Alteration of Transcriptional Regulator Rob In Vivo: Enhancement of Promoter DNA Binding and Antibiotic Resistance in the Presence of Nucleobase Amino Acids.

The identification of proteins that bind selectively to nucleic acid sequences is an ongoing challenge. We previously synthesized nucleobase amino acids designed to replace proteinogenic amino acids; these were incorporated into proteins to bind specific nucleic acids predictably. An early example involved selective cell free binding of the hnRNP LL RRM1 domain to its i-motif DNA target via Watson-Crick-like H-bonding interactions. In this study, we employ the X-ray crystal structure of transcriptional regulator Rob bound to its micF promoter, which occurred without DNA distortion. Rob proteins modified in vivo with nucleobase amino acids at position 40 exhibited altered DNA promoter binding, as predicted on the basis of their Watson-Crick-like H-bonding interactions with promoter DNA A-box residue Gua-6. Rob protein expression ultimately controls phenotypic changes, including resistance to antibiotics. Although Rob proteins with nucleobase amino acids were expressed in Escherichia coli at levels estimated to be only a fraction of that of the wild-type Rob protein, those modified proteins that bound to the micF promoter more avidly than the wild type in vitro also produced greater resistance to macrolide antibiotics roxithromycin and clarithromycin in vivo, as well as the ß-lactam antibiotic ampicillin. Also demonstrated is the statistical significance of altered DNA binding and antibiotic resistance for key Rob analogues. These preliminary findings suggest the ultimate utility of nucleobase amino acids in altering and controlling preferred nucleic acid target sequences by proteins, for probing molecular interactions critical to protein function, and for enhancing phenotypic changes in vivo by regulatory protein analogues.

Assessment of Clofazimine and TB47 Combination Activity against Mycobacterium abscessus Using a Bioluminescent Approach.

Mycobacterium abscessus is intrinsically resistant to most antimicrobial agents. The emerging infections caused by M. abscessus and the lack of effective treatment call for rapid attention. Here, we intended to construct a selectable marker-free autoluminescent M. abscessus strain (designated UAlMab) as a real-time reporter strain to facilitate the discovery of effective drugs and regimens for treating M. abscessus The UAlMab strain was constructed using the dif/Xer recombinase system. In vitro and in vivo activities of several drugs, including clofazimine and TB47, a recently reported cytochrome bc1 inhibitor, were assessed using UAlMab. Furthermore, the efficacy of multiple drug combinations, including the clofazimine and TB47 combination, were tested against 20 clinical M. abscessus isolates. The UAlMab strain enabled us to evaluate drug efficacy both in vitro and in live BALB/c mice in a real-time, noninvasive fashion. Importantly, although TB47 showed marginal activity either alone or in combination with clarithromycin, amikacin, or roxithromycin, the drug markedly potentiated the activity of clofazimine, both in vitro and in vivo This study demonstrates that the use of the UAlMab strain can significantly facilitate rapid evaluation of new drugs and regimens. The clofazimine and TB47 combination is effective against M. abscessus, and dual/triple electron transport chain (ETC) targeting can be an effective therapeutic approach for treating mycobacterial infections.

Roxithromycin inhibits compound 48/80-induced pseudo-allergy via the MrgprX2 pathway both in vitro and in vivo.

Roxithromycin (ROX) is a macrolide antibiotic with a variety of immunological effects. Mast cells (MCs) play a key role in host defense, mediating hypersensitivity and pseudo-allergic reactions. Mas-related G protein-coupled receptor X2 (MrgprX2) is the main receptor related to pseudo-allergy. In this study, we investigated the anti-pseudo-allergy effect of ROX and its underlying mechanism. The effects of ROX on passive cutaneous anaphylaxis (PCA) and active systemic allergy were examined, degranulation, Ca2+ influx, and cytokine release were studied in vivo and in vitro. Interactions between ROX and MrgprX2 protein were also detected through surface plasmon resonance. The PCA and active systemic allergy induced by compound 48/80 were inhibited by ROX. An intermolecular interaction was detected between the ROX and MrgprX2 protein. In conclusion, ROX could inhibit pseudo-allergic reactions, and this effect involves the Ca2+/PLC/IP3 pathway of MrgprX2. This study provides new insight into the anti-pseudo-allergy effects of ROX.

Drug-drug interaction of acetaminophen and roxithromycin with the cocktail of cytochrome P450 and hepatotoxicity in rats.

Acetaminophen (APAP) and roxithromycin (ROX) are often used in combination in clinical practice. To evaluate their drug-drug interactions (DDIs) and the hepatotoxicity of co-administration, rats were randomly separated into four groups: Control, APAP (50 mg/kg), ROX (5.5 mg/kg) and APAP-ROX (50 mg/kg and 5.5 mg/kg, respectively). The pharmacokinetic parameters between APAP and ROX were assayed by HPLC, and a cocktail method was used to evaluate the activities of cytochrome (CYP) 450. The liver microsome CYP2E1 protein was detected using Western blot. The levels of plasma parameters, mRNA levels of inflammatory factors (TNF-α, INF-γ, VCAM-1, CXCL-1 and STAT-3) and antioxidant factors (Nrf-2, GSTA, GCLC-1, HO-1 and NQO1) were determined using real-time PCR, along with the observation on histopathological changes in the liver tissue. APAP and ROX co-treatment significantly increased CYP2E1 activity, decreased CYP2D6 activity and prolonged APAP and ROX clearance. Co-treatment increased mRNA expressions of TNF-α, NQO1 and MDA while decreasing GPX and SOD levels. Histopathological evidence showed the changes of liver tissues in terms of structure, size and tight arrangement. This study confirmed that a combination of APAP and ROX inhibited APAP metabolism and that the peak concentration of ROX was delayed. The resulting high level of CYP2E1 may induce oxidative stress and cause liver damage.

[The role of S100A8/RAGE and Caveolin-1 and the effect of roxithromycin on their expression in a rat model of neutrophilic asthma].

Objective: To explore the role of S100A8, the receptor for advanced glycation endproducts (RAGE) and Caveolin-1 in neutrophilic asthmatic rats, and to further study the intervention of roxithromycin and the possible mechanisms. Methods: Male Brown Norway rats were randomly assigned to a control group, an asthma group and a Roxithromycin group. The asthmatic rat model was established by intraperitoneal injection of ovalbumin (OVA) and Freund's complete adjuvant (FCA) mixture, and aerosol inhalation of OVA. Rats in the Roxithromycin group were given roxithromycin injection 30 mg/kg 30 minutes before each challenge. Rats in the control and the asthma groups were replaced with equal volumes of saline, respectively. Bronchoalveolar lavage fluid (BALF) neutrophil percentage (Neu%) and pathological changes of pulmonary tissue (hematoxylin-eosin, HE staining) were measured to confirm the establishment of asthmatic models. The concentration of inflammatory cytokines and S100A8 were quantified by enzyme-linked immunosorbent assay (ELISA), and the expression of Caveolin-1 and RAGE at protein levels were detected by immunohistochemistry and Western blot. Results: Neu% in BALF of the asthma group was significantly higher than those of the control group, and Neu% in the Roxithromycin group was lower than the asthma group (all P<0.01). Pulmonary histology revealed that there were a large number of inflammatory cells infiltrated in the bronchial and perivascular, pulmonary interstitial and alveolar spaces, and the bronchial wall and smooth muscles were thickened obviously in the asthma group. Rats in the Roxithromycin group showed milder inflammation and airway remodeling change than the asthma group. There was no obvious pathological damage in the control group. The concentration of IL-6 and IL-17 in BALF and serum of rats in the asthma group were significantly higher than those in the control group (P<0.01), and Roxithromycin inhibited the high expression of these cytokines (P<0.05). The expression of S100A8 and RAGE in the asthma group were significantly higher than those in the control group [(20.6±4.4) vs (7.1±2.0) ng/L; (885±118) vs (462±102) ng/L; (14.2±1.7) vs (7.6±1.8) ng/L; (774±166) vs (406±69) ng/L, all P<0.05], and Roxithromycin inhibited the high expression of these proteins [(14.3±3.7) vs (20.6±4.4) ng/L; (650±53) vs (885±118) ng/L; (10.4±1.2) vs (14.2±1.7) ng/L; (560±64) vs (728±72) ng/L] (all P<0.05). Meanwhile, the expression of Caveolin-1 in the asthma group was significantly lower than that in the control group (P<0.01), and Roxithromycin up-regulated its expression (P<0.01). Correlation analysis showed that there was a significantly positive correlation between the expression of S100A8 and RAGE (r=0.706, P<0.01), while there was a significantly negative correlation between the expression of S100A8 and Caveolin-1 (r=-0.775, P<0.01), and between the expression of Caveolin-1 and RAGE (r=-0.919, P<0.01). Conclusion: S100A8 and Caveolin-1 may play an important role in neutrophilic asthma via RAGE, and Roxithromycin may exerts anti-inflammatory effects and inhibition of airway remodeling partly through this signaling pathway.

The role of HDAC2 in cigarette smoke-induced airway inflammation in a murine model of asthma and the effect of intervention with roxithromycin.

BACKGROUND: Cigarette smoke is well known to worsen asthma symptoms in asthmatic patients and to make them refractory to treatment, but the underling molecular mechanism is unclear. We hypothesized that cigarette smoke can reduce the expression of HDAC2 in asthma and the process was achieved by activating the PI3K-δ/Akt signaling pathway. We further hypothesized that roxithromycin (RXM) can alleviate the impacts by cigarette smoke. METHODS: A murine model of asthma induced by ovalbumin (OVA) and cigarette smoke has been established. The infiltration of inflammatory cells and inflammatory factors was examined in this model. Finally, we evaluated the expression of HDAC2, Akt phosphorylation levels, and the effects of RXM treatment on the model described earlier. RESULTS: Cigarette smoke exposure reduced HDAC2 protein expression by enhancing the phosphorylation of Akt in PI3K-δ/Akt signaling pathway. Furthermore, RMX reduced the airway inflammation and improved the level of expression of HDAC2 in the cigarette smoke-exposed asthma mice. CONCLUSIONS: This study provides a novel insight into the mechanism of cigarette smoke exposure in asthma and the effects of RXM treatment on this condition. These results may be helpful for treating refractory asthma and emphasizing the need for a smoke-free environment for asthmatic patients.

Roxithromycin regulates intestinal microbiota and alters colonic epithelial gene expression.

The specialty of gastroenterology will be affected profoundly by the ability to modify the gastrointestinal microbiota through the use of antibiotics. This study investigated the in vivo effect of roxithromycin on gut bacteria and gene expression of colonic epithelial cells (CECs) using microbial 16S rDNA and colonic epithelial cell RNA sequencing, respectively. The results showed that roxithromycin distinctly lowered the microbial diversity in both the small intestine and cecum and altered the compositions of bacteria at both the phylum and genus levels, including the reduction of some bacteria beneficial to the hosts' health. Eight decreased and 8 increased genera in the small intestine and 17 decreased and 4 increased genera of bacteria in the cecum were most affected by roxithromycin consumption. This consumption further altered the CECs' expression of multiple genes. Thirty-one genes, which were significantly enriched in seven KEGG pathways and related to immune response, wound healing, and fibrosis, were significantly affected. Roxithromycin ingestion in healthy hosts, therefore, might lead to some undesirable consequences via affecting hosts' gut microbiota and CECs. Our work offers a more comprehensive understanding of the impact of consuming roxithromycin on human health.

Roxithromycin inhibits VEGF-induced human airway smooth muscle cell proliferation: Opportunities for the treatment of asthma.

Asthma is a chronic respiratory disease characterized by reversible airway obstruction with persistent airway inflammation and airway remodelling, which is associated with increased airway smooth muscle (ASM) mass. Roxithromycin (RXM) has been widely used in asthma treatment; however, its mechanism of action is poorly understood. Vascular endothelial growth factor (VEGF) has been implicated in inflammatory and airway blood vessel remodelling in patients with asthma, and shown to promote ASM cell proliferation. Here, we investigated the effect of RXM on VEGF-induced ASM cell proliferation and attempted to elucidate the underlying mechanisms of action. We tested the effect of RXM on proliferation and cell cycle progression, as well as on the expression of phospho-VEGF receptor 2 (VEGFR2), phospho-extracellular signal-regulated kinase 1/2 (ERK1/2), phospho-Akt, and caveolin-1 in VEGF-stimulated ASM cells. RXM inhibited VEGF-induced ASM cell proliferation and induced cell cycle arrest. Additionally, VEGF-induced ASM cell proliferation was suppressed by inhibiting the activity of ERK1/2, but not that of Akt. Furthermore, RXM treatment inhibits VEGF-induced activation of VEGFR2 and ERK and downregulation of caveolin-1 in a dose-dependent manner. RXM also inhibited TGF-ß-induced VEGF secretion by ASM cells and BEAS-2B cells. Collectively, our findings suggest that RXM inhibits VEGF-induced ASM cell proliferation by suppression of VEGFR2 and ERK1/2 activation and caveolin-1 down-regulation, which may be involved in airway remodelling. Further elucidation of the mechanisms underlying these observations should enable the development of treatments for smooth muscle hyperplasia-associated diseases of the airway such as asthma.

In vitro cytogenetic evaluation of the particular combination of flurbiprofen and roxithromycin.

Flurbiprofen (FLB) (anti-inflammatory and analgesic drug) and roxithromycin (RXM) (antibiotic) were widely used in world wide. This study deals with investigation of genotoxicity, cytotoxicity, and oxidative stress effects of a particular combination of these drugs in human cultured lymphocytes. Also, DNA damaging-protective effects of combination of these drugs were analyzed on plasmid DNA. Human lymphocytes were treated with different concentrations (FLB + RXM; 10 µg/mL + 25 µg/mL, 15 µg/mL + 50 µg/mL, and 20 µg/mL + 100 µg/mL) of the drugs following by study of their genotoxic and cytotoxic effects by analysis of cytokinesis-block micronucleus test and nuclear division index, respectively. The effect of the combination in aspect of anti-oxidative and DNA damaging activity was evaluated on Pet-22b plasmid. According to our results, the combination of FLB and RXM did not show a notable genotoxic effect on cells. Although each of the substances had been shown as a cytotoxic agent by previous researchers, in this research, the combination of these drugs did not exhibit any adverse effect on cell division. FLB had DNA protection effect against H2O2 while in combination with RXM had not the same effect on the plasmid.

Roxithromycin potency quantification in pharmaceutical preparation by applying a validated bioassay method and comparison with HPLC analysis.

Roxithromycin (RXM) is used to treat bacterial infections. An alternative bioassay for the assessment of the potency of this drug in pharmaceutical formulations has not been reported earlier. This study reports the development and validation of cost-effective, simple, sensitive, precise, accurate and reproducible one-level agar diffusion (5+1) bioassay for estimation of potency and bioactivity of RXM in tablet. Among six tested microbial strains, Streptococcus pneumoniae (MTCC-1935) was used as the most susceptible strain against RXM. Bioassay was optimized by investigating buffer pH, inoculums and reference standard concentration. The results of proposed bioassay displayed high linearity, precision, accuracy, robustness and specificity. All potency results were statistically analyzed and found to be linear (R(2)=0.9957) in between 2.0 and 6.0µg/mL, precise with relative standard deviation (RSD) of repeatability intra-assay below 1.5%, and intermediate precision between day RSD 0.39% and accurate (100.68%). The bioassay and previously validated HPLC methods were compared, which indicated that there was no significant difference between these two methods. The results demonstrated the validity of the proposed microbial assay, which allows reliable quantitation of RXM in pharmaceutical samples and therefore can be used as a substitute alternative methodology for the routine quality control of this medicine, in situation when HPLC is not affordable in the laboratory.

Roxithromycin treatment inhibits TGF-ß1-induced activation of ERK and AKT and down-regulation of caveolin-1 in rat airway smooth muscle cells.

BACKGROUND: Roxithromycin (RXM) has been widely used in asthma treatment; however, the mechanism has not been fully understood. The aim of our study was to investigate the underlying mechanism of RXM treatment in mediating the effect of transforming growth factor (TGF)-ß1 on airway smooth muscle cells (ASMCs) proliferation and caveolinn-1 expression. METHODS: Firstly, the rat ovalbumin (OVA) model was built according to the previous papers. Rat ASMCs were prepared and cultured, and then TGF-ß1 production in ASMCs was measured by enzyme-linked immunosorbent assay (ELISA). Moreover, the proliferation of ASMCs was determined using cell counting kit (CCK-8) assay. Additionally, the expressions of caveolin-1, phosphorylated-ERK1/2 (p-ERK1/2) and phosphorylated-AKT (p-AKT) in ASMCs treated with or without PD98059 (an ERK1/2 inhibitor), wortannin (a PI3K inhibitor), ß-cyclodextrin (ß-CD) and RXM were measured by Western blot. Finally, data were evaluated using t-test or one-way ANOVA, and then a P value < 0.05 was set as a threshold. RESULTS: Compared with normal control, TGF-ß1 secretion was significantly increased in asthmatic ASMCs; meanwhile, TGF-ß1 promoted ASMCs proliferation (P < 0.05). However, ASMCs proliferation was remarkably inhibited by RXM, ß-CD, PD98059 and wortmannin (P < 0.05). Moreover, the expressions of p-ERK1/2 and p-AKT were increased and peaked at 20 min after TGF-ß1 stimulation, and then suppressed by RXM. Further, caveolin-1 level was down-regulated by TGF-ß1 and up-regulated by inhibitors and RXM. CONCLUSION: Our findings demonstrate that RXM treatment inhibits TGF-ß1-induced activation of ERK and AKT and down-regulation of caveolin-1, which may be the potential mechanism of RXM protection from chronic inflammatory diseases, including bronchial asthma.

Effect of low-dose, long-term roxithromycin on airway inflammation and remodeling of stable noncystic fibrosis bronchiectasis.

BACKGROUND: Noncystic fibrosis bronchiectasis (NCFB) is characterized by airway expansion and recurrent acute exacerbations. Macrolide has been shown to exhibit anti-inflammatory effects in some chronic airway diseases. OBJECTIVE: To assess the efficacy of roxithromycin on airway inflammation and remodeling in patients with NCFB under steady state. METHODS: The study involved an open-label design in 52 eligible Chinese patients with NCFB, who were assigned to control (receiving no treatment) and roxithromycin (receiving 150 mg/day for 6 months) groups. At baseline and 6 months, the inflammatory markers such as interleukin- (IL-)8, neutrophil elastase (NE), matrix metalloproteinase- (MMP)9, hyaluronidase (HA), and type IV collagen in sputum were measured, along with the detection of dilated bronchus by throat computed tomography scan, and assessed the exacerbation. RESULTS: Forty-three patients completed the study. The neutrophil in the sputum was decreased in roxithromycin group compared with control (P < 0.05). IL-8, NE, MMP-9, HA, and type IV collagen in sputum were also decreased in roxithromycin group compared with the control group (all P < 0.01). Airway thickness of dilated bronchus and exacerbation were reduced in roxithromycin group compared with the control (all P < 0.05). CONCLUSIONS: Roxithromycin can reduce airway inflammation and airway thickness of dilated bronchus in patients with NCFB.

Roxithromycin and rhEGF Co-loaded Reactive Oxygen Species Responsive Nanoparticles for Accelerating Wound Healing.

BACKGROUND: Bacterial infection can delay wound healing and is therefore a major threat to public health. Although various strategies have been developed to treat bacterial infections, antibiotics remain the best option to combat infections. The inclusion of growth factors in the treatment approach can also accelerate wound healing. The co-delivery of antibiotics and growth factors for the combined treatment of wounds needs further investigation. OBJECTIVE: Here we aimed to develop antibiotic and growth factor co-loaded nanoparticles (NPs) to treat Staphylococcus aureus-infected wounds. METHODS: By using our previously prepared reactive oxygen species-responsive material (Oxi-αCD), roxithromycin (ROX)-loaded NPs (ROX/Oxi-αCD NPs) and recombinant human epidermal growth factor (rhEGF)/ROX co-loaded NPs (rhEGF/ROX/Oxi-αCD NPs) were successfully fabricated. The in vivo efficacy of this prepared nanomedicine was evaluated in mice with S. aureus-infected wounds. RESULTS: ROX/Oxi-αCD NPs and rhEGF/ROX/Oxi-αCD NPs had a spherical structure and their particle sizes were 164 ± 5 nm and 190 ± 8 nm, respectively. The in vitro antibacterial experiments showed that ROX/Oxi-αCD NPs had a lower minimum inhibitory concentration than ROX. The in vivo animal experiments demonstrated that rhEGF/ROX/Oxi-αCD NPs could significantly accelerate the healing of S. aureus-infected wounds as compared to the free ROX drug and ROX/Oxi-αCD NPs (P < 0.05). CONCLUSION: ROX and rhEGF co-loaded NPs can effectively eliminate bacteria in wounds and accelerate wound healing. Our present work could provide a new strategy to combat bacteria-infected wounds.

The effect of extracellular polymeric substances on the distribution and transmission of antibiotic resistance genes treating antibiotic wastewater via microbial electrolysis cells.

Microbial electrolysis cells (MEC) have emerged as a prominent technology for the treatment of antibiotics-containing wastewater in recent years. However, there remains a dearth of comprehensive exploration regarding the influence of extracellular polymers substances (EPS) on the distribution and transmission of antibiotic resistance genes (ARGs) in MEC. In this study, we quantified the distribution of ARGs in MEC by Fluorescence quantitative polymerase chain reaction and explored with emphasis on impact of EPS component on ARGs transmission at under different concentrations of roxithromycin. Results showed that the absolute abundance of ARGs in the electrode biofilm was 1-2 orders of magnitude higher than that in the anolyte. Specifically, EPS-associated ARGs accounted for 2.31%-11.18% of ARGs in electrode biofilm. The presence of elevated roxithromycin concentration led to electroactive microorganisms (Geobacter and Geothrix) as potential hosts of ARGs. In addition, both protein and polysaccharide content in the electrode biofilm increased with increasing roxithromycin concentration and showed positive correlations with EPS-associated ARGs. Fluorescence quenching experiments further elucidated that tryptophan and tyrosine residues in EPS could bind to ARGs effectively, contributing the hindering the ARGs transmission between hosts. Therefore, increased EPS content within electrode biofilm could reduce the concentration of ARGs present in anolyte while also influencing ARGs distribution throughout MEC. This study provides valuable insights into the distribution of ARGs in MEC systems and the role of EPS in regulating ARGs migration.

Antibacterial and Osteogenic Dual-Functional Micronano Composite Scaffold Fabricated via Melt Electrowriting and Solution Electrospinning for Bone Tissue Engineering.

The utilization of micronano composite scaffolds has been extensively demonstrated to confer the superior advantages in bone repair compared to single nano- or micron-sized scaffolds. Nevertheless, the enhancement of bioactivities within these composite scaffolds remains challenging. In this study, we propose a novel approach to combine melt electrowriting (MEW) and solution electrospinning (SES) techniques for the fabrication of a composite scaffold incorporating hydroxyapatite (HAP), an osteogenic component, and roxithromycin (ROX), an antibacterial active component. Scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy (FTIR) confirmed the hierarchical architecture of the nanofiber-microgrid within the scaffold, as well as the successful loading of HAP and ROX. The incorporation of HAP enhanced the water absorption capacity of the composite scaffold, thus promoting cell adhesion and proliferation, as well as osteogenic differentiation. Furthermore, ROX resulted in effective antibacterial capability without any observable cytotoxicity. Finally, the scaffolds were applied to a rat calvarial defect model, and the results demonstrated that the 20% HAP group exhibited superior new bone formation without causing adverse reactions. Therefore, our findings present a promising strategy for designing and fabricating bioactive scaffolds for bone regeneration.

Blockage of hERG current and the disruption of trafficking as induced by roxithromycin.

Roxithromycin is an oral macrolide antibiotic agent that has been repeatedly reported to provoke excessive prolongation of the Q-T interval and torsades de pointes in clinical settings. To investigate the mechanisms underlying the arrhythmogenic side effects of roxithromycin, we studied the molecular mechanisms of roxithromycin on human ether-à-go-go-related gene (hERG) K(+) channels expressed in human embryonic kidney (HEK293) cells. Roxithromycin was found to inhibit wild-type (WT) hERG currents in a concentration-dependent manner with a half-maximum block concentration (IC50) of 55.8 ± 9.1 µmol/L. S6 residue hERG mutants (Y652A and F656C) showed reduced levels of hERG current blockage attributable to roxithromycin. Roxithromycin also inhibited the trafficking of hERG protein to the cell membrane, as confirmed by Western blot analysis and confocal microscopy. These findings indicate that roxithromycin may cause acquired long-QT syndrome via direct inhibition of hERG current and by disruption of hERG protein trafficking. Mutations in drug-binding sites (Y652A or F656C) of the hERG channel were found to attenuate hERG current blockage by roxithromycin, but did not significantly alter the disruption of trafficking.

A eukaryotic molecular target candidate of roxithromycin: fungal differentiation as a sensitive drug target analysis system.

Roxithromycin (RXM), active against prokaryotes, has beneficial side effects such as anti-cancer activities on mammalian cells, but the mechanisms underlying these effects remain unclear. We found that RXM inhibited the cellular differentiation of the rice blast fungus Magnaporthe oryzae. Hence, we screened the targets of RXM by the T7 phage display method with fungal genomic DNA, and identified MoCDC27 (M. oryzae Cell Division Cycle 27) as a candidate. We generated mocdc27 knockdown mutants that the appressoria formation was less affected by RXM. A complemented mutant restored sensitivity against RXM to the level of the wild type. These results suggest that MoCDC27 was involved in the inhibition of appressorium formation by RXM, and that the complex of RXM-MoCDC27 affected another molecule involved in appressorium formation. The T7 phage display method with fungal genomic DNA can be a useful tool in the quest for drug target.

[Functional role of caveolin-1 in airway smooth muscle cells proliferation and regulatory effect of roxithromycin].

OBJECTIVE: To explore the functional role of caveolin-1 in airway smooth muscle cells (ASMCs) proliferation and examine the regulatory effect of roxithromycin. METHODS: The rat model of bronchial asthma was established. Electron microscope was employed to observe the status of caveolae and light microscope for the histological changes in pulmonary tissues. The primarily cultured ASMCs were divided into 5 groups: control (group A), asthmatic ASMCs (group B), PD98059 (group C), roxithromycin (group D) and methyl-ß-cyclodextrin (group E). Cell proliferation was detected by Cell Counting Kit-8 (CCK-8). And the expressions of caveolin-1, extracellular regulated protein kinases (ERK) and monocyte chemotactic protein (MCP)-1 were detected by Western blot and reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The cell proliferation of asthmatic ASMCs (0.68 ± 0.15, 0.63 ± 0.13) in groups C and D were significantly less than those in group B (0.96 ± 0.14) (both P < 0.05) while group E was more than group B (1.26 ± 0.11 vs 0.96 ± 0.14, P < 0.05). The content of caveolin-1 (0.392 ± 0.064, 0.332 ± 0.057) in groups C and D were higher than those in group B (0.237 ± 0.032) (both P < 0.05) while ERK1/2 protein level in groups C and D (0.241 ± 0.017, 0.268 ± 0.007) were less than those in group B (0.346 ± 0.009) (both P < 0.01). And MCP-1 protein level in groups C and D (0.198 ± 0.015, 0.286 ± 0.019) were less than those in group B (0.482 ± 0.026) (both P < 0.01). The ERK mRNA level in groups C and D (0.277 ± 0.043, 0.338 ± 0.026) were less than those in group B (0.591 ± 0.022) (both P < 0.01). And also MCP-1 mRNA in groups C and D (0.212 ± 0.042, 0.249 ± 0.032) were less than those in group B (0.676 ± 0.053) (all P < 0.01) CONCLUSIONS: Caveolin-1 preventing the proliferation of asthmatic ASMCs is most likely mediated by ERK1/2 signal pathway and a down-regulation of MCP-1 expression. And roxithromycin reduces the proliferation of asthmatic ASMCs through up-regulating the expression of caveolin-1 and inhibiting the expression of MCP-1.

Novel mechanisms of macrolide resistance revealed by in vitro selection and genome analysis in Mycoplasma pneumoniae.

Mycoplasma pneumoniae is an important pathogen causing upper and lower respiratory tract infections in children and other age groups. Macrolides are the recommended treatments of choice for M. pneumoniae infections. However, macrolide resistance in M. pneumoniae is increasing worldwide, which complicates the treatment strategies. The mechanisms of macrolide resistance have been extensively studied focusing on the mutations in 23S rRNA and ribosomal proteins. Since the secondary treatment choice for pediatric patients is very limited, we decided to look for potential new treatment strategies in macrolide drugs and investigate possible new mechanisms of resistance. We performed an in vitro selection of mutants resistant to five macrolides (erythromycin, roxithromycin, azithromycin, josamycin, and midecamycin) by inducing the parent M. pneumoniae strain M129 with increasing concentrations of the drugs. The evolving cultures in every passage were tested for their antimicrobial susceptibilities to eight drugs and mutations known to be associated with macrolide resistance by PCR and sequencing. The final selected mutants were also analyzed by whole-genome sequencing. Results showed that roxithromycin is the drug that most easily induces resistance (at 0.25 mg/L, with two passages, 23 days), while with midecamycin it is most difficult (at 5.12 mg/L, with seven passages, 87 days). Point mutations C2617A/T, A2063G, or A2064C in domain V of 23S rRNA were detected in mutants resistant to the 14- and 15-membered macrolides, while A2067G/C was selected for the 16-membered macrolides. Single amino acid changes (G72R, G72V) in ribosomal protein L4 emerged during the induction by midecamycin. Genome sequencing identified sequence variations in dnaK, rpoC, glpK, MPN449, and in one of the hsdS (MPN365) genes in the mutants. Mutants induced by the 14- or 15-membered macrolides were resistant to all macrolides, while those induced by the 16-membered macrolides (midecamycin and josamycin) remained susceptible to the 14- and 15-membered macrolides. In summary, these data demonstrated that midecamycin is less potent in inducing resistance than other macrolides, and the induced resistance is restrained to the 16-membered macrolides, suggesting a potential benefit of using midecamycin as a first treatment choice if the strain is susceptible.