tetracycline and Hypoxia

Topics: Adrenocorticotropic Hormone; Adult; Age Factors; Animals; Birth Weight; Body Height; Body Weight; Brain; Child Development; Child, Preschool; Cytomegalovirus Infections; DNA; DNA Replication; Embryonic and Fetal Development; Female; Gestational Age; Growth; Growth Disorders; Growth Hormone; Humans; Hypoxia; Infant; Infant, Newborn; Maternal-Fetal Exchange; Nutrition Disorders; Pregnancy; Rats; RNA; Rubella; Tetracycline

Bacterial pathogens are confronted with a range of challenges at the site of infection, including exposure to antibiotic treatment and harsh physiological conditions, that can alter the fitness benefits and costs of acquiring antibiotic resistance. Here, we develop an experimental system to recapitulate resistance gene acquisition by Staphylococcus aureus and test how the subsequent evolution of the resistant bacterium is modulated by antibiotic treatment and oxygen levels, both of which are known to vary extensively at sites of infection. We show that acquiring tetracycline resistance was costly, reducing competitive growth against the isogenic strain without the resistance gene in the absence of the antibiotic, for S. aureus under hypoxic but not normoxic conditions. Treatment with tetracycline or doxycycline drove the emergence of enhanced resistance through mutations in an RluD-like protein-encoding gene and duplications of

Topics: Anti-Bacterial Agents; Bacterial Proteins; Humans; Hypoxia; Methicillin-Resistant Staphylococcus aureus; Staphylococcal Infections; Staphylococcus aureus; Tetracycline

Minocycline, a tetracycline-derived compound, mitigates damage caused by ischemia/reperfusion (I/R) injury. Here, 19 tetracycline-derived compounds were screened in comparison to minocycline for their ability to protect hepatocytes against damage from chemical hypoxia and I/R injury. Cultured rat hepatocytes were incubated with 50μM of each tetracycline-derived compound 20 min prior to exposure to 500μM iodoacetic acid plus 1mM KCN (chemical hypoxia). In other experiments, hepatocytes were incubated in anoxic Krebs-Ringer-HEPES buffer at pH6.2 for 4h prior to reoxygenation at pH7.4 (simulated I/R). Tetracycline-derived compounds were added 20 min prior to reperfusion. Ca(2+) uptake was measured in isolated rat liver mitochondria incubated with Fluo-5N. Cell killing after 120 min of chemical hypoxia measured by propidium iodide (PI) fluorometry was 87%, which decreased to 28% and 42% with minocycline and doxycycline, respectively. After I/R, cell killing at 120 min decreased from 79% with vehicle to 43% and 49% with minocycline and doxycycline. No other tested compound decreased killing. Minocycline and doxycycline also inhibited mitochondrial Ca(2+) uptake and suppressed the Ca(2+)-induced mitochondrial permeability transition (MPT), the penultimate cause of cell death in reperfusion injury. Ru360, a specific inhibitor of the mitochondrial calcium uniporter (MCU), also decreased cell killing after hypoxia and I/R and blocked mitochondrial Ca(2+) uptake and the MPT. Other proposed mechanisms, including mitochondrial depolarization and matrix metalloprotease inhibition, could not account for cytoprotection. Taken together, these results indicate that minocycline and doxycycline are cytoprotective by way of inhibition of MCU.

Topics: Animals; Anti-Bacterial Agents; Calcium; Calcium Channels; Doxycycline; Hepatocytes; Hypoxia; Iron; Male; Minocycline; Mitochondria, Liver; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tetracycline

Inducible gene expression systems are particularly useful for the functional characterization of genes with putative toxic properties. In the course of studying the role of hypoxia-regulated gene expression on cell survival using the tetracycline-inducible (tet-on) system, the author noted that exposure to the inducing ligand doxycycline (dox) inhibited caspase-3 cleavage in control samples. To limit this confounding off-target effect, he devised an in vitro pulse dose, delayed-injury protocol testing both dox and a novel tetracycline analog 9-t-butyl doxycycline (9-TB). Although 9-TB induced higher transgene levels compared to matched concentrations of dox, continuous exposure to both drugs inhibited caspase-3 cleavage in hypoxic samples. Conversely, a 6-h pulse dose of 9-TB followed by a 40-h washout period prior to hypoxic challenge activated robust transgene expression and lessened the inhibitory effects on caspase-3 processing. It is anticipated that these protocol modifications will improve the performance of tet-regulated genetic screens, particularly in situations where cell death is used as a primary end point.

Topics: Animals; Anti-Bacterial Agents; Apoptosis; Caspase 3; Cell Line; Doxycycline; Gene Expression Regulation; Genetic Techniques; Genetic Vectors; Hypoxia; Mice; Promoter Regions, Genetic; Tetracycline

Expression of vascular endothelial growth factor (VEGF) is tightly regulated, particularly at the level of its mRNA stability, which is essentially mediated through the 3'-untranslated region (3'-UTR) of VEGF mRNA. To identify new protein partners regulating VEGF mRNA stability, we screened a cDNA expression library with an RNA probe corresponding to the entire VEGF mRNA 3'-UTR. We identified the "poly(A)-binding protein-interacting protein 2" (PAIP2) as a new VEGF mRNA 3'-UTR interacting protein. By RNA electromobility shift assays, we showed that PAIP2 binds to two distinct regions of a domain encompassing base 1 to 1280 of the VEGF 3'-UTR. Such in vitro interaction was confirmed using cell extracts in which PAIP2 expression is induced by tetracycline (Tet-on cells). Moreover, we demonstrated by RNA affinity purification as well as by ribonucleoprotein complexes immunoprecipitation, that PAIP2 interacts with VEGF mRNA in vivo. Using an in vitro RNA degradation assay, the half-life of VEGF 3'-UTR was found to be increased by overexpressing PAIP2. PAIP2 stabilizes endogenous VEGF mRNA in Tet-on cells, leading to increased VEGF secretion. Moreover, RNAi-mediated knock-down of PAIP2 significantly reduces the steady-state levels of endogenous VEGF mRNA. We also showed, by in vitro protein-protein interactions and co-immunoprecipitation experiments, that PAIP2 interacts with HuR, an already known VEGF mRNA-binding protein, suggesting cooperation of both proteins for VEGF mRNA stabilization. Hence, PAIP2 appears to be a crucial regulator of VEGF mRNA and as a consequence, any variation in its expression could modulate angiogenesis.

Topics: 3' Untranslated Regions; Animals; Binding, Competitive; Blotting, Western; Carrier Proteins; Cell Line, Tumor; Cloning, Molecular; DNA, Complementary; Enzyme-Linked Immunosorbent Assay; Gene Library; Glutathione Transferase; HeLa Cells; Humans; Hypoxia; Mice; Microscopy, Fluorescence; Models, Genetic; Plasmids; Precipitin Tests; Protein Binding; Protein Biosynthesis; Protein Structure, Tertiary; Recombinant Fusion Proteins; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA Interference; RNA-Binding Proteins; RNA, Messenger; Tetracycline; Time Factors; Tissue Distribution; Vascular Endothelial Growth Factor A

Fatty tetracycline-induced steatosis is shown to reduce levels of mitochondrial and microsomal cytochromes. A decrease of microsomal cytochromes in the liver of hypoxia-resistant animals was similar to that of hypoxia-sensitive ones. This decrease was different for hypoxia-resistant and hypoxia-sensitive animals: for the former only cytochrome a declined, in the latter case cytochromes c+, c1 and a.

Topics: Acute Disease; Animals; Cytochromes; Fatty Liver; Hypoxia; Male; Microsomes, Liver; Mitochondria, Liver; Rats; Tetracycline

Topics: Adult; Disseminated Intravascular Coagulation; Female; Humans; Hypoxia; Psittacosis; Tetracycline

Tetracycline was used as a fluorescent test-antibiotic for Ca2+ ions in rat liver mitochondria. Incubation of the isolated mitochondria under anaerobic conditions at 20 degrees C resulted in a rapid (in 30-min) loss by the mitochondria of the property to accumulate Ca2+. Disturbances of the mitochondrial Ca2+-accumulating property during the survival of the liver developed much more slowly (it took over 2 hours) and were not monotonous; the maximal values were recorded during the 5th-10th and the 60th minutes of survival.

Topics: Animals; Calcium; Fluorescence; Hypoxia; In Vitro Techniques; Mitochondria, Liver; Rats; Tetracycline

Topics: Carbon Dioxide; Humans; Hydrogen-Ion Concentration; Hypoxia; Lung Diseases; Lung Diseases, Obstructive; Oxygen; Tetracycline

Topics: Adrenal Cortex Hormones; Bronchial Spasm; Bronchodilator Agents; Central Nervous System Stimulants; Coronary Disease; Digitalis Glycosides; Guaiacol; Haemophilus Infections; Humans; Hypercapnia; Hypoxia; Injections, Intramuscular; Injections, Intravenous; Metaproterenol; Oxygen Inhalation Therapy; Phenethylamines; Physical Therapy Modalities; Resorcinols; Respiratory Insufficiency; Tetracycline; Time Factors

Topics: Ampicillin; Angina Pectoris; Anti-Bacterial Agents; Bacteriological Techniques; Diagnosis, Differential; Drug Therapy; Emergencies; Genital Diseases, Female; Humans; Hydrocortisone; Hypoxia; Metaraminol; Sepsis; Shock; Shock, Septic; Surgical Wound Infection; Tetracycline