Gene Ther. 1997 Sep;4(9):901-8.
Tetracycline-responsive gene expression in mouse brain after amplicon-mediated gene transfer.

Fotaki ME, Pink JR, Mous J.

F Hoffmann-La Roche Ltd, Basel, Switzerland.

Amplicon vectors incorporate genetic elements from Herpes simplex virus (HSV) in a plasmid form which is packaged into virions in the presence of a replication-defective helper virus. We constructed a new amplicon vector, pHermes-tet-lacZ, that carries the bacterial beta-galactosidase (lacZ) gene under the control of a minimal promoter preceded by a heptameric tetracycline operator. The minimal promoter element is activated by a tetracycline-responsive hybrid protein, the gene for which is also present in the vector. This amplicon was propagated in parallel in two different permissive cell lines, E5 and 2-2, in the presence of two helper viruses, d120 and 5dl1.2, respectively. The viral stocks produced were injected into the hippocampal region of the mouse brain, where strong localized expression of the transgene developed in the granular cell layer of the dentate gyrus with limited cytotoxicity. The transgene expression could be repressed by a factor of 10 after administration of tetracyclines. The repression level depended on the helper virus present in the injected viral stock. The in vivo regulation of transgene expression conferred by the tetracycline responsive element improves the flexibility of amplicon vectors as tools for gene transfer into the brain.

Source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9349426&dopt=Abstract antibiotics, tetracycline




Biotechnol Prog. 1997 Nov-Dec;13(6):733-40.
Autoregulated multicistronic expression vectors provide one-step cloning of regulated product gene expression in mammalian cells.

Fussenegger M, Moser S, Mazur X, Bailey JE.

Institute of Biotechnology, ETH Zurich, Switzerland.

Regulated expression of a cloned gene often provides much higher final expression of the gene product. Also, regulated expression of an activity can enable optional metabolic engineering and simplify functional genomic research. We constructed di-, tri-, and quattrocistronic mammalian expression vectors which allow the simultaneous, coordinated, and adjustable expression of up to two product genes. A single, tetracycline-regulatable promoter, PhCMV*-1, drives high-level expression of a multicistronic expression unit, containing the product gene(s), the gene for tetracycline-responsive transactivator (tTA), and, in the case of pQuattro-tTA, also the neomycin resistance gene. This autoregulatory genetic configuration retains a very low basal transcription activity in the presence of tetracycline, thereby reducing or eliminating possible toxic effects of tTA expression. However, upon withdrawal of tetracycline, a positive feedback regulation loop is activated which leads to higher levels of tTA expression and consequently also to higher expression levels of all other cistrons encoded on the multicistronic expression unit. Since such multicistronic expression vectors combine all genetic elements necessary for high-level expression as well as regulation in a single multicistronic expression unit, they alleviate limitations of previously reported tetracycline-regulatable vector systems and allow straightforward, one-step genetic engineering of eucaryotic cells to give an adjustable phenotype under strict control of the external stimulus, here tetracycline. Because the expression vectors described here were used for the expression for several heterologous product genes such as the green fluorescent protein and the tumor suppressor gene p21 in several cell lines (CHO-K1, BHK-21, and HeLa), we expect these multicistronic, positive feedback regulation vectors to function in a wide variety of eucaryotic cells and to be useful for basic as well as for applied research applications. Other vectors based upon the same autoregulation and multicistronic expression concepts can be constructed using other regulator gene-regulated promoter elements.

Source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9413131&dopt=Abstract antibiotics, tetracycline




Am J Vet Res. 1998 Jan;59(1):44-7.
Antimicrobial susceptibility of Fusobacterium necrophorum isolated from bovine hepatic abscesses.

Lechtenberg KF, Nagaraja TG, Chengappa MM.

Department of Animal Sciences, Kansas State University, Manhattan 66506-1600, USA.

OBJECTIVE: To determine the resistance and susceptibility to antimicrobial compounds of Fusobacterium necrophorum isolates from bovine hepatic abscesses. PROCEDURE: 37 isolates of F necrophorum (21 subsp necrophorum and 16 subsp funduliforme) isolated from bovine hepatic abscesses were obtained from cultures grown and maintained in anaerobic brain heart infusion broth. A broth dilution method was used as an initial screening to determine general susceptibility to 31 antimicrobial compounds. The minimal inhibitory concentrations (MIC) of 19 of the antimicrobial compounds that inhibited growth in the initial test were determined by use of the broth microdilution method. RESULTS: Fusobacterium necrophorum isolates were generally susceptible to penicillins, tetracyclines (chlortetracycline and oxytetracycline), lincosamides (clindamycin and lincomycin), and macrolides (tylosin and erythromycin), and were resistant to aminoglycosides (kanamycin, neomycin, gentamicin, and streptomycin), ionophores (except narasin), and peptides (avoparcin, polymyxin, and thiopeptin). The 5 antimicrobials (bacitracin, chlortetracycline, oxytetracycline, tylosin, and virginiamycin) that have FDA approval for prevention of liver abscesses in feedlot cattle were inhibitory to F necrophorum. Differences in antimicrobial susceptibility patterns were observed between the 2 subspecies only for clindamycin and lincomycin. The MIC of F necrophorum isolates from antibiotic-fed cattle were similar to those for isolates from nonantibiotic-fed cattle. CONCLUSIONS: The MIC of FDA-approved antibiotics were not reflective of the efficacy of antibiotics in preventing liver abscesses in feedlot cattle. Also, continuous feeding of tylosin did not appear to select resistant F necrophorum.

Source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9442241&dopt=Abstract antibiotics, tetracycline




West Indian Med J. 1997 Dec;46(4):107-10.
Antibiotic susceptibility of Neisseria gonorrhoeae in Trinidad and Tobago.

Swanston WH, Ali C, Mahabir BS, Prabhakar P, Basraj S, George J.

Faculty of Medical Sciences, University of the West Indies, Trinidad.

Treatment failures with standard doses of penicillin have been observed in the Sexually Transmitted Diseases (STD) clinics in Trinidad and Tobago. In the absence of an ongoing surveillance system, the antimicrobial susceptibility of 518 Neisseria gonorrhoeae strains was determined in order to guide treatment. 39 (7.6%) strains were resistant to penicillin, including 27 (5.2%) positive for beta-lactamase; that is penicillinase-producing Neisseria gonorrhoeae (PPNG). 51 (10%) strains were resistant to tetracycline, with 26 (5.0%) of these exhibiting high levels of resistance compatible with tetracycline resistant Neisseria gonorrhoeae (TRNG). Six strains showed evidence of having both PPNG and TRNG plasmids, and five strains showed chromosomally-mediated resistance to both penicillin and tetracycline. The overall resistance rate to penicillin and tetracycline was 17.7%. There was no resistance to spectinomycin, cefuroxime, ceftriaxone and norfloxacin. The resistance rates demonstrated in this study are sufficiently significant to preclude the use of penicillin and tetracycline in the STD clinics and to justify the use of newer antimicrobials. It is essential that resistance patterns be monitored by continued surveillance.

PIP: Neisseria gonorrhoeae from many areas of the world have developed resistance to a wide variety of antibiotics over the past 2 decades. The incidence of treatment failures with standard doses of penicillin observed in Trinidad and Tobago's sexually transmitted disease (STD) clinics suggests that such resistance also exists in Trinidad. 518 Neisseria gonorrhoeae strains were isolated from 1502 male and female patients attending the 7 STD clinics throughout Trinidad during May-October 1992, and tested to identify their antimicrobial susceptibility. 39 strains were resistant to penicillin, including 27 which were positive for beta-lactamase, indicating infection with penicillinase-producing Neisseria gonorrhoeae (PPNG). 51 strains were resistant to tetracycline, of which 26 exhibited high levels of resistance compatible with tetracycline-resistant Neisseria gonorrhoeae (TRNG). 6 strains showed evidence of having both PPNG and TRNG plasmids, and 5 strains showed chromosomally-mediated resistance to both penicillin and tetracycline. The overall resistance rate to penicillin and tetracycline was 17.7%, and there was resistance to neither spectinomycin, cefuroxime, ceftriaxone, nor norfloxacin. The resistance rates observed in this study are significant enough to warrant the cessation of penicillin and tetracycline use in STD clinics, and the use of newer antimicrobials. Neisseria gonorrhoeae resistance patterns demand ongoing monitoring.

Source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9494404&dopt=Abstract antibiotics, tetracycline

leeds.ac.uk

A genetic basis for tetracycline resistance in cutaneous propionibacteria was suggested by comparing the nucleotide sequences of the 16S rRNA genes from 16 susceptible and 21 resistant clinical isolates and 6 laboratory-selected tetracycline-resistant mutants of a susceptible strain. Fifteen clinical isolates resistant to tetracycline were found to have cytosine instead of guanine at a position cognate with Escherichia coli 16S rRNA base 1058 in a region important for peptide chain termination and translational accuracy known as helix 34. Cytosine at base 1058 was not detected in the laboratory mutants or the tetracycline-susceptible strains. The apparent mutation was recreated by site-directed mutagenesis in the cloned E. coli ribosomal operon, rrnB, encoded by pKK3535.E. coli strains carrying the mutant plasmid were more resistant to tetracycline than those carrying the wild-type plasmid both in MIC determinations and when grown in tetracycline-containing liquid medium. These data are consistent with a role for the single 16S rRNA base mutation in clinical tetracycline resistance in cutaneous propionibacteria.

Source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9661007&dopt=Abstract antibiotics, tetracycline




Biol Pharm Bull. 1998 Jul;21(7):678-81.
Screening of an inhibitor of the tetracycline efflux pump in a tetracycline-resistant clinical-isolate of Staphylococcus aureus 743.

Hirata T, Wakatabe R, Nielsen J, Satoh T, Nihira S, Yamaguchi A.

Nippon Roche Research Center, Kamakura, Kanagawa, Japan.

Clinically-isolated methicillin-resistant Staphylococcus aureus (MRSA) strain 743 exhibited resistance to tetracycline as judged from the active efflux of the drug. The efflux of tetracycline was inhibited by an uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and minocycline. Inhibitors of the efflux pump were examined in this strain to determine the cellular accumulation of tetracycline. Out of seven compounds examined, three caused a significant increase in the cellular concentration of tetracycline by inhibiting the efflux pump. Two of them seem to be energy inhibitors. Ro 07-3149 inhibited the efflux pump without affecting the energy state, and exhibited very low antibacterial activity but showed weak synergy with tetracycline.

Source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9703248&dopt=Abstract antibiotics, tetracycline




J Vasc Surg. 1998 Dec;28(6):1082-93.
Pharmacologic suppression of experimental abdominal aortic aneurysms: acomparison of doxycycline and four chemically modified tetracyclines.

Curci JA, Petrinec D, Liao S, Golub LM, Thompson RW.

Department of Surgery, Washington University School of Medicine, St Louis, MO 63110, USA.

BACKGROUND: Matrix metalloproteinases (MMPs) likely contribute to the degradation of medial elastin in abdominal aortic aneurysms (AAAs), and tetracycline antibiotics exhibit MMP-inhibiting properties. The purpose of this study was to compare the effects of doxycycline and several non-antibiotic chemically modified tetracyclines (CMTs) in a rat model of elastase-induced AAA. METHODS: Fifty-two male Wistar rats underwent intraluminal perfusion of the abdominal aorta with porcine pancreatic elastase. The rats then were treated for 7 days with subcutaneous injections of saline solution, different doses of doxycycline, or 1 of 4 different CMTs. The aortic diameters were measured with microcalipers, and the fixed tissues were examined by means of light microscopy. Gelatin zymography was used to assess the MMP activity in the aortic tissue extracts. RESULTS: The mean aortic diameter in the control group increased by 126% +/- 14% on day 7 (from 1.57 +/- 0.04 mm to 3.54 +/- 0.27 mm; P <.05), and 5 of 6 animals (83%) had AAAs. Doxycycline appeared to inhibit aortic dilatation in a dose-dependent manner, and AAAs did not develop in any animals. Half-maximal effects were observed at a dose of approximately 6 mg/kg/day, and maximal effects were noted at greater than 30 mg/kg/day. No AAAs were observed in the animals that were treated with CMTs at 15 mg/kg/day. Each of the following CMTs exhibited an efficacy that was similar to that of doxycycline (percent inhibition of aortic dilatation vs control; all P <.05): CMT-3 (47.6%), CMT-4 (38.9%), CMT-7 (47.6%), CMT-8 (54.0%), and doxycycline (51.6%). Tissues from saline solution-treated controls exhibited a transmural inflammatory response and marked destruction of the medial elastic lamellae. Tetracycline derivatives limited the disruption of medial elastin without appearing to alter either the inflammatory response or the rat aortic wall production of metallogelatinases. CONCLUSION: Tetracycline derivatives suppress the development of AAAs after elastase-induced aortic injury in the rat. The aneurysm-suppressing effects of doxycycline appear to be dose-dependent and distinct from its antibiotic activities, and they coincide with the structural preservation of medial elastin fibers. Further studies are needed to explore the potential of MMP-inhibiting tetracyclines as a novel pharmacologic strategy for the suppression of aortic aneurysms.

Source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9845660&dopt=Abstract antibiotics, tetracycline




J Mol Biol. 1999 Jan 15;285(2):455-61.
Crystal structure of the tet repressor in complex with a novel tetracycline, 9-(N,N-dimethylglycylamido)- 6-demethyl-6-deoxy-tetracycline.

Orth P, Schnappinger D, Sum PE, Ellestad GA, Hillen W, Saenger W, Hinrichs W.

Freie Universitat Berlin, Takustr. 6, Berlin, D-14195, Germany.

The tetracycline analog 9-(N, N-dimethylglycylamido)-6-demethyl-6-deoxy-tetracycline (9glyTc) belongs to a new group of tetracyclines called glycylcyclines. They are strong antibiotics showing reduced sensitivity against the major tetracycline resistance mechanisms. We have determined the crystal structure of 9glyTc in complex with Tet repressor class D, TetR(D), at 2.4 A resolution. Sterical hindrance at the entrance of the tetracycline binding tunnel of TetR by the bulky and charged glycyl amido substituent interferes with conformational changes required for the mechanism of induction, and leads to decreased induction efficiency as observed for point mutations of amino acid residues located in the neighbourhood to the glycylamido moiety of bound 9glyTc. Copyright 1999 Academic Press.

Source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9878420&dopt=Abstract antibiotics, tetracycline