Effects of alkali metal ions on some virulence traits of<Emphasis Type="Italic">Candida albicans</Emphasis>

The effects of the alkali metal ions (Li+, Na+ and K+) on the growth and on certain virulence factors (adhesion, cell-surface hydrophobicity and germinating ability) of Candida albicans were determined. High concentrations of these ions displayed an inhibitory effect on the growth of the Candida cells; preincubation in their presence showed a negative effect on all virulence factors studied. The changes induced during the preincubation remained there even when high concentration of the ions was removed from the cell suspension. In contrast, a considerable growth was found at high Na+ and K+ concentrations. Although alkali metal ions significantly decreased certain virulence traits of the fungus they did not totally inhibit adhesion and germ-tube formation. This suggests that C. albicans may represent a health hazard even at a high salt concentration.     

Mg2+-dependent doxycycline-induced inhibition of leukocyte adhesion

The method for estimating in vitro leukocyte adhesion was modified in relation to investigation of the effect of antibiotics and other pharmacological agents on cell activation. The adhesion intensity was estimated in standard 96-cavity microplates from the content of beta-hexosaminidase in the adherent cells. That made it possible to easily automatize the method by using the equipment for enzyme immunoassay. It was shown that spontaneous and induced adhesion of guinea pig leukocytes depended on temperature and bivalent ions. The level of spontaneous adhesion mainly depended on Mg ions. Doxycycline, 0.1 to 100 micrograms/ml, did not induce degranulation of leukocytes and had no cytotoxic action. Doxycycline in supratherapeutic concentrations (greater than 10 micrograms/ml) inhibited the spontaneous leukocyte adhesion. In special experiments it was found that the doxycycline's inhibitory effect was partially reversible and associated with ++chelation of Mg ions rather than Ca ones.     

Adhesion of Mytilus edulis foot protein 1 on silica: ionic effects on biofouling

To determine the effect of the ionic environment on the marine adhesion molecule Mytilus edulis foot protein 1 (Mefp-1), atomic force microscopy (AFM) was used to measure the adhesion between Mefp-1 and a silica substrate under a range of ionic conditions. Both ion strength and type were varied on the basis of the ions present in natural seawater. Salts containing monovalent ions (NaCl, KCl) increased adhesion only slightly, but salts containing divalent ions (MgCl(2), CaCl(2), Na(2)SO(4)) induced multiple jumpouts in the decompression curve similar to other biological systems and an increase in hydrodynamic radius as observed by light scattering. This behavior may be due to metal complexation between 3,4-dihydroxyphenyl-L-alanine and o-quinone catechol groups on Mefp-1. The addition of a salt containing a trivalent ion (FeCl(3)) resulted in the highest adhesion. The strong effect of salt type and concentration suggests that the ionic composition of the environment within the mussel byssus may be tailored in order to achieve maximum adhesion and minimum curing time.     

Doxycycline reduces the migration of tuberous sclerosis complex‐2 null cells ‐ effects on RhoA‐GTPase and focal adhesion kinase

Lymphangioleiomyomatosis (LAM) is associated with dysfunction of the tuberous sclerosis complex (TSC) leading to enhanced cell proliferation and migration. This study aims to examine whether doxycycline, a tetracycline antibiotic, can inhibit the enhanced migration of TSC2‐deficient cells, identify signalling pathways through which doxycycline works and to assess the effectiveness of combining doxycycline with rapamycin (mammalian target of rapamycin complex 1 inhibitor) in controlling cell migration, proliferation and wound closure. TSC2‐positive and TSC2‐negative mouse embryonic fibroblasts (MEF), 323‐TSC2‐positive and 323‐TSC2‐null MEF and Eker rat uterine leiomyoma (ELT3) cells were treated with doxycycline or rapamycin alone, or in combination. Migration, wound closure and proliferation were assessed using a transwell migration assay, time‐lapse microscopy and manual cell counts respectively. RhoA‐GTPase activity, phosphorylation of p70S6 kinase (p70S6K) and focal adhesion kinase (FAK) in TSC2‐negative MEF treated with doxycycline were examined using ELISA and immunoblotting techniques. The enhanced migration of TSC2‐null cells was reduced by doxycycline at concentrations as low as 20 pM, while the rate of wound closure was reduced at 2–59 μM. Doxycycline decreased RhoA‐GTPase activity and phosphorylation of FAK in these cells but had no effect on the phosphorylation of p70S6K, ERK1/2 or AKT. Combining doxycycline with rapamycin significantly reduced the rate of wound closure at lower concentrations than achieved with either drug alone. This study shows that doxycycline inhibits TSC2‐null cell migration. Thus doxycycline has potential as an anti‐migratory agent in the treatment of diseases with TSC2 dysfunction.     

Synthesis, characterization, and antibacterial activity of three palladium(II) complexes of tetracyclines

Pd(II) complexes with three antibiotics of the tetracycline family (tetracycline, doxycycline and chlortetracycline) were synthesized and characterized by elemental, thermogravimetric, and conductivity analyses, and infrared spectroscopy. The interactions between Pd(II) ions and tetracycline were investigated in aqueous solution by (1)H NMR. All the tetracyclines studied form 1:1 complexes with Pd(II) via the oxygen of the hydroxyl group at ring A and that of the amide group. The effect of the three complexes on the growth of bacterial strains sensitive and resistant to tetracycline was studied. The Pd(II) complex of tetracycline is practically as efficient as tetracycline in inhibiting the growth of two Escherichia coli (E. coli) sensitive bacterial strains and 16 times more potent against E. coli HB101/pBR322, a bacterial strain resistant to tetracycline. Pd(II) coordination to doxycycline also increased its activity in the resistant strain by a factor of 2.     

Tetracyclines and host defence mechanisms. Doxycyclinepolymethaphosphate sodium complex (DMSC) compared to doxycycline with regard to influence on some host defence mechanisms.

A new doxycycline preparation, doxycycline polymethaphosphate sodium complex (DMSC), was compared to doxycycline in a cross-over study on six volunteers. The serum levels attained were of the same magnitude, but DMSC was shown to influence the serum bactericidal effect to a lesser degree. In vitro investigations of the influence on chemotaxis showed that DMSC even in high concentrations did not interfere with the spontaneous or induced migration to the same extent as doxycycline.     

Molecular effects of doxycycline treatment on pterygium as revealed by massive transcriptome sequencing

Pterygium is a lesion of the eye surface which involves cell proliferation, migration, angiogenesis, fibrosis, and extracellular matrix remodelling. Surgery is the only approved method to treat this disorder, but high recurrence rates are common. Recently, it has been shown in a mouse model that treatment with doxycycline resulted in reduction of the pterygium lesions. Here we study the mechanism(s) of action by which doxycycline achieves these results, using massive sequencing techniques. Surgically removed pterygia from 10 consecutive patients were set in short term culture and exposed to 0 (control), 50, 200, and 500 μg/ml doxycycline for 24 h, their mRNA was purified, reverse transcribed and sequenced through Illumina's massive sequencing protocols. Acquired data were subjected to quantile normalization and analyzed using cytoscape plugin software to explore the pathways involved. False discovery rate (FDR) methods were used to identify 332 genes which modified their expression in a dose-dependent manner upon exposure to doxycycline. The more represented cellular pathways included all mitochondrial genes, the endoplasmic reticulum stress response, integrins and extracellular matrix components, and growth factors. A high correlation was obtained when comparing ultrasequencing data with qRT-PCR and ELISA results. Doxycycline significantly modified the expression of important cellular pathways in pterygium cells, in a way which is consistent with the observed efficacy of this antibiotic to reduce pterygium lesions in a mouse model. Clinical trials are under way to demonstrate whether there is a benefit for human patients.     

Effect of rifampicin and doxycycline on the chemiluminescence of leukocytes]

The effect of rifampicin and doxycycline on spontaneous and zymosan-induced chemiluminescence of polymorphonuclear leukocytes was studied on guinea pigs. The cells were incubated in the presence of the antibiotics, washed and stimulated by zymosan. Under such conditions rifampicin in therapeutic doses of 0.1 to 10 micrograms/kg and doxycycline in a dose of 100 micrograms/kg potentiated the leukocyte chemiluminescence. Investigation of the antibiotics effect on the cells without washing failed because of the direct interference of rifampicin and doxycycline with the cell-independent stage of the chemiluminescent reaction.     

Combined action of doxycycline and mytilan on the immune response to tularemia antigen]

Combined action of doxycycline and mytilan, a natural polysaccharide, on the primary immune response to the antigen of the tularemia vaccinal strain in CBA mice was studied. The polysaccharide was used to compensate the immunosuppressive effect of doxycycline high doses on the humoral immune response. The maximum stimulation of the antibody titers as compared to the controls (more than 250 per cent) was observed when mytilan was administered simultaneously with or prophylactically 3 days prior to the antibiotic in doses of 2.5 and 25 mg/kg. The use of mytilan in combination with doxycycline high doses made it possible to compensate the antibiotic-induced decrease of DTH and even to stimulate it as compared to the controls. The highest levels of DTH (150 per cent against the control) were observed when mytilan was administered prophylactically in doses of 2.5 and 11.25 mg/kg 3 days prior to immunization. Mytilan had the highest stimulating effect on antibody production. The combined use of doxycycline and mytilan was characterized by significant stimulation of antibody production and DTH when the dose/time regimens were rational.     

Histidine-capped copper nanoclusters for in situ amplified fluorescence monitoring of doxycycline through inner filter effect

There is a significant need to accurately measure doxycycline concentrations in view of the adverse effects of an overdose on human health. A fluorescence (FL) detection method was adopted and copper nanoclusters (CuNCs) were synthesized using chemical reduction technology. Based on FL quenching with doxycycline, the prepared CuNCs were used to explore a fluorescent nanoprobe for doxycycline detection. In an optimal sensing environment, this FL nanosensor was sensitive and selective in doxycycline sensing and displayed a linear relationship in the range 0.5–200 μM with a detection limit of 0.092 μΜ. A characterization test demonstrated that CuNCs offered active functional groups for identifying doxycycline using electrostatic interaction and hydrogen bonds. Static quenching and the inner filter effect (IFE) resulted in weakness in the FL of His@CuNCs with doxycycline with great efficiency. This suggested nanosensor was revealed to be a functional model for simple and rapid detection of doxycycline in real samples with very pleasing accuracy.     

泛素连接酶LNX对上皮细胞间连接的影响

为阐明泛素连接酶LNX在维持上皮细胞之间连接的作用,构建了四环素诱导表达LNX的MDCK细胞株;以免疫荧光法观察细胞连接蛋白E-钙黏素和ZO-1在细胞内的分布,发现LNX的表达使E-钙黏素和ZO-1在细胞内的分布发生明显改变,大量E-钙黏素和ZO-1聚集在胞浆中,而在细胞膜上的分布则明显减少;透射电镜观察上皮细胞间连接的超微结构显示,LNX的表达导致紧密连接和黏附连接的正常结构消失;用钙离子转换实验检测黏附连接的形成发现,表达LNX的MDCK细胞间的黏附连接形成的速度明显滞后于正常细胞.上述结果表明,LNX的表达影响了E-钙黏素在细胞膜上的正常分布,从而延迟黏附连接复合体的形成,导致上皮细胞间连接结构的异常.     

Effect of doxycycline on immune response in a multifactorial experiment]

Multifactorial analysis was applied to studies on the effect of doxycycline on the primary immune response to sheep erythrocytes. The experimental factors were the following: the antibiotic dose, the antigen dose and the time of the onset of the antibiotic therapy with respect to the antigen action. Polynomial statistic models describing the delayed hypersensitivity and antibody titers within wide ranges of factor values were designed by the experimental data. It was shown that the prophylactic use of doxycycline prior to the antigenic stimulus markedly lowered the high-dose tolerance induced by high doses of the antigen.     

State of phagocytic immunity in white mice treated with doxycycline and pentoxyl for experimental staphylococcal sepsis]

When albino mice with experimental staphylococcal sepsis were treated by doxycycline for 10 days, it was noted that the antibiotic had an inhibitory effect on the absorbing capacity of the peritoneal leucocytes with respect to Staph aureus. The decreased digesting capacity of the leucocytes was observed only during the first 5 days from the moment of infection and treatment. The combined use of doxycycline and pentoxyl stimulated the activity and intensity of phagocytosis. The activity values of completed phagocytosis did not change, while the coefficient of the phagocytosis completeness increased as compared to the same values in the animal group treated with doxycycline alone.     

Effect of ethylenediaminetetraacetic acid disodium salt and doxycycline on drug resistant bacteria]

Effect of Na2EDTA and doxycycline applied alone and in combination in the treatment of experimentally induced dermatitis in rabbits with Staphylococcus aureus resistant to tetracyclines was studied. The rabbits were divided into three groups. The animals of group I were treated locally with the ointment containing 1% doxycycline or 1% Na2EDTA. In group II topical treatment was applied by means of injections of Na2EDTA solution in doses of 12.5 and 6.25 mg Na2EDTA per 1 kg/body weight. Group III was also treated locally with Na2EDTA solution like in group II, but additionally doxycycline in a dose of 50 micrograms per 1 kg/body weight was given i.v. Favourable therapeutic results were observed in the case of local, simultaneous application of Na2EDTA and doxycycline, or local application of Na2EDTA, and intravenous administration of doxycycline. However, the best therapeutic effect was seen in the case of local, simultaneous application of the ointment containing Na2EDTA and doxycycline.     

Comparative toxicity of oxytetracycline and its semisynthetic derivatives, methacycline and doxycycline

The acute toxicity parameters of oxytetracycline and its semisynthetic derivatives was determined on laboratory animals of various species. The three antibiotics were described as belonging to the group of low toxic compounds according to the classification adopted in industrial toxicology. However, the absolute values of LD50 indicated that doxycycline was 5.6 or 2.8 times more toxic than oxyteyracycline or methacycline respectively. In addition, doxycycline had the most pronounced hepatoxic effect.     

Doxycycline inhibits the cancer stem cell phenotype and epithelial-to-mesenchymal transition in breast cancer

Experimental evidence suggest that breast tumors originate from breast cancer stem cells (BCSCs), and that mitochondrial biogenesis is essential for the anchorage-independent clonal expansion and survival of CSCs, thus rendering mitochondria a significant target for novel treatment approaches. One of the recognized side effects of the FDA-approved drug, doxycycline is the inhibition of mitochondrial biogenesis. Here we investigate the mechanism by which doxycycline exerts its inhibitory effects on the properties of breast cancer cells and BCSCs, such as mammosphere forming efficiency, invasion, migration, apoptosis, the expression of stem cell markers and epithelial-to-mesenchymal transition (EMT) related markers of breast cancer cells. In addition, we explored whether autophagy plays a role in the inhibitory effect of doxycycline on breast cancer cells. We find that doxycyline can inhibit the viability and proliferation of breast cancer cells and BCSCs, decrease mammosphere forming efficiency, migration and invasion, and EMT of breast cancer cells. Expression of stem cell factors Oct4, Sox2, Nanog and CD44 were also significantly downregulated after doxycycline treatment. Moreover, doxycycline could down-regulate the expression of the autophagy marker LC-3BI and LC-3BII, suggesting that inhibiting autophagy may be responsible in part for the observed effects on proliferation, EMT and stem cell markers. The potent inhibition of EMT and cancer stem-like characteristics in breast cancer cells by doxycycline treatment suggests that this drug can be repurposed as an anti-cancer drug in the treatment of breast cancer patients in the clinic.     

Doxycycline improves cage activity,but not exercised,supraspinatus tendon and muscle in a rat model

The objective of this study was to investigate the effects of doxycycline, a broad-spectrum MMP inhibitor, on cage activity and exercised supraspinatus tendon and muscle using a Sprague-Dawley rat model of non-injurious exercise. Because exercise may alter muscle and tendon MMP activity and matrix turnover, we hypothesized that doxycycline would abolish the beneficial adaptations found with exercise but have no effect on cage activity muscle and tendon properties. Rats were divided into acute or chronic exercise (EX) or cage activity (CA) groups, and half of the rats received doxycycline orally. Animals in acute EX groups were euthanized 24 h after a single bout of exercise (10 m/min, 1 h) on a flat treadmill. Animals in chronic EX groups walked on a flat treadmill and were euthanized at 2 or 8 week time points. Assays included supraspinatus tendon mechanics and histology and muscle fiber morphologic and type analysis. Doxycycline improved tendon mechanical properties and collagen organization in chronic cage activity groups, which was not consistently evident in exercised groups. Combined with exercise, doxycycline decreased average muscle fiber cross-sectional area. Results of this study suggest that administration of doxycycline at pharmaceutical doses induces beneficial supraspinatus tendon adaptations without negatively affecting the muscle in cage activity animals, supporting the use of doxycycline to combat degenerative processes associated with underuse; however, when combined with exercise, doxycycline does not consistently produce the same beneficial adaptations in rat supraspinatus tendons and reduces muscle fiber cross-sectional area, suggesting that doxycycline is not advantageous when combined with activity.     

血小板生成素基因在NIH/3T3细胞中的表达与调控

应用 Tet- On基因表达系统 ,调控血小板生成素 (TPO)基因在 NIH/3T3细胞中的表达时间与水平 .籍脂质体介导的基因转移方法 ,p Tet- On质粒转染 NIH/3T3细胞株 ,得到稳定细胞株NIH/3T3- Tet- On.p TRE/TPO与 p TK- Hyg质粒共转染 NIH/3T3- Tet- On细胞株 ,得到双稳定细胞株 NIH/3T3- Tet- On- TPO.在培养基中加入或不加强力霉素 ,RT- PCR、Western印迹及 ELISA法检测培养上清 TPO表达 .结果表明 ,当培养基中不加强力霉素时 ,TPO无明显表达 (0 .1 μg/L) ;当培养基中加入 2 mg/L强力霉素时 ,TPO表达明显增高 (1 0 .8μg/L) .TPO表达水平与强力霉素浓度有关 ,随强力霉素浓度增高 ,TPO表达增加 .TPO表达水平还与强力霉素作用时间有关 ,加入强力霉素 6 h后 ,TPO表达明显增加 (1 .2μg/L) ,随培养时间延长 ,TPO表达增加 ,2 4 h达到峰值(1 0 .8μg/L) ,而且这种诱导作用是可逆的 .为进一步进行 TPO基因表达调控的体内研究奠定基础 ,有望为 TPO基因治疗提供一条可控的安全途径     

How to make tetracycline-regulated transgene expression go on and off

Tetracycline-regulated gene expression systems are widely used to allow temporal and quantitative control of transgene expression in cultured cells and transgenic animals. While working with the Tet-Off system, where tetracycline or the analogue doxycycline suppresses expression, we noted a considerable variability in induced transgene expression after removal of doxycycline. Variable expression of the transgene could not be explained by clonal variation since it was noted when working with clonal cell lines. Instead we found that doxycycline bound nonspecifically to cells and extracellular matrix and was slowly released after it had been removed from tissue culture media. The released doxycycline reached sufficiently high levels to completely suppress transgene expression. The effect was not dependent on cell type or the nature of the transgene. However, robust and rapid transgene expression could be induced if released doxycycline were removed by washing cells 3h after the initial removal of doxycycline. The use of different vector systems, harboring the tetracycline-regulatable components, yielded similar results. These results not only help explain why tetracycline-regulatable transgene expression systems sometimes are variable but also provide simple ways to substantially improve the efficiency, utility, and reliability of these widely used expression systems.