SC and TK, by single- and double-label immunofluorescence (IF) at various times after oral inoculation with Eimeria acervulina. The duodenum intraepithelial T lymphocytes (IEL) expressing the CD8 antigen (cytotoxic/suppressor T cells) increased in the SC and TK chickens following primary infection. However, a significant increase in the duodenum CD8+ IEL occurred in the SC chickens which manifested as a lower level of oocyst production compared to the TK chickens following primary and challenge infections.

Use of recombinant vaccinia virus vectors for cell biology.

This chapter describes the use of several of the recombinant vaccinia expression systems, focuses on the systems that are most useful for cell biologists, and discusses their advantages and limitations. Vaccinia-mediated expression can be used for assessing cellular localization, posttranslational modifications, oligomerization, and transport and turnover rates. The system provides a rapid method for screening mutant proteins for expression and targeting.

Comparison of Telazol, Telazol-ketamine, Telazol-xylazine, and Telazol-ketamine-xylazine as chemical restraint and anesthetic induction combination in swine.

The use of Telazol (T, tiletamine and zolazepam, 4.4 mg T/kg) alone, Telazol-ketamine (TK, 4.4 mg T/kg and 2.

Thermodynamics of monosaccharide binding to concanavalin A, pea (Pisum sativum) lectin, and lentil (Lens culinaris) lectin.

Titration calorimetry measurements of the binding of methyl alpha-D-mannopyranoside (Me alpha Man), D-mannopyranoside (Man), methyl alpha-D-glucopyranoside (Me alpha Glu), and D-glucopyranoside (Glu) to concanavalin A (Con A), pea lectin, and lentil lectin were performed at 281 and 292 K in 0.01 M dimethylglutaric acid-NaOH buffer (pH 6.9) containing 0.

Detection of silent temporal gaps between narrow-band noise makers having second-formantlike properties of voiceless stop/vowel combinations.

Temporal gap detection thresholds were measured in narrow-band noise-burst markers having acoustic characteristics representative of isolated steady-state second-formant (F2) properties for/p,t,k/paired separately with/i,ae,u,o/. The results revealed that gap detection threshold increased systematically as the difference was increased between the simulated stop and vowel F2 frequencies. A strong positive correlation (r = 0.

Transduction of a signal for arachidonic acid metabolism by untriggered CSF-1 receptor induces an opposite effect to that induced by CSF-1 receptor and its ligand: separate regulation of phospholipase A2 and cyclooxygenase by CSF-1 receptor/CSF-1.

The mouse hematopoietic cell line, 32D, was transfected with c-fms, which encodes for the CSF-1 receptor, a tyrosine kinase (TK). In the absence of CSF-1, transfected cells show moderate levels of arachidonic acid (AA) release and produce a substantial amount of prostaglandin E2 (PGE2) in comparison with the original cell line. Exposure of transfected cells to CSF-1, while inducing a substantial increase in arachidonate release, nevertheless resulted in inhibition of PGE2 production.

Identification of the site phosphorylated by casein kinase II in smooth muscle caldesmon.

Phosphorylation of avian gizzard caldesmon by casein kinase II was investigated. The enzyme incorporates about 1 mol of phosphate per mol of caldesmon. All sites of phosphorylation are located in short chymotryptic peptides with Mr 25-27 kDa or in the short N-terminal peptide formed after cleavage of chicken gizzard caldesmon at Cys153.

Mutations in the thymidine kinase gene that allow expression of the enzyme in quiescent (G0) cells.

Thymidine kinase (TK) is a nucleotide salvage pathway enzyme whose activity is highly dependent on the growth state and cell cycle phase of a cell. Cells in the resting or quiescent (G0) phase express very low levels of TK mRNA and protein. When quiescent cells are stimulated to enter the cell cycle by the addition of serum, TK mRNA, activity and polypeptide increase coordinately after about 10-15 h, at the beginning of S phase.

Tumor metastasis and nm23: current concepts.

Reduced expression and/or somatic allelic deletion of nm23 is associated with high metastatic potential in several types of rodent tumors and human breast and colorectal carcinomas. Transfection of murine nm23-1 cDNA into highly metastatic murine K-1735 TK melanoma cells results in a reduced incidence of primary tumor formation, significant reduction in tumor metastatic potential, and altered responsiveness to the cytokine, transforming growth factor beta. Here we discuss emerging concepts concerning nm23, such as its varied pattern of alteration/expression in tumor metastasis, its effect on tumorigenesis, and its possible biochemical functions.

Cell cycle regulation of thymidine kinase: residues near the carboxyl terminus are essential for the specific degradation of the enzyme at mitosis.

The level of human thymidine kinase (TK) polypeptide is subject to cell cycle regulation. The enzyme is barely detectable in G1 phase but increases 10- to 20-fold by M phase. The low level of human TK in G1 phase is due primarily to the specific degradation of the protein during cell division.

Reduced tumor incidence, metastatic potential, and cytokine responsiveness of nm23-transfected melanoma cells.

Reduced expression of the nm23 gene in certain rodent model systems and human breast tumors has been correlated with high tumor metastatic potential. To investigate the functional effects of nm23 expression, we have transfected a constitutive murine nm23-1 expression construct into highly metastatic K-1735 TK murine melanoma cells. TK clones expressing the exogenous nm23-1 construct exhibited a reduced incidence of primary tumor formation, significant reductions in tumor metastatic potential independent of tumor cell growth, and altered responses to the cytokine transforming growth factor beta 1 in soft agar colonization assays, compared with control-transfected TK clones.

Selecting an efficient design for assessing exposure-disease relationships in an assembled cohort.

We develop approximate methods to compare the efficiencies and to compute the power of alternative potential designs for sampling from a cohort before beginning to collect exposure data. Our methods require only that the cohort be assembled, meaning that the numbers of individuals Nkj at risk at pairs of event times tk and tj greater than or equal to tk are available. To compute Nkj, one needs to know the entry, follow-up, censoring, and event history, but not the exposure, for each individual.

Chemical mutagenesis at the thymidine kinase locus in L5178Y mouse lymphoma cells: results for 31 coded compounds in the National Toxicology Program.

Experimental data from the testing of 31 chemicals for mutagenicity at the TK locus in L5178Y mouse lymphoma cells are presented and evaluated. If mutagenic activity was not obtained for the chemical added to suspension cultures for 4 hr, then the testing was repeated in the presence of hepatic S9 mix prepared from Aroclor 1254-induced male Fischer 344 rats. Multiple trials were performed for each chemical, and mutagenic treatments were analyzed for the induction of small and large mutant colony populations.

Species-specific lens activation of the thymidine kinase promoter by a single copy of the mouse alpha A-CRYBP1 site and loss of tissue specificity by multimerization.

One copy of the mouse alpha A-crystallin gene alpha A-CRYBP1 site activated the thymidine kinase (tk) promoter in a mouse lens epithelial cell line but not in primary chicken lens cells; multiple copies further activated the tk promoter and extended expression to fibroblasts, B cells, and chicken lens cultures. The loss of lens specificity by multimerization may place selective constraints on the number of alpha A-CRYBP1 sites in the alpha A-crystallin promoter.

Expression of the tyrosine kinase receptor gene trkB is confined to the murine embryonic and adult nervous system.

We have examined the expression of the trkB gene, which encodes a member of the family of protein tyrosine kinase (TK) transmembrane receptors, during mouse embryogenesis using in situ hybridization and Northern analysis. Transcripts were first detected in the neuroepithelium and in the neural crest of 9.5 day embryos with regions of high expression in the neural folds and at the lateral neuroepithelium.

Contrasting effects of hypoxia on tension in rat pulmonary and mesenteric arteries.

The effects of hypoxia on resting and K-stimulated tension were tested on small rings of rat pulmonary and mesenteric resistance arteries (SPA and SMA, respectively) and on the large branches of the main pulmonary artery (LPA). Reduction of PO2 from approximately 135 Torr to less than 40 Torr slowly increased SPA and LPA resting tension but did not affect SMA tension. The increases in SPA and LPA tension during hypoxia were reversible and were dependent on external Ca2+.

Expression of the trk proto-oncogene is restricted to the sensory cranial and spinal ganglia of neural crest origin in mouse development.

We have cloned and characterized the mouse homolog of the human trk proto-oncogene, a member of the protein tyrosine kinase (TK) receptor gene family. Here, we present the first report of a trk-encoded mRNA species in vivo. In situ hybridization analysis in the mouse embryo reveals a striking temporal and spatial regulation of trk transcription, with expression confined to the sensory cranial (trigeminal, superior, jugular) and dorsal root ganglia (DRG) of neural crest origin.

The replication of viral and cellular DNA in human herpesvirus 6-infected cells.

Human herpesvirus 6 (HHV-6) is a newly identified lymphotropic herpesvirus. We have analyzed viral and host DNA replication in peripheral blood lymphocytes infected in the absence of drugs or infected in the presence of phosphonoacetic acid (PAA) or acyclovir (ACV). The results revealed the following: (i) Infection with HHV-6 resulted in the shutoff of host DNA replication.

Delineation of an enhancerlike positive regulatory element in the interleukin-2 receptor alpha-chain gene.

We have delineated a positive regulatory element in the interleukin-2 receptor alpha-chain gene (IL-2R alpha) between positions -299 and -243 that can potently activate a heterologous (herpesvirus thymidine kinase [tk]) promoter in phorbol myristate acetate (PMA)-induced Jurkat T cells and is functional when cloned in either orientation. This enhancerlike element contains a site (-268/-257) that can bind NF-kappa B; however, unlike the immunoglobulin kappa gene kappa B enhancer element, the IL-2R alpha kappa B-like site alone can only weakly activate a heterologous promoter. Adjacent 5' and 3' sequences also weakly activate the tk-CAT vector, but constructs combining the IL-2R alpha kappa B-like site plus adjacent 5' and 3' sequences potently activate gene expression.

Differential regulation by varicella-zoster virus (VZV) and herpes simplex virus type-1 trans-activating genes.

Transient expression assays were performed in Vero cells in order to compare varicella-zoster virus (VZV)-encoded trans-activating proteins [defined by the products of open reading frames (ORF) 4 and 62] with herpes simplex virus type-1 (HSV-1) trans-activating proteins, ICP4 and ICP0, with respect to activation of gene expression. We demonstrate that the product of VZV ORF4 and ORF62 (which are the HSV-1 analogs of ICP27 and ICP4, respectively) stimulate a variety of viral and cellular gene promoters, including the HSV-1 thymidine kinase (tk) promoter. On the other hand, expression of a recombinant vector containing the VZV tk promoter could not be stimulated, by HSV-1 infection or by the HSV-1 ICP4 or ICP0 proteins expressed during cotransfection experiments.

Mapping of two varicella-zoster virus-encoded genes that activate the expression of viral early and late genes.

A transient assay system was used to identify varicella-zoster virus (VZV)-encoded genes whose products are able to activate the expression of an early gene promoter, the thymidine kinase (tk) promoter, and a late gene promoter, and the glycoprotein I (gpI) promoter. Vero cells were cotransfected with individual cloned DNA fragments spanning the entire VZV genome and with the recombinant construct p1tkCAT which contained the chloramphenicol acetyl transferase (CAT) gene under the control of putative regulatory sequences. Five- to 20-fold increases in the expression p1tkCAT was observed in cotransfections with plasmids containing VZV open reading frame (ORF)4 (map location 0.

The chronic hepatic or renal toxicity of di(2-ethylhexyl) phthalate, acetaminophen, sodium barbital, and phenobarbital in male B6C3F1 mice: autoradiographic, immunohistochemical, and biochemical evidence for levels of DNA synthesis not associated with carcinogenesis or tumor promotion.

Male B6C3F1 mice, 6 weeks of age, were fed diets or water containing di(2-ethylhexyl) phthalate (DEHP) at 12,000 or 6000 ppm, acetaminophen (ACT) at 10,000 or 5000 ppm, sodium barbital (BBS) at 1000 ppm, or phenobarbital (PB) at 500 ppm for 40 weeks. Groups of six mice were terminated at 2, 8, 24, and 40 weeks for evaluation of liver and kidney weights, histopathology, and thymidine kinase (TK) activity in liver and kidney and levels of DNA synthesis, measured by tritiated thymidine [( 3H]T) autoradiography or bromodeoxyuridine (BrdU) immunohistochemistry. Liver weights, as percentage of body weight, were significantly elevated at most time intervals for mice exposed to all chemicals at each dose.