tetracycline and thiazolyl-blue

Bioglass(®)-based scaffolds for bone tissue engineering have been developed, which can also serve as carriers for drug delivery. For this, P(3HB) microspheres (PMSs) loaded with tetracycline were fabricated and immobilised on the scaffold surfaces by a modified slurry dipping technique. The sustained drug delivery ability in simulated body fluid was confirmed by using UV-Vis absorption spectroscopy measurements. The MTT assay using mouse fibroblast cells provided evidence that the tetracycline loaded microspheres produced in this study show limited cytotoxicity. The scaffolds developed in this work provide mechanical support, adequate 3D surface roughness, bioactivity and controlled drug delivery function, and are thus interesting candidates for bone tissue engineering applications.

Topics: Animals; Anti-Bacterial Agents; Bone and Bones; Bone Substitutes; Ceramics; Coated Materials, Biocompatible; Drug Carriers; Drug Delivery Systems; Fibroblasts; Glass; Materials Testing; Mice; Microspheres; Tetracycline; Tetrazolium Salts; Thiazoles; Tissue Engineering; Tissue Scaffolds

Gene therapy holds great potential for treating neurological disorders. However, delivering gene vectors to the brain has been either invasive or inefficacious in most studies to date. The aim of this study was to develop a safe and efficacious strategy for delivering gene vectors to the brain. A tetracycline-inducible replication-defective herpes simplex virus type-1 vector, QR9TO-LacZ, was administered to rats intranasally. QR9TO-LacZ could infect primary cortical neurons and express the reporter gene without detectable replication. QR9TO-LacZ was observed in the olfactory bulb, hippocampus, striatum, cortex, medulla, cerebellum, ventricles, and nasal septum after intranasal administration. Expression of the reporter gene could be controlled effectively by tetracycline. In vitro, introduction of QR9TO-LacZ did not change the structure of transfected neurons. In vivo, QR9TO-LacZ did not increase apoptosis in neurons and did not alter levels of interleukin 6 and tumor necrosis factor α in the brain after intranasal delivery. Our data suggest that intranasally applied QR9TO-LacZ has a wide distribution and expresses the reporter gene in the brain under the control of tetracycline with less cytotoxicity than intravenous or stereotactic delivery methods.

Topics: Analysis of Variance; Animals; beta-Galactosidase; Brain; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Viral; Genetic Vectors; Herpesvirus 1, Human; In Situ Nick-End Labeling; Male; Microscopy, Electron, Transmission; Neurons; Rats; Rats, Sprague-Dawley; Tetracycline; Tetrazolium Salts; Thiazoles; Time Factors; Transfection

Abstract Alterations in glutathione (GSH) metabolism are associated with neurodegeneration in Alzheimer's disease (AD), and GSH depletion follows application of exogenous fibrillar amyloid beta (Abeta) peptides in experimental systems; these results are commonly cited as evidence of oxidative damage in AD. We used MC65 human neuroblastoma cells that conditionally express carboxy-terminal fragments of the Abeta precursor protein (Abeta/CTFs) to directly test the hypothesis that GSH is part of the cellular response to stressors associated with Abeta/CTF accumulation and not simply a marker of oxidative damage. Our data showed that Abeta/CTFs accumulated by post-translational processes and were associated with progressive increases in oxidative damage and cytotoxicity. Ethycrinic acid (EA) or diethyl maleate (DEM), reagents that deplete GSH through non-specific thiol adduction, gave rise to dose-dependent cytotoxicity that was independent of Abeta/CTF expression and minimally responsive to alpha-tocopherol (AT). In contrast, buthionine sulfoximine (BSO), a selective inhibitor of GSH synthase, not only augmented Abeta/CTF-associated cell death but unexpectedly potentiated Abeta/CTF accumulation; both outcomes were completely suppressed by AT. These data suggest that antioxidants may serve as 'Abeta targeting' therapies that suppress toxic protein aggregation rather than simply acting as downstream radical scavengers.

Topics: alpha-Tocopherol; Amyloid beta-Protein Precursor; Analysis of Variance; Antioxidants; Blotting, Western; Buthionine Sulfoximine; Cell Line, Tumor; Cell Survival; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Enzyme Inhibitors; Extracellular Space; F2-Isoprostanes; Gene Expression Regulation, Neoplastic; Glutathione; Humans; Neuroblastoma; Peptide Fragments; Protein Synthesis Inhibitors; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tetracycline; Tetrazolium Salts; Thiazoles; Time Factors; Tocopherols

To examine the roles for NF-kappaB family proteins in hematopoiesis, we first expressed dominant negative Rel/NF-kappaB(IkappaBSR) in a factor-dependent cell line, Ba/F3. Although IkappaBSR neither affected thrombopoietin-dependent nor gp130-mediated growth, it suppressed interleukin-3- and erythropoietin-dependent growth at low concentrations. In addition, IkappaBSR enhanced factor-deprived apoptosis through the accumulation of reactive oxygen species (ROS). When expressed in normal hematopoietic stem/progenitor cells, IkappaBSR induced apoptosis even in the presence of appropriate cytokines by accumulating ROS. We also expressed IkappaBSR in an inducible fashion at various stages of hematopoiesis using the OP9 system, in which hematopoietic cells are induced to develop from embryonic stem cells. When IkappaBSR was expressed at the stage of Flk-1(+) cells (putative hemangioblasts), IkappaBSR inhibited the development of primitive hematopoietic progenitor cells by inducing apoptosis through the ROS accumulation. Furthermore, when IkappaBSR was expressed after the development of hematopoietic progenitor cells, it inhibited their terminal differentiation toward erythrocytes, megakaryocytes, and granulocytes by inducing apoptosis through the ROS accumulation. These results indicate that NF-kappaB is required for preventing apoptosis at multiple steps of hematopoiesis by eliminating ROS.

Topics: Animals; Apoptosis; Blotting, Northern; Cell Line; Cell Separation; Cell Survival; Cells, Cultured; Coloring Agents; Culture Media, Conditioned; Cytokines; Erythropoietin; Flow Cytometry; Glutathione; Granulocytes; Hematopoiesis; Hematopoietic Stem Cells; I-kappa B Proteins; Immunoblotting; Interleukin-3; Mice; Mice, Inbred BALB C; NF-kappa B; Plasmids; Reactive Oxygen Species; Retroviridae; Reverse Transcriptase Polymerase Chain Reaction; Tetracycline; Tetrazolium Salts; Thiazoles; Time Factors

kpm is a human serine/threonine kinase that is homologous to Drosophila tumor suppressor warts/lats and its mammalian homologue LATS1. In order to define the biological function of kpm, we generated stable transfectants of wild-type kpm (kpm-wt), a kinase-dead mutant of kpm (kpm-kd), and luciferase in HeLa Tet-Off cells under the tetracycline-responsive promoter. Western blot analysis showed that high levels of expression of kpm-wt as well as kpm-kd with an apparent mass of 150 kDa were induced after the removal of doxycycline. Induction of kpm-wt expression resulted in a marked decline in viable cell number measured by both trypan blue dye exclusion and MTT assay, whereas that of kpm-kd or luciferase had no effect. We then analyzed the cell cycle progression and apoptosis upon induction of kpm expression. 2-3 days after removal of doxycycline, cells underwent G(2)/M arrest, demonstrated by flow cytometric analysis of propidium iodide incorporation and MPM-2 reactivity. In vitro kinase assay showed that induction of kpm-wt led to down-regulation of kinase activity of the Cdc2-cyclin B complex, which was accompanied by an increase in the hyperphosphorylated form of Cdc2 and a change of phosphorylation status of Cdc25C. Furthermore, both DAPI staining and TUNEL assay showed that the proportion of apoptotic cells increased as kpm expression was induced. Taken together, these results indicate that kpm negatively regulates cell growth by inducing G(2)/M arrest and apoptotic cell death through its kinase activity.

Topics: Anti-Bacterial Agents; Apoptosis; Blotting, Western; cdc25 Phosphatases; Cell Cycle; Cell Cycle Proteins; Cell Division; Cell Survival; Coloring Agents; Cyclin B; Dose-Response Relationship, Drug; Down-Regulation; Doxycycline; Drosophila Proteins; Flow Cytometry; G2 Phase; HeLa Cells; Humans; In Situ Nick-End Labeling; Mitosis; Phosphorylation; Promoter Regions, Genetic; Propidium; Protein Kinases; Protein Serine-Threonine Kinases; Tetracycline; Tetrazolium Salts; Thiazoles; Time Factors; Transfection; Trypan Blue; Tumor Suppressor Proteins

The ability to accurately predict the phototoxic potential of personal and skin care products remains a key element in assessing the safety of premarketed products. To find a reliable in vitro alternative test for photoirritancy, the European Commission and the European Cosmetic Association are conducting a 3-year, European validation study. Based on the results of this study, an in vitro photoirritancy method will be selected for incorporation into new international guidelines for photoirritancy testing. As a part of this study, Skin2, a cultured human skin system, was used to evaluate the phototoxic potential of chemicals with known photoirritative properties. The Skin2 ZK1351, a 3-dimensional co-culture system, consists of dermal fibroblasts and a multilayered epidermis comprising differentiated keratinocytes. This product line has previously been used to evaluate the irritative potential of topically applied ingredients and products. In this study, various concentrations of the test chemicals were applied to the epidermal side of the Skin2 tissue for contact times of 1 h or 24 h and then the tissue was exposed to 2.9 J/cm2 of ultraviolet A (UVA) radiation. Treated but nonirradiated tissues were also assayed to predict the cytotoxic potential of the test chemicals, which could mask the phototoxic reaction. After exposure, the tissue substrates were rinsed free of test chemicals and allowed to recover for 24 h. Following this incubation, the MTT reduction assay was used to assess cytotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Benzophenones; Cell Survival; Coloring Agents; Culture Techniques; Dermatitis, Phototoxic; Epidermal Cells; Fibroblasts; Humans; Irritants; Keratinocytes; Models, Biological; Promethazine; Skin; Tetracycline; Tetrazolium Salts; Thiazoles; Ultraviolet Rays