tetracycline and 4-epianhydrotetracycline

The combined pollution of antibiotics and heavy metals has attracted a worldwide attention in the recent years. 4-epianhydrotetracycline (EATC) is the major degradation product of tetracycline (TC), which has been detected frequently in environment and its concentration is even higher than TC under some circumstances. Cadmium (Cd) is a common heavy metal contaminant and has highly toxic to organisms, plants and humans even at low doses. In the present study, zebrafish (Danio rerio) embryo toxicity test was performed to investigate the single and combined effects of EATC and Cd on aquatic organisms. Exposure to EATC and Cd at environmentally relevant concentrations had a series of hazardous impacts on the embryonic development, including lethality, hatching rate, heart rate and teratogenic effects. Compared to the contaminant existed alone, combined pollution produced stronger toxicity, which appeared as the decreasing of heart rate and hatching rate, and the increasing of malformation of zebrafish embryos. After 96 h exposure, the reactive oxygen species (ROS) levels in zebrafish embryos were increased significantly, revealing that EATC-Cd co-exposure resulted in potential oxidative stress-induced damage. Acridine orange (AO) staining showed that combined exposure resulted in stronger cell apoptosis. The potential health risks of the combined pollution of EATC and Cd should be paid more attention to higher level vertebrates and humans.

Topics: Animals; Anti-Bacterial Agents; Cadmium; Embryo, Nonmammalian; Metals, Heavy; Oxidative Stress; Tetracycline; Tetracyclines; Water Pollutants, Chemical; Zebrafish

Tetracyclines (TCs) are an important class of antibiotics threatened by an emerging new resistance mechanism─enzymatic inactivation. These TC-inactivating enzymes, also known as tetracycline destructases (TDases), inactivate all known TC antibiotics, including drugs of last resort. Combination therapies consisting of a TDase inhibitor and a TC antibiotic represent an attractive strategy for overcoming this type of antibiotic resistance. Here, we report the structure-based design, synthesis, and evaluation of bifunctional TDase inhibitors derived from anhydrotetracycline (aTC). By appending a nicotinamide isostere to the C9 position of the aTC D-ring, we generated bisubstrate TDase inhibitors. The bisubstrate inhibitors have extended interactions with TDases by spanning both the TC and presumed NADPH binding pockets. This simultaneously blocks TC binding and the reduction of FAD by NADPH while "locking" TDases in an unproductive FAD "out" conformation.

Topics: Anti-Bacterial Agents; Heterocyclic Compounds; NADP; Oxidation-Reduction; Protein Synthesis Inhibitors; Tetracycline; Tetracyclines

Inactivation of tetracycline antibiotics by tetracycline destructases (TDases) remains a clinical and agricultural threat. TDases can be classified as type 1 Tet(X)-like TDases and type 2 soil-derived TDases. Type 1 TDases are widely identified in clinical pathogens. A combination therapy of tetracycline and a TDase inhibitor is much needed to rescue the clinical efficacy of tetracyclines. Anhydrotetracycline is a pan-TDase inhibitor that inhibits both type 1 and type 2 TDases. Here, we present structural, biochemical, and phenotypic evidence that anhydrotetracycline binds in a substrate-like orientation and competitively inhibits the type 1 TDase Tet(X6) to rescue tetracycline antibiotic activity as a sacrificial substrate. Anhydrotetracycline interacting residues of Tet(X6) are conserved within type 1 TDases, indicating a conserved binding mode and mechanism of inhibition. This mode of binding and inhibition is distinct from anhydrotetracycline's inhibition of type 2 TDases. This study forms the framework for development of next-generation therapies to counteract enzymatic tetracycline resistance.

Topics: Anti-Bacterial Agents; Tetracycline; Tetracyclines

Developing treatments for antibiotic resistant bacterial infections is among the highest priority public health challenges worldwide. Tetracyclines, one of the most important classes of antibiotics, have fallen prey to antibiotic resistance, necessitating the generation of new analogs. Many tetracycline analogs have been accessed through both total synthesis and semisynthesis, but key C-ring tetracycline analogs remain inaccessible. New methods are needed to unlock access to these analogs, and heterologous biosynthesis in a tractable host such as

Topics: Alcohol Oxidoreductases; Anti-Bacterial Agents; Fungal Proteins; Hydroxylation; Mixed Function Oxygenases; NADH, NADPH Oxidoreductases; Oxidation-Reduction; Saccharomyces cerevisiae; Tetracycline; Tetracyclines

Tetracycline (TC) as an emerging contaminant has raised serious concerns about its toxicity and removal in wastewater treatment processes. The more toxic transformation products of TC, 4-epitetracycline (ETC), anhydrotetracycline (ATC) and 4-epianhydrotetracycline (EATC) are also widely detected. This study investigated the antibacterial and bactericidal activity of TC, ETC, ATC, EATC against Shewanella sp, using Escherichia coli and Pseudomonas aeruginosa strains as quality controls. Further, batch assays were conducted to investigate the inhibition of these antibiotics on the phosphorus removal of the Shewanella strain, and removal mechanisms of TC and its transformation products (TCs). The inhibition on phosphorus removal by the Shewanella strain at 20 mg L

Topics: Adsorption; Anti-Bacterial Agents; Heterocyclic Compounds; Phosphorus; Shewanella; Tetracycline; Tetracyclines; Wastewater

The transcriptional inducer anhydrotetracycline (aTc) and the bacteriostatic antibiotic tetracycline (Tc) are commonly used in all fields of biology for control of transcription or translation. A drawback of these and other small molecule inducers is the difficulty of their removal from cell cultures, limiting their application for dynamic control. Here, we describe a simple method to overcome this limitation, and show that the natural photosensitivity of aTc/Tc can be exploited to turn them into highly predictable optogenetic transcriptional- and growth-regulators. This new optogenetic class uniquely features both dynamic and setpoint control which act via population-memory adjustable through opto-chemical modulation. We demonstrate this method by applying it for dynamic gene expression control and for enhancing the performance of an existing optogenetic system. We then expand the utility of the aTc system by constructing a new chemical bandpass filter that increases its aTc response range. The simplicity of our method enables scientists and biotechnologists to use their existing systems employing aTc/Tc for dynamic optogenetic experiments without genetic modification.

Topics: Cloning, Molecular; Dose-Response Relationship, Drug; Escherichia coli; Gene Expression; Genes, Reporter; Genetic Vectors; Luminescent Proteins; Optogenetics; Photolysis; Protein Biosynthesis; Protein Synthesis Inhibitors; Recombinant Proteins; Red Fluorescent Protein; Tetracycline; Tetracyclines; Transcription, Genetic; Ultraviolet Rays

Topics: Anti-Bacterial Agents; Antioxidants; Chlorella vulgaris; Fresh Water; Lipid Peroxidation; Oxidative Stress; Tetracycline; Tetracyclines

The synthesis and biological evaluation of semisynthetic anhydrotetracycline analogues as small molecule inhibitors of tetracycline-inactivating enzymes are reported. Inhibitor potency was found to vary as a function of enzyme (major) and substrate-inhibitor pair (minor), and anhydrotetracycline analogue stability to enzymatic and nonenzymatic degradation in solution contributes to their ability to rescue tetracycline activity in whole cell Escherichia coli expressing tetracycline destructase enzymes. Taken collectively, these results provide the framework for the rational design of next-generation inhibitor libraries en route to a viable and proactive adjuvant approach to combat the enzymatic degradation of tetracycline antibiotics.

Topics: Anti-Bacterial Agents; Enzyme Inhibitors; Escherichia coli; Escherichia coli Proteins; Tetracycline; Tetracyclines

Determination of the effect of physicochemical parameters on the removal of tetracycline (TC) and degradation products is important because of the importance of the removal of antibiotics in Wastewater Treatment Plant (WWTP). Therefore, the purpose of this study was to investigate the relationships between removals of TC and degradation products and physicochemical parameters in Municipal Wastewater Treatment Plant (MWWTP). For this aim, (i) the removals of physicochemical parameters in a MWWTP located in Elazığ city (Turkey) were determined (ii) the removals of TC and degradation products in MWWTP were determined (iii) the relationships between removals of TC and degradation products and physicochemical parameters were investigated. TC, 4-epitetracycline (ETC), 4-epianhydrotetracycline (EATC), anhydrotetracycline (ATC), and physicochemical parameters (pH, temperature, electrical conductivity (EC), suspended solids (SS), BOD5, COD, total organic carbon (TOC), NH4(+)-N, NO2(-)-N, NO3(-)-N and O-PO4(-3)) were determined. The calculation of the correlation coefficients of relationships between the physicochemical parameters and TC, EATC, ATC showed that, among the investigated parameters, EATC and SS most correlated. The removals of other physicochemical parameters were not correlated with TC, EATC and ATC.

Topics: Anti-Bacterial Agents; Chemical Phenomena; Environmental Monitoring; Hydrogen-Ion Concentration; Tetracycline; Tetracyclines; Turkey; Waste Disposal, Fluid; Wastewater

The aims of this study are to investigate the fate of tetracycline (TC) and degradation products (DPs) in municipal biological wastewater treatment plant (MBWWTP) located in Elazığ City (Turkey) and to determine the occurrence and transport of TC and DPs in surface water (SW) (Kehli Stream) which the effluents of the plant discharged. The aqueous phase removal of TC, 4-epitetracycline (ETC), 4-epianhydrotetracycline (EATC), and anhydrotetracycline (ATC) in the studied treatment plant was 39.4 ± 1.9, 31.8 ± 1.5, 15.1 ± 0.7, and 16.9 ± 0.8%, respectively. According to the analyses' results of SW samples taken from downstream at every 500-m distance, TC and DPs decreased by the increase in the distance. In downstream, at 2000 m, TC, ETC, EATC, and ATC were 4.12 ± 0.20, 6.70 ± 0.33, 8.31 ± 0.41, and 3.57 ± 0.17 μg/L, respectively. As a result, antibiotic pollution in the SW that takes the effluent of MBWWTP exists.

Topics: Anti-Bacterial Agents; Cities; Environmental Monitoring; Tetracycline; Tetracyclines; Turkey; Waste Disposal, Fluid; Wastewater; Water Pollutants, Chemical

We report the construction of a tetracycline inducible expression vector that allows regulated gene expression in the enteric pathogen Vibrio cholerae. The expression vector, named pXB300, contains the tetracycline regulatory elements from Tn10, a multiple cloning site downstream of the tetA promoter and operator sequences, a ColE1 origin of replication, a β-lactamase resistance gene for positive selection, and the hok/sok addiction system for selection in the absence of antibiotic. The function of the tetracycline expression system was demonstrated by cloning lacZ under control of the tetA promoter and quantifying β-galactosidase expression in Escherichia coli and V. cholerae. The utility for pXB300 was documented by complementation of V. cholerae virulence mutants during growth under virulence inducing conditions. The results showed that pXB300 allowed high-level expression of recombinant genes with linear induction in response to the exogenous concentration of the inducer anhydrotetracycline. We further show that pXB300 was reliably maintained in V. cholerae during growth in the absence of antibiotic selection.

Topics: Antiporters; Bacterial Proteins; Cholera Toxin; DNA-Binding Proteins; Gene Expression Regulation, Bacterial; Genetic Complementation Test; Genetic Vectors; Mutation; Plasmids; Promoter Regions, Genetic; Tetracycline; Tetracyclines; Transcription Factors; Vibrio cholerae; Virulence Factors

Tetracycline (TC) derivatives are extensively used as antibiotics in human and animal medicine and, very recently, they have been screened as anti-amyloidogenic drugs. Anhydrotetracycline (AHTC) is one of the major degradation products of TC that has been linked to several side effects of the drug. We evaluated the interaction of AHTC with bovine serum albumin (BSA), one of the main carriers of amphiphilic molecules in blood, using three complementary analytical methods: fluorescence spectroscopy, isothermal titration calorimetry and differential scanning calorimetry. AHTC bound to BSA with an association constant in the order of 10(5) M(-1). Drug binding was enthalpically and entropically driven and seemed to involve hydrophobic interactions. AHTC fluorescence enhancement and hypsochromic shifts observed upon binding suggested a low-polarity location excluded from water for the bound drug. Our data are useful for evaluating the biodisponibility of the pharmacophore and the dynamic distribution of the toxic derivative.

Topics: Animals; Calorimetry; Cattle; Circular Dichroism; Hydrophobic and Hydrophilic Interactions; Protein Binding; Serum Albumin, Bovine; Spectrometry, Fluorescence; Tetracycline; Tetracyclines; Thermodynamics

Inducible expression is a valuable approach for the elucidation of gene functions. Here, we present new configurations of the tetracycline-dependent gene regulation (tet) system for Staphylococcus aureus. To provide improved and expanded modes of control, strains and plasmids were constructed for the constitutive expression of tetR or a variant allele, rev-tetR(r2). The encoded regulators respond differently to the effector anhydrotetracycline (ATc), which causes target gene expression to be induced with TetR or repressed with rev-TetR. To quantify and compare regulation mediated by episomal or chromosomal (rev-)tetR constructs, expression from a chromosomal P(xyl/tet)-gfpmut2 fusion was measured. Chromosomally encoded TetR showed tight repression and allowed high levels of dose-dependent gene expression in response to ATc. Regulatory abilities were further verified using a strain in which a native S. aureus gene (zwf) was put under tet control in its native chromosomal location. Tight repression was reflected by transcript amounts, which were barely detectable under repressed conditions and high in ATc-treated cells. In reporter gene assays, this type of control, termed Tet-on, was more efficient than Tet-off regulation, in which addition of ATc causes downregulation of a target gene. The latter was achieved and quantified by direct rev-TetR control of P(xyl/tet)-gfpmut2. Additionally, TetR was used in trans to control the expression of antisense RNA for posttranscriptional gene silencing. Induction of antisense RNA expression of the fabI gene caused pronounced growth retardation lasting several hours. These results demonstrate the efficiency of the new tet systems and their flexible use for different purposes.

Topics: Alleles; Bacterial Proteins; Gene Expression; Gene Expression Regulation, Bacterial; Gene Silencing; Genes, Reporter; Genetic Vectors; Hemolysin Proteins; Histone Deacetylases; Plasmids; Promoter Regions, Genetic; Repressor Proteins; RNA, Antisense; Staphylococcus aureus; Tetracycline; Tetracyclines; Transcriptional Activation; Transduction, Genetic; Transfection

Molecular-dynamics simulations have been used to investigate the mechanism of induction of a mutant (revTetR) of the tetracycline repressor protein (TetR) that shows the reverse phenotype (i.e., it is induced in the absence of tetracyclines and not in their presence). Low-frequency, normal-mode analyses demonstrate that the reverse phenotype is reproduced by the simulations on the basis of criteria established for wild-type TetR. The reverse phenotype is caused by the fact that the DNA-binding heads in revTetR are closer than the ideal distance needed for DNA-binding when no inducer is present. This distance increases on binding an inducer. Whereas this distance increase makes the interhead distance too large in wild-type TetR, it increases to the ideal value in revTetR. Thus, the mechanism of induction is the same for the two proteins, but the consequences are reversed because of the smaller interhead distance in revTetR when no inducer is present.

Topics: Computer Simulation; DNA; Models, Biological; Models, Molecular; Molecular Conformation; Mutation; Protein Structure, Secondary; Repressor Proteins; Structure-Activity Relationship; Tetracycline; Tetracyclines; Time Factors

Tetracycline (TC) and 4-epitetracycline (4eTC) degradation, as well as anhydrotetracycline (ATC) and 4-epianhydrotetracycline (4eATC) formation, has been evaluated in thermally treated chicken breast, pig loin, and pig loin with added back-fat. Samples containing TC and 4eTC residues were submitted to microwave or boiling heating, extracted with a mixture of McIlvaine buffer/methanol (75:25), and analyzed by high-performance liquid chromatography-diode array detection on a phenyl-hexyl reverse phase chromatographic column. The formation of ATC and 4eATC, as well as of two unidentified compounds, was described for the first time in edible meat samples submitted to mild thermal treatments, similar to those applied at home to cook foods. Degradation of TC and 4eTC and formation of ATC and 4eATC versus time of treatment fitted satisfactorily a first-order kinetic. Even if the potential toxic effects of these breakdown compounds should be further investigated, their formation in cooked meat should be taken into account when maximum residue limits are established.

Topics: Animals; Chickens; Chromatography, High Pressure Liquid; Cooking; Hot Temperature; Meat; Swine; Tetracycline; Tetracyclines; Tetrodotoxin

The binding motif (pharmacophore) for induction and the changes in the structure of the binding site that accompany induction have been determined from molecular-dynamics simulations on the tetracycline-repressor signal-transduction protein. The changes and the induction mechanism are discussed and compared with conclusions drawn from earlier X-ray structures. The differences in inducer strength of tetracycline and 5a,6-anhydrotetracycline are discussed with respect to their interaction in the MD simulations.

Topics: Allosteric Regulation; Anti-Bacterial Agents; Binding Sites; Crystallography, X-Ray; Models, Molecular; Protein Binding; Protein Conformation; Protein Synthesis Inhibitors; Repressor Proteins; Tetracycline; Tetracyclines

Topics: Doxycycline; Gene Expression Regulation; HeLa Cells; Humans; Luciferases, Firefly; Luciferases, Renilla; Models, Molecular; Molecular Structure; Recombinant Fusion Proteins; Repressor Proteins; Tetracycline; Tetracyclines; Trans-Activators; Transcriptional Activation

Inducible expression systems are powerful tools for studying gene function. Though several inducible expression systems are now available for mycobacteria, none have been used to modulate bacterial gene expression during an animal infection. A tetracycline-inducible expression system from Streptomyces coelicolor was successfully adapted for use in mycobacteria. To prevent baseline expression without induction, S. coelicolor tetR gene was overexpressed using the acetamidase promoter and regulatory gene block. Target gene expression was controlled by the S. coelicolor tcp830 promoter and operator allele. The -10 promoter consensus sequence of the tcp830 promoter was modified to better resemble known strong mycobacterial promoters. Using this system, induction of tetR fully repressed tcp830-dependent expression of green fluorescent protein (GFP) to baseline levels. Addition of anhydrotetracycline led to a 62-fold induction of GFP expression in vitro and 15-fold induction in a mouse mycobacterial peritonitis model in the presence of maximal tetR expression. Chemically regulatable gene expression during animal infection may be a useful tool in studying mycobacterial pathogenesis.

Topics: Acetamides; Animals; Mice; Mycobacterium Infections, Nontuberculous; Mycobacterium smegmatis; Promoter Regions, Genetic; Regulatory Elements, Transcriptional; Repressor Proteins; Streptomyces coelicolor; Tetracycline; Tetracyclines; Transformation, Genetic

Reversible tetracycline-dependent gene regulation allows induction of expression with the tetracycline repressor (TetR) or gene silencing with the newly developed reverse mutant revTetR. We report here the implementation of both approaches with full regulatory range in gram-positive bacteria as exemplified in Bacillus subtilis. A chromosomally located gene is controlled by one or two tet operators. The precise adjustment of regulatory windows is accomplished by adjusting tetR or revtetR expression via different promoters. The most efficient induction was 300-fold in the presence of 0.4 microM anhydrotetracycline obtained with a Pr-xylA-tetR fusion. Reversible 500-fold gene knockouts were obtained in B. subtilis after adjusting expression of revTetR by synthetically designed promoters. We anticipate that these tools will also be useful in many other gram-positive bacteria.

Topics: Bacillus subtilis; Bacterial Proteins; Base Sequence; Gene Expression Regulation, Bacterial; Gene Silencing; Molecular Sequence Data; Operator Regions, Genetic; Promoter Regions, Genetic; Repressor Proteins; Tetracycline; Tetracyclines

Overexpression of the receptor tyrosine kinase HER-2/neu is associated with poor prognosis in patients with breast and ovarian cancer. Recent excitement has surrounded the therapeutic effects of HER-2-blocking therapy strategies and has rekindled interest on the molecular mechanisms of HER-2/neu in tumor biology. To study the role of HER-2/neu overexpression in vivo, we used a murine fibroblast cell line (NIH3T3-her2) conditionally expressing human HER-2/neu under control of a tetracycline-responsive promoter. Expression of HER-2 could be down-regulated below detection limit (>625-fold dilution) by exposure of NIH3T3-her2 cells to anhydrotetracycline (ATc). Subcutaneous injection of NIH3T3-her2 cells into nude mice resulted in rapid tumor growth. Mice with mean tumor volumes of 0.2, 0.8, 1.9, and 14.9 cm(3) were treated daily with 10 mg/kg ATc to switch off HER-2/neu expression, producing reductions in tumor size of 100, 98.1, 81.4, and 74.2%, respectively, by 7 days after onset of ATc administration (P = 0.005, Kruskal-Wallis test). Different long-term effects of HER-2 down-regulation were observed when mice with small (0.2 cm(3); n = 7), intermediate (0.8-1.2 cm(3); n = 10) and large (> or =1.9 cm(3); n = 11) tumors received ATc for up to 40 days. Complete remission was observed for 100, 40, and 18% of the small-, intermediate-, and large-sized tumors, respectively (P = 0.003). However, after 20-45 days of ATc administration, recurrent tumor growth was observed for all mice, even in those with previous complete remissions. The time periods for which mean tumor volume could be suppressed to volumes <0.1 cm(3) under ATc administration were 34, 22, 8, and 0 days for tumors with initial volumes of 0.2, 0.8, 1.9 and 14.9 cm(3), respectively (P = 0.005, Kruskal-Wallis test). Interestingly, HER-2 remained below the detection limit in recurrent tumor tissue, suggesting that initially HER-2-dependent tumors switched to HER-2 independence. The "second hits" leading to HER-2-independent tumor growth have not yet been identified. The rapid regression of tumors after down-regulation of HER-2 was explained by two independent mechanisms: (a) a block in cell cycle progression, as evidenced by a decrease in Ki-67 antigen expression from 40% before ATc treatment to 8.3% after 7 days of ATc treatment; and (b) induction of apoptosis as demonstrated by caspase-3 activation and by the terminal deoxynucleotidyltransferase (Tdt)-mediated nick end labeling assay (TUNEL). In con

Topics: Animals; Apoptosis; Cell Cycle; Cell Division; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Nude; Neoplasms, Experimental; NIH 3T3 Cells; Promoter Regions, Genetic; Receptor, ErbB-2; Tetracycline; Tetracyclines

Fluorescence enhancement of anhydrotetracycline hydrochloride and iso-tetracycline has been described. The fluorescence intensities of anhydrotetracycline hydrochloride and iso-tetracycline with cetyltrimethylammonium bromide (CTMAB) enhanced by micellar solution have been examined. It is found that fluorescence enhancement of anhydrotetracycline hydrochloride and iso-tetracycline depends on the concentration of CTMAB and pH of the solution. It can be used to develop sensitive methods for the determination of tetracycline hydrochloride and its decomposition product.

Topics: Cetrimonium; Cetrimonium Compounds; Fluorescence; Protein Isoforms; Spectrometry, Fluorescence; Surface-Active Agents; Tetracycline; Tetracyclines

A set of single Trp mutants of class B Tet repressor (TetR), in which Trp residues are located from positions 159 to 167, has been engineered to investigate the dynamics of the loop joining the alpha-helices 8 and 9. The fluorescence anisotropy decay of most mutants can be described by the sum of three exponential components. The longest rotational correlation time, 30 ns at 10 degrees C, corresponds to the overall rotation of the protein. The shortest two components, on the subnanosecond and nanosecond time scale, are related to internal motions of the protein. The initial anisotropy, in the 0.16-0.22 range, indicates the existence of an additional ultrafast motion on the picosecond time scale. Examination of physical models for underlying motions indicates that librational motions of the Trp side chain within the rotameric chi(1) x chi(2) potential wells contribute to the picosecond depolarization process, whereas the subnanosecond and nanosecond depolarization processes are related to backbone dynamics. In the absence of inducer, the order parameters of these motions, about 0.90 and 0.80 for most positions, indicate limited flexibility of the loop backbone. Anhydrotetracycline binding to TetR induces an increased mobility of the loop on the nanosecond time scale. This suggests that entropic factors might play a role in the mechanism of allosteric transition.

Topics: Bacterial Proteins; Energy Transfer; Fluorescence Polarization; Peptide Fragments; Protein Conformation; Protein Structure, Secondary; Repressor Proteins; Spectrometry, Fluorescence; Tetracycline; Tetracyclines; Thermodynamics; Tryptophan

Anhydrotetracycline (AHTC) is a toxic decomposition product of the widely used antibiotic tetracycline (TC). The side effects of AHTC have been attributed to the conformational changes in the ring system. In the present study a systematic conformational analysis has been carried out using the semiempirical quantum mechanical AM1 model. The conformational pH dependence has been analyzed through the study of all the ionized species. The results obtained showed two distinct families of conformation, referred to as A and B, with the interconversion process involving a rotation around the C4a-C12a bond. The solvent effect has been considered using the continuum model COSMO. From the population analysis in the gas phase, we conclude that form A should be dominant for the LH3+ and LH2 +/- species and B is the preferred conformer for the L2- ionized form (97.54%). For the LH- derivative, we predict that both conformations should be present in the equilibrium mixture in the gas phase, with the relative concentration found to be 68.47% (A) and 31.53% (B). The inclusion of the solvent does not change the A/B equilibrium for the LH3+ and LH2 +/- species. However, for the LH- form, the equilibrium is shifted to conformer A in water solution. The population analysis in water solution for the L2- suggest the following relative concentrations: A (34.46%) and B (65.54%). The biological activity of the TC parent compound is attributed to the zwitterionic species, which should adopt a twisted conformation. According to the results obtained in the present study, the most abundant form of the LH2 +/- zwitterionic species for the AHTC molecule is the extended one (100% in both the gas phase and water solution). Therefore, from a pharmacodynamic point of view, this conformational difference should be taken into account in order to explain the toxic effects of the anhydrous derivative. Another point related to the structure-activity relationship was analyzed through the investigation of the tautomerization process LH2(0)-->LH2 +/-. The result obtained suggests that the LH2(0) tautomer should be dominant in the gas phase (nonpolar solvent) and adopt a conformation classified as B. In water solution, the tautomer LH2 +/- is present as conformer A (96%). This result is in agreement with the conformation changes involved in the tautomerization process for the OTC active derivative.

Topics: Anti-Bacterial Agents; Hydrogen-Ion Concentration; Ions; Models, Chemical; Molecular Conformation; Molecular Structure; Tetracycline; Tetracyclines

A liquid chromatographic method of tetracycline and its major degradation products on a C8-reversed phase column with acidic mobile phase and fluorescence detection is described. The quantification limit, measured as the amount of sample that gave a signal ten times the peak-to-peak noise of the baseline, was: 0.25 ng for tetracycline (TC) and epitetracycline (ETC), 25 ng for and 4-epianhydrotetracycline (EATC) and 50 ng for anhydrotetracycline (ATC) of injected standard. By means of this liquid chromatography (LC) assay TC, ETC, EATC and ATC as main degradation products of tetracycline, can be separated and determined with good sensitivity and specificity within 15 min.

Topics: Anti-Bacterial Agents; Chromatography, Liquid; Models, Chemical; Reproducibility of Results; Spectrometry, Fluorescence; Tetracycline; Tetracyclines

The tetracycline-regulatable system (TRS) has become a widely adopted tool for modification of gene expression and analysis of gene function in mammalian cells, plants and transgenic animals. We have studied the potential application of the TRS in gene therapy, using a single vector containing both the tetracycline-controlled transactivator (tTA) and the tTA-responsive promoter (tRP) transcribing mouse GM-CSF. Stable 293 cells established using this vector were used to study the kinetics of the TRS in response to various tetracycline analogues. Dose-response studies show that doxycycline is the most potent-analogue in abolishing tTA activity. Kinetic studies indicate that, at 1,000 ng/ml, all the analogues have similar efficiencies in down-regulating the system in given time. In contrast, following the removal of the analogues, there is a temporal, dose-dependent delay in resumption of the tRP activity. The time taken for resumption of near-optimal tRP activity is approximately 48 h for tetracycline, 144 h for anhydrotetracycline, 192 h for minocycline and 216 h for doxycycline when cells were pretreated with 1000 ng/ml of these antibiotics. This property of the analogues can be employed in planning a desired course of transgene regulation.

Topics: Animals; Anti-Bacterial Agents; Cell Line; Dose-Response Relationship, Drug; Doxycycline; Gene Expression Regulation; Genetic Therapy; Genetic Vectors; Granulocyte-Macrophage Colony-Stimulating Factor; Mice; Minocycline; Promoter Regions, Genetic; Tetracycline; Tetracyclines; Time Factors; Transgenes

In the present study we describe the reversible transformation of NIH3T3 fibroblasts by overexpression of the HER2/c-erbB2 receptor tyrosine kinase. Cell lines expressing HER2 under control of a tetracycline-responsive promoter were isolated. Induction of HER2 expression resulted in cellular transformation in vitro as depicted by growth in soft agar and focus formation in tissue culture. Subsequent treatment of these cells with the effector anhydrotetracyline switched-off HER2 expression and induced morphological and functional changes characteristic for non-transformed cells. Subcutaneous transplantation of cells in nude mice resulted in the formation of solid tumors. Interestingly tumor formation was completely suppressed by treatment of the animals with anhydrotetracyline. Our findings indicate that overexpression of HER2 induces the transformed phenotype of NIH3T3 cells and is required for tumor formation and progression in nude mice. By linking the expression of the marker gene secreted placental alkaline phosphatase to the expression of HER2, a sensitive monitoring of tumor development in nude mice was feasible.

Topics: 3T3 Cells; Alkaline Phosphatase; Animals; Antineoplastic Agents; Cell Line, Transformed; Cell Transformation, Neoplastic; Female; Gene Expression Regulation, Neoplastic; Mice; Mice, Nude; Neoplasms, Experimental; Phenotype; Receptor, ErbB-2; Repressor Proteins; Tetracycline; Tetracyclines; Trans-Activators; Transcription, Genetic; Transplantation, Heterologous

Ruggedness tests were performed on the United States Pharmacopoeia assay for tetracycline.HCl to examine problems previously reported in the literature. The experiments were performed on nine different columns. The effects of four factors selected from the procedure were examined on qualitative responses by performing half-fraction factorial designs at three different ages on each column. The influence of column ageing was separately evaluated by injections under nominal method conditions at different ages. The C-8 columns gave a separation that was as good as, or better than, the C-18 ones and were less influenced by ageing. The normalized effects of each of the factors on a response were compared and found to be more or less equal for most of the columns and to remain constant with time. The responses were most affected by the pH of the mobile phase and by the content of the organic modifier. In general it was found that C-8 columns were preferred for this assay.

Topics: Chlortetracycline; Chromatography, High Pressure Liquid; Dimethylformamide; Hydrogen-Ion Concentration; Osmolar Concentration; Oxalates; Pharmacopoeias as Topic; Tetracycline; Tetracyclines; United States

Topics: Animals; Antiporters; Dictyostelium; Gene Expression Regulation; HeLa Cells; Humans; Luciferases; Repressor Proteins; Saccharomyces cerevisiae; Schizosaccharomyces; Tetracycline; Tetracyclines

A spectrofluorimetric method, involving alkaline degradation and formation of a magnesium complex, is described for the determination of tetracycline (TC) and anhydrotetracycline (ATC) in their mixed solution. Tetracycline is degraded and determined in alkaline solution. This treatment of ATC produces almost no fluorescence, but a fluorescent magnesium complex forms at pH 7.5. Several synthetic samples of TC and ATC, with TC:ATC ratios ranging from 50:1 to 1:50, were analysed. The recoveries of TC and ATC are about 71-76 and 61-63% in serum, respectively, and are all about 100% in urine.

Topics: Humans; Spectrometry, Fluorescence; Tetracycline; Tetracyclines

A differential pulse polarographic method is described for the determination of the antibiotic tetracycline HCl in the presence of its degradation product anhydrotetracycline. The method utilizes the large difference in their differential pulse polarograms at a peak potential of -1.39 V in 0.1 M phosphate buffer as the base electrolyte (pH 6.8). The assay was evaluated using synthetic mixtures and applied to the analysis of commercial tetracycline HCl samples. The results obtained with this method are in close agreement with those from the spectrophotometric absorbance ratio method.

Topics: Polarography; Spectrophotometry, Ultraviolet; Tetracycline; Tetracyclines

We inserted the Tn10 tetracycline resistance determinant (tet) into the multicopy plasmid pACYC177, and we examined the phenotype of Escherichia coli K-12 strains harboring these plasmids. In agreement with others, we find that Tn10 tet exhibits a negative gene dosage effect. Strains carrying multicopy Tn10 tet plasmids are 4- to 12-fold less resistant to tetracycline than are strains with a single copy of Tn10 in the bacterial chromosome. In addition, we find that multicopy tet strains are 30- to 100-fold less resistant to the tetracycline derivative 5a,6-anhydrotetracycline than are single-copy tet strains. Multicopy tet strains are, in fact, 10- to 25-fold more sensitive to anhydrotetracycline than are strains that lack tet altogether. The hypersensitivity of multi-copy strains to anhydrotetracycline is correlated with the effectiveness of anhydrotetracycline as an inducer of tet gene expression, rather than its effectiveness as an inhibitor of protein synthesis. Anhydrotetracycline is 50- to 100-fold more effective than tetracycline as an inducer of tetracycline resistance and as an inducer of beta-galactosidase in strains that harbor tet-lac gene fusions. In contrast, anhydrotetracycline appears to be two- to fourfold less effective than tetracycline as an inhibitor of protein synthesis. Both anhydrotetracycline and tetracycline induce synthesis of tet polypeptides in minicells harboring multicopy tet plasmids. Differences between E. coli K-12 backgrounds influence the tetracycline and anhydrotetracycline sensitivity of multicopy strains; ZnCl2 enhances the tetracycline and anhydrotetracycline sensitivity of these strains two- to threefold. We propose that the overexpression of one or more Tn10 tet gene products inhibits the growth of multicopy tet strains and accounts for their relative sensitivity to inducers of tet gene expression.

Topics: Bacterial Proteins; beta-Galactosidase; Chlorides; DNA Transposable Elements; Drug Resistance, Microbial; Escherichia coli; Gene Expression Regulation; Genes, Bacterial; Plasmids; Tetracycline; Tetracyclines; Zinc; Zinc Compounds

The analysis of tetracycline epimers in tetracycline preparations by high-performance liquid chromatography is described. The method uses a microparticulate phenyl column (3.9 mm i.d. X 30 cm) with a step gradient of 12-22% acetonitrile in 0.2 M phosphate buffer at pH 2.2. The analysis takes 22 min. The relative standard deviations of the method (2 sigma, n = 6) for the analysis of chlortetracycline, 4-epitetracycline, 4-epianhydrotetracycline, and anhydrotetracycline in tetracycline were +/- 3.68, +/- 4.47, +/- 7.60, and +/- 2.77 %, respectively.

Topics: Capsules; Chemical Phenomena; Chemistry; Chlortetracycline; Chromatography, High Pressure Liquid; Drug Contamination; Powders; Stereoisomerism; Tetracycline; Tetracyclines

Topics: Anti-Bacterial Agents; Chemistry, Pharmaceutical; Chromatography; Research; Tetracycline; Tetracyclines