ABSTRACT
The aim of this paper is to provide information about the neurophysiologic model of tinnitus and Tinnitus Retraining Therapy (TRT). With this overview of the model and therapy, professionals may discern with this basic foundation of knowledge whether they wish to pursue learning and subsequently implement TRT in their practice. This paper provides an overview only and is insufficient for the implementation of TRT.
Subject(s)
Hyperacusis/therapy , Tinnitus/therapy , Hair Cells, Auditory/physiopathology , Humans , Hyperacusis/diagnosis , Loudness Perception/physiology , Tinnitus/diagnosis , Tinnitus/physiopathologyABSTRACT
The effects of an extract from Ginkgo biloba, EGb 761, on tinnitus were tested using an animal model of tinnitus. Daily oral administration of EGb 761 in doses from 10 to 100 mg/ kg/day began 2 weeks before behavioral procedures and continued until the end of the experiment. Tinnitus was induced by daily administration of 321 mg/kg sodium salicylate s.c. (corresponding to 275 mg/kg/day of salicylate acid) in fourteen groups of pigmented rats, 6 animals/group. The results from salicylate- and EGb-761-treated animals were compared to control groups receiving either salicylate, saline, or EGb 761 only in doses of 100 mg/kg. Administration of EGb 761 resulted in a statistically significant decrease of the behavioral manifestation of tinnitus for doses of 25, 50 and 100 mg/kg/ day.
Subject(s)
Free Radical Scavengers/therapeutic use , Plant Extracts/therapeutic use , Salicylates/adverse effects , Tinnitus/chemically induced , Tinnitus/drug therapy , Administration, Oral , Animals , Dose-Response Relationship, Drug , Ginkgo biloba , Male , RatsABSTRACT
A method of identifying thymidylate synthase (TS) at the cellular level was developed using anti-TS monoclonal antibody (M-TS-4), a monoclonal antibody created against purified TS from a HeLa cell line. In HeLa cells and four human glioma cell lines (U-251, U-87, 343-MGA, and SF-188), TS was identified primarily in the cytoplasm. Autoradiographic and flow cytometric studies showed that TS appeared mainly in the G1 phase and subsided early in the S phase; thus, the G1 phase can be divided into TS-positive and -negative fractions. Nuclear TS was not demonstrated unequivocally with M-TS-4, and the relationship between nuclear TS and DNA synthesis could not be determined. Although the percentage of TS-positive cells was larger than the S-phase fraction measured by autoradiography after a pulse of tritiated thymidine or by the immunoperoxidase method using BUdR, the ratios were within a similar range (1.2-1.4) in all cell lines studied. Therefore, the S-phase fraction can be estimated indirectly from the percentage of TS-positive cells measured by M-TS-4. Because the emergence of TS detected by our method is cell cycle dependent, M-TS-4 may be useful for biochemical studies of TS and for cytokinetic analysis.
Subject(s)
Antibodies, Monoclonal , Cell Cycle/physiology , Thymidylate Synthase/analysis , Autoradiography , Bromodeoxyuridine , Cell Line , DNA/biosynthesis , Flow Cytometry , Glioma , HeLa Cells , Humans , Immunoenzyme Techniques , Staining and Labeling , Thymidylate Synthase/immunologyABSTRACT
A rapid procedure is described for assaying chloramphenicol acetyltransferase (CAT, EC 2.3.1.28) enzyme activity in intact animals following transfection of the RSV CAT plasmid into mouse bone marrow cells by electroporation. The reconstituted mice were injected with [14C]chloramphenicol and ethyl acetate extracts of 24-h urine samples were analyzed by TLC autoradiography for the excretion of 14C-labeled metabolites. CAT expression in vivo can be detected by the presence of acetylated 14C-labeled metabolites in the urine within 1 week after bone marrow transplantation and, under the conditions described, these metabolites can be detected for at least 3 months. CAT expression in intact mice as monitored by the urine assay correlates with the CAT expression in the hematopoietic tissues assayed in vitro. This method offers a quick mode of screening for introduced CAT gene expression in vivo without sacrificing the mice.
Subject(s)
Acetyltransferases/genetics , Genes , Transfection , Acetyltransferases/analysis , Animals , Avian Sarcoma Viruses/genetics , Bone Marrow/enzymology , Chloramphenicol/urine , Chloramphenicol O-Acetyltransferase , Mice , Mice, Transgenic , Transcription, GeneticABSTRACT
Electroporation was used to introduce high-molecular-weight DNA into murine hematopoietic cells and NIH3T3 cells. CCRF-CEM cells were stably transfected with SV2NEO plasmid and the genomic DNA from G-418-resistant clones (greater than 65 kb) was introduced into mouse bone marrow and NIH3T3 cells by electroporation. NEO sequences and expression were detected in the hematopoietic tissues of lethally irradiated mice, with 24% of individual spleen colonies expressing NEO. The frequency of genomic DNA transfer into NIH3T3 cells was 0.25 X 10(-3). Electroporation thus offers a powerful mode of gene transfer not only of cloned genes but also of high-molecular-weight DNA into cells.
Subject(s)
Bone Marrow Cells , DNA/genetics , Genetic Engineering/methods , Animals , Cell Line , Drug Resistance/genetics , Electricity , Gene Expression Regulation , Hematopoietic Stem Cells/metabolism , Kanamycin Kinase , Mice , Molecular Weight , Phosphotransferases/genetics , Plasmids , TransfectionABSTRACT
The introduction of 2'-deoxyuridine 5'-monophosphate and its analog, 5-fluoro-2'-deoxyuridine 5'-monophosphate, into intact CCRF-CEM and NIH3T3 cells was achieved by electroporation. Following electroporation, cells were shown to be fully functional as monitored by the incorporation of deoxyuridylate, after conversion to thymidylate, into DNA. Pretreatment of cells with fluorodeoxyuridine completely abolished this effect. In contrast, introduction of the fluoro analog into cells by electroporation markedly inhibited both DNA synthesis and cell growth in a time-dependent manner. Thus, electroporation offers a powerful tool to permeabilize cells to a variety of cellular metabolites and antimetabolites.
Subject(s)
Deoxyuracil Nucleotides/pharmacology , Fluorodeoxyuridylate/pharmacology , Cell Line , Cell Membrane Permeability , Cell Survival/drug effects , DNA/metabolism , Deoxyuridine/metabolism , Fluorodeoxyuridylate/metabolism , Thymidylate Synthase/antagonists & inhibitorsABSTRACT
Mouse bone marrow cells were subjected to electroporation in the presence of RSVCAT and SV2NEO plasmids. CAT activity was detected in the G-418 resistant granulocyte-macrophage colonies. RSVCAT electroporated into primary bone marrow cells, repopulated lethally irradiated mice as demonstrated by the persistence of CAT activity in the hematopoietic tissues showing that electroporation can offer a powerful mode of gene transfer into bone marrow cells.
Subject(s)
Acetyltransferases/genetics , Bone Marrow Cells , Hematopoietic Stem Cells/enzymology , Transfection , Acetyltransferases/metabolism , Animals , Chloramphenicol O-Acetyltransferase , Colony-Forming Units Assay , Electricity , Female , Granulocytes/enzymology , Macrophages/enzymology , Mice , Mice, Inbred CBA , PlasmidsABSTRACT
A simple method, employing high-voltage electric discharge (electroporation), was developed to introduce phosphorylated nucleosides into the cytoplasm of viable cells. HL-60 leukemia cells permeabilized by this technique remained viable and incorporated deoxyribonucleoside triphosphates into nuclear DNA. Furthermore, DNA synthesis was depressed for at least 24 h in HL-60 cells made permeable to 1-beta-D-arabinosylcytosine 5'-triphosphate by this methodology. Electroporation was found to be applicable to the permeabilization of a wide variety of cell lines in culture to nucleotides, suggesting that this methodology may be useful for the introduction into intact cells of a wide variety of molecules that are not normally transported effectively.
Subject(s)
Deoxyribonucleotides/administration & dosage , Cell Line , Cell Membrane Permeability , DNA/biosynthesis , Electricity , HumansABSTRACT
A reliable method for the introduction of foreign DNA into epidermal cells is described. Electroporation of murine BALB/c MK-1 epidermal cells with pSV2-CAT resulted in the transient expression of chloramphenicol-acetyltransferase (0.03 to 0.05 nmoles acetylchloramphenicol per mg protein per min) in the transfected cells. Transfection of MK-1 cells with pSV2-neo led to the appearance of approximately eight G418 resistant clones per 10(-6) cells per microgram of plasmid DNA. Distinct patterns of integration of SV2-neo were detected in three different resistant clones.
Subject(s)
Epidermis/physiology , Genetic Engineering/methods , Transfection , Acetyltransferases/genetics , Animals , Cell Line , Cell Membrane Permeability , Chloramphenicol O-Acetyltransferase , Cloning, Molecular , Electricity , Epidermal Cells , Mice , Time FactorsABSTRACT
Altered mouse dihydrofolate reductase gene (DHFRR) was expressed in murine cells using Abelson murine leukemia provirus genome as a prototype vector. A cDNA clone of DHFRR was inserted into a plasmid structure containing retroviral transcriptional as well as packaging signals. The recombinant plasmid was transfected into psi-2 ecotropic cells and the transient virus was used to infect amphotropic PA-12 cells. Recombinant virus (ABL-DHFRR) was detected in the culture medium of transfected PA-12 cells and was free of helper virus. The ABL-DHFRR was capable of conferring methotrexate (MTX) resistance to a variety of cells in culture. The titer of ABL-DHFRR virus was at least tenfold higher than other DHFR retroviruses. The ABL-DHFRR virus titer was increased by selection at increasing concentrations of MTX. The presence of the DHFRR in the virus-infected cells was confirmed by assays which showed reduced inhibition of enzyme activity by MTX. A helper-virus-free, amphotropic, high-titer retrovirus containing the altered DHFR was obtained which may be of use as a dominant selectable marker in infecting hematopoietic progenitor cells.
Subject(s)
Abelson murine leukemia virus/genetics , Genetic Vectors , Leukemia Virus, Murine/genetics , Tetrahydrofolate Dehydrogenase/genetics , Animals , Cloning, Molecular , Drug Resistance , Helper Viruses/genetics , Methotrexate/pharmacology , Mice , PlasmidsABSTRACT
Activity of thymidylate synthase was measured in situ in leukemia cells by tritium release from [5-3H]dUrd. Aphidicolin, an inhibitor of DNA polymerase alpha, but not thymidylate synthase, caused a time dependent inhibition of the enzyme when added to the cells after [5-3H]dUrd. Cells treated with hydroxyurea and aphidicolin in sequence before addition of [5-3H]dUrd had a high initial thymidylate synthase activity that decreased with time. This pattern indicates that thymidylate synthase activity is linked to DNA synthesis; however, its inhibition by drugs that inhibit DNA synthesis may be due to accumulation of thymidine nucleotide(s), rather than to an allosteric interaction in the replitase complex.
Subject(s)
DNA Replication/drug effects , Methyltransferases/antagonists & inhibitors , Multienzyme Complexes/metabolism , Thymidylate Synthase/antagonists & inhibitors , Animals , Aphidicolin , Cell Line , Diterpenes/pharmacology , Humans , Leukemia L1210/enzymology , Mice , Thymidine/metabolismABSTRACT
Monoclonal antibodies against electrophoretically pure thymidylate synthase from HeLa cells have been produced. Antibodies (M-TS-4 and M-TS-9) from hybridoma clones were shown by enzyme-linked immunoassay to recognize thymidylate synthase from a variety of human cell lines, but they did not bind to thymidylate synthase from mouse cell lines. The strongest binding of antibodies was observed to enzyme from HeLa cells. These two monoclonal antibodies bind simultaneously to different antigenic sites on thymidylate synthase purified from HeLa cells, as reflected by a high additivity index and results of cross-linked radioimmunoassay. Both monoclonal antibodies inhibit the activity of thymidylate synthase from human cell lines. The strongest inhibition was observed with thymidylate synthase from HeLa cells. Monoclonal antibody M-TS-9 (IgM subclass) decreased the rate of binding of [3H]FdUMP to thymidylate synthase in the presence of 5,10-methylenetetrahydrofolate while M-TS-4 (IgG1) did not change the rate of ternary complex formation. These data indicate that the antibodies recognize different epitopes on the enzyme molecule.
Subject(s)
Antibodies, Monoclonal , Methyltransferases/metabolism , Thymidylate Synthase/metabolism , Binding Sites, Antibody , Cell Line , Enzyme-Linked Immunosorbent Assay , HeLa Cells/enzymology , Humans , Hybridomas/immunology , Kinetics , Structure-Activity Relationship , Thymidylate Synthase/analysis , Thymidylate Synthase/immunologyABSTRACT
Magnesium ions variably influenced activity of highly purified thymidylate synthetase preparations from different mouse tumors, activating the enzyme from Ehrlich ascites carcinoma (EAC) cells and inhibiting the enzyme from L1210 and L5178Y cells and from 5-fluorodeoxyuridine (FdUrd)-resistant EAC cells. In the presence of Mg2+ in a concentration resulting in either maximum activation or inhibition (25-30 mM) the enzymes from both the sensitive and FdUrd-resistant EAC lines and L5178Y cells were activated by ATP. Under the same conditions of Mg2+ concentration ADP and AMP inhibited the enzyme from the parental but not from the FdUrd-resistant EAC cells.
Subject(s)
Adenine Nucleotides/pharmacology , Carcinoma, Ehrlich Tumor/enzymology , Leukemia, Experimental/enzymology , Magnesium/pharmacology , Methyltransferases/metabolism , Thymidylate Synthase/metabolism , Animals , Cell Line , Drug Resistance , Enzyme Activation/drug effects , Floxuridine/pharmacology , Magnesium Chloride , Mice , Thymidylate Synthase/antagonists & inhibitorsABSTRACT
Thymidylate synthetase from 5-fluorodeoxyuridine-resistant Ehrlich ascites carcinoma cells was purified to a state close to electrophoretical homogeneity (sp. act. = 1.3 mumoles/min/mg protein) and studied in parallel with the homogeneous preparation of the enzyme from the parental Ehrlich ascites carcinoma cells. The enzyme from the resistant cells compared to that from the parental cells showed: (i) a higher turnover number (at least 91 against 31 min-1), (ii) a higher inhibition constant (19 against 1.9 nM) for FdUMP (a tight-binding inhibitor of both enzymes), (iii) a lower activation energy at temps above 36 degrees (1.37 against 2.59 kcal/mole), and (iv) a lower inhibition constant (26 against 108 microM) for dTMP, inhibiting both enzymes competitively vs dUMP.
Subject(s)
Carcinoma, Ehrlich Tumor/enzymology , Floxuridine/pharmacology , Methyltransferases/isolation & purification , Thymidylate Synthase/isolation & purification , Animals , Cell Line , Drug Resistance , Electrophoresis, Polyacrylamide Gel , Humans , Kinetics , Methotrexate/pharmacology , Molecular Weight , Temperature , Thymidine Kinase/analysis , Thymidylate Synthase/antagonists & inhibitors , Thymidylate Synthase/metabolismABSTRACT
A subline of Ehrlich ascites carcinoma (EAC) cells resistant to 5-fluoro-2'-deoxy-uridine (FdUrd) was developed by continuous exposure to progressively increasing concentrations of the drug (35-75 mg/kg per day) during 15 passages through mice. Since then, the EAC cells have been retransplanted more than 80 times through drug-untreated mice and continue to be resistant. After adaptation to growth in suspension culture the drug-adapted cells were 1000 times more resistant to FdUrd in comparison with parental ones, and remained near-tetraploid with doubling time longer than in parental line. The activity of thymidine kinase was deeply depressed (100-fold) whereas that of thymidylate synthetase several-fold increased in the resistant EAC cells, both grown in vivo and in vitro.