Bicyclol attenuates tetracycline-induced fatty liver associated with inhibition of hepatic ER stress and apoptosis in mice.

Endoplasmic reticulum (ER) stress is known to be involved in the development of several metabolic disorders, including non-alcoholic fatty liver disease (NAFLD). Tetracycline can cause hepatic steatosis, and ER stress may be involved in tetracycline-induced fatty liver. Our previous study showed that bicyclol has been proven to protect against tetracycline-induced fatty liver in mice, and ER stress may also be involved in bicyclol's hepatoprotective effect. Therefore, this study was performed to investigate the underlying mechanisms associated with ER stress and apoptosis, by which bicyclol attenuated tetracycline-induced fatty liver in mice. Bicyclol (300 mg/kg) was given to mice by gavage 3 times. Tetracycline (200 mg/kg, intraperitoneally) was injected at 1 h after the last dose of bicyclol. At 6 h and 24 h after single dose of tetracycline injection, serum ALT, AST, TG, CHO and hepatic histopathological examinations were performed to evaluate liver injuries. Hepatic steatosis was assessed by the accumulation of hepatic TG and CHO. Moreover, hepatic apoptosis and ER stress related markers were determined by TUNEL, real-time PCR, and western blot. As a result, bicyclol significantly protected against tetracycline-induced fatty liver as evidenced by the decrease of elevated serum transaminases and hepatic triglyceride, and the attenuation of histopathological changes in mice. In addition, bicyclol remarkably alleviated hepatic apoptosis and the gene expression of caspase-3, and increased the gene expression of XIAP. The gene expressions of ER stress-related markers, including CHOP, GRP78, IRE-1α, and ATF6, which were downregulated by bicyclol pretreatment in tetracycline-injected mice. These results suggested that bicyclol protected tetracycline-induced fatty liver partly due to its ability of anti-apoptosis associated with ER stress.

Dual stimulation with bacterial and viral components increases the expression of hepcidin in human monocytes.

Hepcidin belongs to the antimicrobial peptide (AMP) family and is the key regulator of iron metabolism. It modulates iron homeostasis by binding to, and degrading the iron exporter molecule, ferroportin, thus inhibiting cellular iron efflux. Many antimicrobial peptides have a dual function; some are able to act directly as an antimicrobial agent as well as having an immunoregulatory role in the host. Toll-like receptors (TLRs) bind to components of microorganisms, activate cellular signal transduction pathways and stimulate innate immune responses. The effect of TLR3 (poly I:C) and TLR9 (CpG) co-stimulation of THP-1-derived monocytes using purified TLR ligands showed that 24 h after exposure poly I:C and CpG ligands in combination, hepcidin expression was significantly increased (10-fold) when compared to the untreated control. This combination of TLR ligands mimics simultaneous bacterial and viral infections, thus suggesting a potential key role for hepcidin in combined infections. Additionally, using a chequerboard assay, we have shown that hepcidin has an antagonistic effect in combination with the antibiotics rifampicin and tetracycline against Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus pyogenes, evidenced by a fractional inhibitory concentration index (FICI) > 4. This finding has important implications for future treatment regimens especially in an era of increasing antimicrobial resistance.

Tetra- and pentacyclic triterpene acids from the ancient anti-inflammatory remedy frankincense as inhibitors of microsomal prostaglandin E(2) synthase-1.

The microsomal prostaglandin E2 synthase (mPGES)-1 is the terminal enzyme in the biosynthesis of prostaglandin (PG)E2 from cyclooxygenase (COX)-derived PGH2. We previously found that mPGES-1 is inhibited by boswellic acids (IC50 = 3-30 µM), which are bioactive triterpene acids present in the anti-inflammatory remedy frankincense. Here we show that besides boswellic acids, additional known triterpene acids (i.e., tircuallic, lupeolic, and roburic acids) isolated from frankincense suppress mPGES-1 with increased potencies. In particular, 3α-acetoxy-8,24-dienetirucallic acid (6) and 3α-acetoxy-7,24-dienetirucallic acid (10) inhibited mPGES-1 activity in a cell-free assay with IC50 = 0.4 µM, each. Structure-activity relationship studies and docking simulations revealed concrete structure-related interactions with mPGES-1 and its cosubstrate glutathione. COX-1 and -2 were hardly affected by the triterpene acids (IC50 > 10 µM). Given the crucial role of mPGES-1 in inflammation and the abundance of highly active triterpene acids in frankincence extracts, our findings provide further evidence of the anti-inflammatory potential of frankincense preparations and reveal novel, potent bioactivities of tirucallic acids, roburic acids, and lupeolic acids.

Effect of milk on antibacterial activity of tetracycline against Escherichia coli and Staphylococcus aureus isolated from bovine mastitis.

The susceptibility of mastitis-causing Escherichia coli and Staphylococcus aureus to two commonly used antibiotics, tetracycline and penicillin G, was tested in raw milk and in Muller-Hinton (MH) broth by introducing a pH indicator, bromocresol purple, which was shown to be a simple, sensitive, and rapid method. The minimum inhibitory concentration (MIC) of penicillin G in milk was the same as those in MH broth, whereas the MIC of tetracycline in milk was 4 to 32 times that in MH. An irreversible binding between tetracycline and large molecules of milk, which might be due to a hydrophobic interaction, was demonstrated by a dialysis test, suggesting the observed impairing effect was due to the action of milk on the tetracycline being tested. Further investigation revealed that much of the reduction of tetracycline's activity in milk was attributable to the milk protein casein, while other heat-sensitive components in milk also play some roles.

Identification of a stability determinant on the edge of the Tet repressor four-helix bundle dimerization motif.

Isofunctional tetracycline repressor (TetR) proteins isolated from different bacteria show a sequence identity between 38 and 88% of the residues. Their active state is a homodimer formed by a four-alpha-helix bundle as the main interaction motif. We utilize this sequence variation of isofunctional proteins to determine residues contributing to the stability of the four-helix bundle. The thermodynamic stabilities of two TetR proteins with 63% sequence identity were determined by urea-induced reversible denaturation followed by fluorescence and circular dichroism. Both methods yield identical results. The deltaG(o)U (H2O) values are 60 and 75 kJ x mol(-1). We have constructed TetR hybrid proteins derived from these wild types to identify the determinant leading to the 15 kJ x mol(-1) stability difference. Successive size reduction of the exchanged portion yielded two single residues affecting the overall protein stability. The P184Q exchange leads to a more stable protein, whereas the G181D exchange located at the solvent's exposed edge of the four-helix bundle is solely responsible for the reduced stability. Additional mutants based on crystal structures of TetR do not reveal any hint for steric interference of the Asp181 side chain with neighboring residues. Thus, this is an example for the role played by surface-exposed turn residues for the stability of four-helix bundles. We assume that the larger conformational flexibility of Gly and the reduction of the negative surface charge could favor formation of the turn on the edge of the four-helix bundle.

PCR monitoring for tetracycline resistance genes in subgingival plaque following site-specific periodontal therapy. A preliminary report.

BACKGROUND: The selection of antibiotic resistance genes during antibiotic therapy is a critical problem complicated by the transmission of resistance genes to previously sensitive strains via conjugative plasmids and transposons and by the transfer of resistance genes between gram-positive and gram-negative bacteria. The purpose of this investigation was to monitor the presence of selected tetracycline resistance genes in subgingival plaque during site specific tetracycline fiber therapy in 10 patients with adult periodontitis. METHOD: The polymerase chain reaction (PCR) was used in separate tests for the presence of 3 tetracycline resistance genes (tetM, tetO and tetQ) in DNA purified from subgingival plaque samples. Samples were collected at baseline, i.e., immediately prior to treatment, and at 2 weeks, and 1, 3, and 6 months post-fiber placement. The baseline and 6-month samples were also subjected to DNA hybridization tests for the presence of 8 putative periodontal pathogenic bacteria. RESULTS: PCR analysis for the tetM resistance gene showed little or no change in 5 patients and a decrease in detectability in the remaining 5 patients over the 6 months following tetracycline fiber placement. The results for tetO and tetQ were variable showing either no change in detectability from baseline through the 6-month sampling interval or a slight increase in detectability over time in 4 of the 10 patients. DNA hybridization analysis showed reductions to unmeasurable levels of the putative periodontal pathogenic bacteria in all but 2 of the 10 patients. CONCLUSIONS: These results complement earlier studies of tet resistance and demonstrate the efficacy of PCR monitoring for the appearance of specific resistance genes during and after antibiotic therapy.

[Changes in the properties of the plasma membrane of Acholeplasma laidlawii under the action of tetracycline]. / Izmenenie svoistv plazmaticheskoi membrany Acholeplasma laidlawii pri deistvii tetratsiklina.

The role of physico-chemical rearrangements in the cell plasmalemma of Acholeplasma laidlawii in the development of resistance to tetracycline was investigated. The cells of A.laidlawii were shown to be tolerant to tetracycline and to preserve a rather high titre of the cells even at a concentration of the antibiotic in the inoculation medium much higher than the MIC. The results of the investigation of the structural rearrangements in the plasmatic membrane of the cells grown in the presence of tetracycline revealed changes in the lipid flow in the surface layer and an increase in the cholesterol and phospholipid contents. The size of the changes depended on the time of tetracycline addition and the phase of the culture growth.

Fast large-scale purification of tetracycline repressor variants from overproducing Escherichia coli strains.

We constructed a plasmid for overexpression of Tn10 Tet repressor (TetR) by placing a synthetic tetR gene under control of the Pc promoter. Active TetR is expressed up to 30% of the total soluble cell protein. A protocol containing anion-exchange, cation-exchange, and size-exclusion chromatography steps is described for the large-scale purification of milligram amounts of TetR in three days. Cation-exchange chromatography already yields almost homogenous TetR. Purification of about fifty TetR mutants demonstrates that this protocol is generally applicable. No correlation between net charge of TetR variants and elution behaviour was detected for the anion-exchange column. On the other hand, TetR mutants with increased negative charge in their DNA binding domain eluted at lower NaCl concentration from the cation-exchange column. The applicability of this purification protocol to the wide variety of TetR variants suggests that it can be used for the rapid purification of other DNA binding proteins as well.

[Drug-resistant tropical malaria in Angola]. / Lekarstvenno-ustoichivaia tropicheskaia malariia v Angole.

Three antimalarial treatment regimens by the complete standard WHO tests were examined in 105 Plasmodium falciparum-infected patients who were nonimmune newcomers treated at the Russian hospital in Luanda in 1991-1992, 61% showed chloroquine resistance and 40% fansidar resistance. All 59 patients with high rates of parasitemia were successfully cured with quinine in combination with tetracycline. Thick, if required thin, blood smears were microscopically examined. The findings suggest that Fansidar should be a drug of first-line therapy in Angola, though in the neighbouring countries quinine continues preserving its efficacy, but there is a delayed elimination of the parasites within 7 days of initiation of the therapy, making it necessary to prolong therapy with this drug up to 10 days.

[The effect of tetracycline and silibore on lipid peroxidation in the liver od rats of various age]. / Vliianie tetratsiklina i silibora na perekisnoe okislenie lipidov pecheni krys raznogo vozrasta.

As shown by accumulation of malonic dialdehyde and chemiluminescence rate monitoring tetracycline activated both NADPH- and ascorbate-dependent lipid peroxidation in liver tissue of 1, 3, 12 and 24 months old rats. The most distinct induction of lipid peroxidation was observed in old animals, which appears to occur due to a decrease in efficiency of NADPH-GSH-dependent enzymatic antioxidant system. Silibore prevented the stimulating effect of tetracycline on enzymatic and nonenzymatic lipid peroxidation, being apparently involved in hepatoprotective effect.

[The role of the RS1 sequence of Vibrio cholerae in the amplification of a segment of plasmid DNA carrying the tetracycline resistance gene and cholera toxin genes]. / Rol' RS1-posledovatel'nosti kholernogo vibriona v amplifikatsii segmenta plazmidnoi DNK, nesushchego gen rezistentnosti k tetratsiklinu i geny kholernogo toksina.

The clones with 4 to 30-fold increased level of tetracycline resistance (TcR) were selected from the strain of Escherichia coli K-12 carrying the pCO107 plasmid. The plasmid is the cointegrate formed from the plasmids pOX38 (F-derivative) and pCT105 (pBR322-derivative carrying the vct operon of Vibrio cholerae eltor and the RSI sequence). pCO107 contains RSI at the junctions of two plasmid genomes. Using restriction analysis and Southern blot hybridization technique it was shown that increased tetracycline resistance is accompanied by amplification of the pCT105 segment of pCO107 and depends upon the presence of direct repeats of flanking RSI. Amplification of the pCT105 also resulted in increased production of the cholerae toxin (CT).

The quantification of the neutralization of the action of antibiotics by cationic iron.

This paper describes the reversal of antibiotic action by iron. Crude measurements of the molar ratio of iron to various antibiotics at reversal indicated that ampicillin, carbenicillin and lincomycin had low ratios of 0.2 to 2.4. With the remainder the ratios lay between 13 and 105; they averaged 46 for the tetracyclines, and 70 for the aminoglycosides. Precise measurement with Fe+++ and tetracycline revealed that the molar ratio for neutralization increased with increasing concentrations of tetracycline; from 1 to 625 mg/l the ratio increased over three-fold from 35 to 118. At the tetracycline concentration of 5 mg/l--usually achieved in plasma during effective therapy--the ratio is of the order of 45.

[Characteristics of the derepressed mutant of the F-like plasmid pAP18-1 coding for tetracycline resistance in Escherichia coli]. / Kharakteristika derepressirovannogo mutanta F-podobnoi plazmidy pAP18-1, kodiruiushchei rezistentnost' k tetratsiklinu u Escherichia coli.

A transfer function derepressed mutant of the F-like plasmid pAP18-1 (Tc, ColV) was induced with the help of N-methyl-N'-nitro-N-nitrozoguanidine. The mutant plasmid pAP18-1drd belongs to the FVII incompatibility group of the F-like plasmids. The plasmid pAP18-1drd is characterized by the loss of the capacity for inhibiting the tra-genes functions of the Flac plasmid and is sensitive to the Tra-function inhibitors of the reference plasmids of the FinV and FinW groups.

The antagonism of tetracycline and ferric iron in vivo.

To test the hypothesis that the in-vivo antibiotic action of tetracycline might be affected by ferric iron and the enhancement of infection by ferric iron by tetracycline, the actions of intraperitoneal antibiotic and local ferric ammonium citrate, given separately and together, were measured in the dorsal skin of guinea-pigs bearing lesions due to staphylococci, streptococci, a Proteus sp., an Erysipelothrix sp., Clostridium perfringens, Pseudomonas aeruginosa, Aeromonas hydrophila and Klebsiella pneumoniae. Tetracycline, given in two intraperitoneal doses of 25 mg/kg at 0 and 2 h after intracutaneous challenge, maintained plasma concentrations of 4-6 micrograms/ml for more than the first 4 h of infection, after which the local lesions had become largely insusceptible to the antibiotic. The intracutaneous injection of Fe 10 micrograms in a volume of 0.1 ml containing the bacteria was sufficient to enhance infection by those strains susceptible to this effect. The in-vivo efficacy of tetracycline was not always related to low MIC; a low MIC was sometimes associated with little action and a high MIC with moderate action. Sixteen organisms were tested. The iron diminished the tetracycline effect only feebly with one staphylococcal strain and the strain of E. rhusiopathiae. In only one case, with a strain of Proteus sp., was the tetracycline action grossly diminished. On the other hand, tetracycline diminished the enhancement effect of iron moderately with three strains of staphylococci and one strain each of K. pneumoniae, P. aeruginosa and C. perfringens, and strongly with two strains of staphylococci, a group-C streptococcus and one strain each of K. pneumoniae, E. rhusiopathiae and A. hydrophila. It is evident that the diminution of tetracycline action by moderate excess of readily available Fe , whether endogenous or administered, is an unlikely event (three instances among the 16 tested) whereas the diminution of the infection-enhancing effect of iron by tetracycline is much more likely (12 instances among the 16). Insofar as a decrease in iron available for enhancement of infection is valid evidence of a diminution of the iron available for necessary physiological processes of the subject treated, our results suggest that these processes might be affected by tetracycline.

[Expression of the resistance genes of plasmid RP4 in Escherichia coli cells grown by continuous cultivation]. / Ekspressiia genov rezistentnost' plazidy RP4 v kletkakh Escherichia coli, vyrashchennykh v usloviiakh nepreryvnogo kul'tivirovaniia.

The resistance to tetracycline decreased in Escherichia coli C600 cells containing plasmid RP4 and grown under the conditions of continuous cultivation. The population of cells containing plasmid RP4 is heterogeneous in the trait of tetracycline resistance, and most cells cannot grow in a selective medium with tetracycline at a concentration of 20 micrograms/ml. The decreased resistance to tetracycline was most pronounced for a glucose-limited chemostat culture and also in the presence of two plasmids, RP4 and pBS94 , in the cells. No decrease was found in the resistance to other drugs determined by plasmid genes.

Intracistronic complementation of the tetracycline resistance membrane protein of Tn10.

The structural gene region for tetracycline resistance on Tn10 consists of two complementation groups, tetA and tetB (M. S. Curiale and S. B. Levy, J. Bacteriol. 151:209-215, 1982). Using a series of deletion mutants, we have determined that the tetA region is 450 to 600 base pairs long and that the tetB region, which is adjacent to tetA, is 600 to 750 base pairs long. Point mutations in either tetA or tetB affected the amount and size of the inducible inner-membrane Tet protein synthesized in Escherichia coli maxicells. Moreover, deletions in these regions led to the synthesis of an appropriately smaller Tet protein. A single tetracycline-inducible RNA of about 1,200 bases was detected that was homologous with the tetracycline resistance structural gene region. These results indicate that the tetA and tetB complementation regions represent two parts of a single gene encoding two domains of the tetracycline resistance protein Tet.

Recombinant DNA risk assessment studies in humans: efficacy of poorly mobilizable plasmids in biologic containment.

Recombinant DNA risk assessment studies quantitated the mobilizability of "safe" plasmid pBR325, in comparison with readily mobilizable plasmid pJBK5 (chloramphenicol and tetracycline resistant). Of 15 volunteers who became colonized after ingestion of 5 X 10(10) Escherichia coli HS-4, a normal human flora strain containing pJBK5 and daily oral tetracycline, nine manifested transfer of pJBK5 to normal flora by means of triparental mating. In contrast, none of 12 other volunteers cocolonized with HS-4 bearing "safe" pBR325 and normal flora showed transfer (P = 0.001), despite ingestion of tetracycline. To accomplish transfer directly, E coli HS-4 containing both pBR325 and a derepressed, conjugative plasmid (F-amp) was fed to two groups of volunteers. Transfer of pBR325 to normal flora occurred in 13 of 18 volunteers taking daily tetracycline but in none of eight who did not (P less than 0.002). Nor were transconjugants detected, despite tetracycline ingestion, in five volunteers who ingested and excreted E coli K12 (pBR325 plus F-amp).

[Seasonal features of the excretory function of the liver in the presence of tetracycline damage and correction of the disorders with antioxidants]. / Sezonnye osobennosti ékskretornoi funktsii pechni pri tetratsiklinovykh porazheniiakh i korrektsiia narushenii antioksidantami.

It was shown on pubertal albino rats that the intensity of excretion with the bile of radioactive Bengal rose was different at different seasons: the maximum and minimum levels were observed in winter and summer, respectively. When the liver was affected with tetracycline, this process was suppressed especially in summer. The use of antioxidants, such as tocopherol acetate in combination with sodium selenite promoted the recovery of liver excretion function in winter, spring and autumn. In summer, the recovery was only partial.

[Induced resistance to kanamycin and tetracycline in Rhizobium japonicum]. / Indutsirovannaia ustoichivost' k kanamitsinu i tetratsiklinu u Rhizobium japonicum.

Spontaneous mutants Km-R and Tc-R of R. japonicum with various levels of resistance to kanamycin (0.8-20 mg/ml) and tetracycline (130-210 micrograms/ml) were isolated. No cross resistance in the mutants was observed. Plasmid R68.45 transferred to the wild strains resistance to 210 micrograms/ml of tetracycline and to 20 mg/ml of kanamycin. This plasmid did not practically increase the resistance to tetracycline in mutants Tc-R. At the same time it markedly increased the resistance to kanamycin in mutants Km-R.