Antitoxin MqsA decreases antibiotic susceptibility through the global regulator AgtR in Pseudomonas fluorescens.

Type II toxin-antitoxin systems are highly prevalent in bacterial genomes and play crucial roles in the general stress response. Previously, we demonstrated that the type II antitoxin PfMqsA regulates biofilm formation through the global regulator AgtR in Pseudomonas fluorescens. Here, we found that both the C-terminal DNA-binding domain of PfMqsA and AgtR are involved in bacterial antibiotic susceptibility. Electrophoretic mobility shift assay (EMSA) analyses revealed that AgtR, rather than PfMqsA, binds to the intergenic region of emhABC-emhR, in which emhABC encodes an resistance-nodulation-cell division efflux pump and emhR encodes a repressor. Through quantitative real-time reverse-transcription PCR and EMSA analysis, we showed that AgtR directly activates the expression of the emhR by binding to the DNA motif [5´-CTAAGAAATATACTTAC-3´], leading to repression of the emhABC. Furthermore, we demonstrated that PfMqsA modulates the expression of EmhABC and EmhR. These findings enhance our understanding of the mechanism by which antitoxin PfMqsA contributes to antibiotic susceptibility.

Proteomic analysis reveals the mechanism of different environmental stress-induced tolerance of Pseudomonas aeruginosa to monochloramine disinfection.

Although drinking water disinfection proved to be an effective strategy to eliminate many pathogens, bacteria can still show disinfection tolerance in drinking water distribution systems. To date, the molecular mechanisms on how environmental stress affects the tolerance of Pseudomonas aeruginosa to monochloramine are not well understood. Here, we investigated how three stress conditions, namely starvation, low temperature, and starvation combined with low temperature, affected the monochloramine tolerance of Pseudomonas aeruginosa, an opportunistic pathogen commonly found in drinking water distribution systems. All stress conditions significantly promoted monochloramine tolerance, among which starvation had the most drastic effects. Proteomic analyses suggested that the three conditions not only triggered a positive antioxidant defense against oxidative damages but also prepared the bacteria to employ a passive defense mechanism against disinfectants via dormancy. Moreover, the expression of antioxidant enzymes reached the maximum under the starvation condition and further low temperature treatment had little effect on bacterial response to oxidative stress. Instead, we found further treatment of the starved cells with low temperature decreased the osmotic stress response and the stringent response, which generally play pivotal roles in disinfection tolerance. Taken together, these findings shed light on how abiotic factors influence the bacterial disinfection tolerance and will aid design of efficient strategies to eliminate Pseudomonas aeruginosa from drinking water.

The Regulator PltZ Regulates a Putative ABC Transporter System PltIJKNOP of Pseudomonas aeruginosa ATCC 27853 in Response to the Antimicrobial 2,4-Diacetylphloroglucinol.

Pseudomonas aeruginosa is an opportunistic pathogen commonly infecting immunocompromised patients with diseases like cystic fibrosis (CF) and cancers and has high rates of recurrence and mortality. The treatment efficacy can be significantly worsened by the multidrug resistance (MDR) of P. aeruginosa, and there is increasing evidence showing that it is easy for this pathogen to develop MDR. Here, we identified a gene cluster, pltZ-pltIJKNOP, which was originally assumed to be involved in the biosynthesis of an antimicrobial pyoluteorin, significantly contributing to the antibiotic resistance of P. aeruginosa ATCC 27853. Moreover, the TetR family regulator PltZ binds to a semi-palindromic sequence in the promoter region of the pltIJKNOP operon and recognizes the antimicrobial 2,4-diacetylphloroglucinol (2,4-DAPG), which in turn induces the expression of the pltIJKNOP operon. Using quantitative proteomics method, it was indicated that the regulator PltZ also plays an important role in maintaining metabolic hemostasis by regulating the transporting systems of amino acids, glucose, metal ions, and bacteriocins.

The antitoxin MqsA homologue in Pseudomonas fluorescens 2P24 has a rewired regulatory circuit through evolution.

The mqsRA operon encodes a toxin-antitoxin pair that was characterized to participate in biofilm and persister cell formation in Escherichia coli. Notably, the antitoxin MqsA possesses a C-terminal DNA-binding domain that recognizes the [5'-AACCT(N)2-4 AGGTT-3'] motif and acts as a transcriptional regulator controlling multiple genes including the general stress response regulator RpoS. However, it is unknown how the transcriptional circuits of MqsA homologues have changed in bacteria over evolutionary time. Here, we found mqsA in Pseudomonas fluorescens (PfmqsA) is acquired through horizontal gene transfer and binds to a slightly different motif [5'-TACCCT(N)3 AGGGTA-3'], which exists upstream of the PfmqsRA operon. Interestingly, an adjacent GntR-type transcriptional regulator, which was termed AgtR, is under negative control of PfMqsA. It was further demonstrated that PfMqsA reduces production of biofilm components through AgtR, which directly regulates the pga and fap operons involved in the synthesis of extracellular polymeric substances. Moreover, through quantitative proteomics analysis, we showed AgtR is a highly pleiotropic regulator that influences up to 252 genes related to diverse processes including chemotaxis, oxidative phosphorylation and carbon and nitrogen metabolism. Taken together, our findings suggest the rewired regulatory circuit of PfMqsA influences diverse physiological aspects of P. fluorescens 2P24 via the newly characterized AgtR.

Two cinnamoyloctopamine antioxidants from garlic skin attenuates oxidative stress and liver pathology in rats with non-alcoholic steatohepatitis.

Hepatic oxidative stress plays a key role in the development of non-alcoholic steatohepatitis (NASH), therefore, treatment approaches that address the antioxidant is helpful in the therapy of patients with NASH. N-trans-coumaroyloctopamine (1) and N-trans-feruloyloctopamine (2) were identified as the primary antioxidant constituents of garlic skin with high antioxidant activities. The aim of this study was to elucidate the protective effect and mechanism of the antioxidants on NASH in rats. The results provide morphological and molecular biological evidences for the protective role of the antioxidant 2 in ameliorating oxidative stress and hepatic apoptosis in experimental NASH for the first time. Mechanism study indicated that the antioxidant 2 significantly reduced the expression of COX-2 mRNA and protein by western blot, RT-PCR and immunohistochemical techniques.

A new norsesquiterpenoid from Ligularia virgaurea.

A new eremophilane norsesquiterpenoid (1), together with a known eremophilane sesquiterpenoid (2), was isolated from the leaves of Ligularia virgaurea. The structure of 1 was elucidated by a combination of spectroscopic analysis (IR, 1D NMR, 2D NMR, and HR-ESI-MS), and its absolute configuration was determined by a single-crystal X-ray diffraction experiment (with copper radiation). The known compound 2 was identified by comparison of its physical and spectral data with those reported in the literature. Compound 1 was assayed for its cytotoxic activities against human cervical carcinoma cell (HeLa) and human small cell lung cancer cell (NCI-446) lines.

Synergistic effects of Akt1 shRNA and paclitaxel-incorporated conjugated linoleic acid-coupled poloxamer thermosensitive hydrogel on breast cancer.

The phosphoinositide 3-kinase/Akt1 signaling pathway has emerged as a target for cancer therapy. In this study, we aimed to develop a strategy to enhance Akt-targeted cancer therapy. We hypothesized that combination of Akt1-targeted therapy with conventional chemotherapy using paclitaxel-incorporated conjugated linoleic acid-coupled poloxamer thermosensitive hydrogel may have synergistic effects in cancer therapeutic efficiency compared with chemotherapy alone. In this study, we found that the combination of shAkt1 with paclitaxel exerted synergistic anti-cancer effects, thus, inhibiting the growth of human breast cancer cells, and breast cancer xenografts in mice as well. The combination therapy demonstrated enhanced anti-cancer effects through inhibiting Akt1 signaling and inducing apoptosis. Our results suggest that the presented strategy of combination of shAkt1 with paclitaxel may have a potential for treatment of breast cancer.

Enhanced anti-cancer effect of 5-fluorouracil loaded into thermo-responsive conjugated linoleic acid-incorporated poloxamer hydrogel on metastatic colon cancer models.

Conjugated linoleic acid-coupled Pluronic F127 (Plu-CLA) is an effective drug delivery system with numerous advantages and anti-cancer activity. 5-FU administered in Plu-CLA hydrogel (P-FU) led to the significant enhancement of tumor growth suppression and cellular apoptosis. Moreover, growth of hepatic and intraperitoneal metastases in vivo was significantly reduced in mice treated with P-FU. Therefore, Plu-CLA could be a potential intraperitoneal carrier for hydrophilic 5-FU for the effective treatment of metastatic colon cancer.

[Study on the content of imperatorin and HPLC fingerprint of Baizhi collected from different areas].

OBJECTIVE: To compare the content of imperatorin and HPLC fingerprint of Baizhi collected from different areas. METHODS: The medicinal materials and seeds of Baizhi from 15 main habitats of 7 provinces and cities were collected, and the seeds were planted in the germplasm resource garden of Suining, Sichuan. The content of imperatorin and HPLC fingerprint were compared using the samples collected from different areas and germplasm resource garden collected and dried at the most suitable harvest date. RESULTS: The differences on the content of imperatorin and HPLC fingerprint were obvious. The content of imperatorin significantly increased and the HPLC fingerprint were trending to coincide when the herbs were planted in the same place. CONCLUSION: The environmental factors and the cultivation technique had great influences on the quality of Baizhi.

Alpha-eleostearic acid suppresses proliferation of MCF-7 breast cancer cells via activation of PPARgamma and inhibition of ERK 1 / 2.

Alpha-eleostearic acid (alpha-ESA) is known to suppress the growth in cancer cells although its underlying molecular mechanisms have not been fully elucidated. The present study was designed to elucidate and evaluate the anticancer mechanism of alpha-ESA on MCF-7 breast cancer cells. Also, an attempt was made to better understand the anticancer mechanism by which alpha-ESA activated PPARgamma and attenuated the ERK1/2 MAPK phosphorylation state. The MCF-7 breast cancer cell-line and nontumorigenic MCF-10A human mammary epithelial cells were treated with alpha-ESA and compared with negative control (without treatment) and positive control groups (treated with rosiglitazone), and changes of apoptosis-related molecules, PPARgamma and pERK1/2 were examined. In MCF-7 cells treated with alpha-ESA, we found that the expression of p53, p21, and Bax was up-regulated whereas expression of Bcl-2 and procaspase-9 was down-regulated. Moreover, nuclear translocation of PPARgamma by alpha-ESA positively correlated with inhibition of ERK1/2 activation. Our data suggest that alpha-ESA can be considered to be a PPARgamma agonist and thus a candidate for a chemotherapeutic agent against breast cancer.

Synergistic anti-tumor activity of paclitaxel-incorporated conjugated linoleic acid-coupled poloxamer thermosensitive hydrogel in vitro and in vivo.

Local delivery of anti-tumor drugs provides a high local concentration and decreases the incidence of side effects commonly observed with systemic therapy. Hydrogel systems are commonly used as a local drug delivery system. In this study, we prepared a novel thermosensitive conjugated linoleic acid (CLA)-coupled poloxamer hydrogel for local delivery of paclitaxel (PTX) to gain the benefits of the pro-drug activity of the CLA-coupled poloxamer and enhanced PTX solubility due to the micellar property of the CLA-coupled poloxamer. To evaluate the anti-tumor activity of the PTX-incorporated CLA-coupled poloxamer hydrogel in vivo, formulations were injected subcutaneously into tumor-bearing mice. Cell cycle and apoptosis markers were examined to determine the mechanism of the anti-tumor activity of PTX. The PTX-incorporated CLA-coupled poloxamer thermosensitive hydrogel showed excellent anti-tumor activity in vivo inducing stronger cell cycle arrest and apoptosis in tumor tissue than the PTX-incorporated poloxamer hydrogel. These results were attributed to the synergistic effect of the anti-tumor property of PTX with released CLA from the CLA-coupled poloxamer as the pro-drug and the enhanced solubility of PTX resulting from the micellar property of the CLA-coupled poloxamer. The CLA-coupled poloxamer designed in this study has great potential as an effective injectable carrier of PTX.

Chitosan microspheres containing Bordetella bronchiseptica antigens as novel vaccine against atrophic rhinitis in pigs.

The immune-stimulating activities of Bordetella bronchiseptica antigens containing dermonecrotoxin (BBD) loaded in chitosan microspheres (CMs) have already been reported in vitro and in vivo with a mouse alveolar macrophage cell line (RAW264.7) and mice. Therefore, this study attempted to demonstrate the successful induction of mucosal immune responses after the intranasal administration of BBD loaded in CMs (BBD-CMs) in colostrum-deprived pigs. The BBD was introduced to the CMs using an ionic gelation process involving tripolyphosphate (TPP). Colostrum-deprived pigs were then directly immunized through intranasal administration of the BBD-CMs. A challenge with a field isolate of B. bronchiseptica was performed ten days following the final immunization. The BBD-specific IgG and IgA titers, evident in the nasal wash and serum from the vaccinated pigs, increased with time (p<0.05). Following the challenge, the clinical signs of infection were about 6-fold lower in the vaccinated pigs compared with the nonvaccinated pigs. The grades for gross morphological changes in the turbinate bones from the vaccinated pigs were also significantly lower than the grades recorded for the nonvaccinated pigs (p<0.001). Therefore, the mucosal and systemic immune responses induced in the current study would seem to indicate that the intranasal administration of BBD-CMs may be an effective vaccine against atrophic rhinitis in pigs.

PEGylated conjugated linoleic acid stimulation of apoptosis via a p53-mediated signaling pathway in MCF-7 breast cancer cells.

The objective of this study was to investigate whether PEGylated conjugated linoleic acid (PCLA), as compared with conjugated linoleic acid (CLA) alone, displays anti-cancer properties in MCF-7 breast cancer cells. To generate PCLA, CLA was simply coupled to poly(ethylene glycol) (PEG) at the melting state of PEG without a solvent or a catalyst. The coupling reaction generated an ester linkage between the carboxyl group of CLA and hydroxyl one of PEG. The half-life of the generated PCLA was 52h at pH 7.4 at 37 degrees C, indicating that PCLA potentially acts as a pro-drug. Apoptosis of MCF-7 breast cancer cells treated with PCLA showed a dose response to PCLA concentration during treatment. In addition, pro-apoptotic proteins such as Bax were up-regulated, whereas anti-apoptotic proteins, such as Bcl-2, were down-regulated by treatment with both CLA and PCLA. The tumor suppressor gene p53 was significantly up-regulated by treatment with increasing concentrations of PCLA, suggesting that PCLA-induced apoptosis is regulated by a p53-mediated signaling pathway. Overall, the anti-cancer effects of PCLA on MCF-7 breast cancer cells may have therapeutic significance.

pH-sensitive and mucoadhesive thiolated Eudragit-coated chitosan microspheres.

The aim of this study was using Eudragit-cysteine conjugate to coat on chitosan microspheres (CMs) for developing an oral protein drug delivery system, having mucoadhesive and pH-sensitive property. Bovine serum albumin (BSA) as a protein model drug was loaded in thiolated Eudragit-coated CMs (TECMs) to study the release character of the delivery system. After thiolated Eudragit coating, it was found that the release rate of BSA from BSA-loaded TECMs was observably suppressed at pH 2.0 PBS solution, while at pH 7.4 PBS solution the BSA can be sustainingly released for several hours. The structural integrity of BSA released from BSA-loaded TECMs was guaranteed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and circular dichroism (CD) spectroscopy. The mucoadhesive property of TECMs was evaluated and compared with CMs and Eudragit-coated chitosan microspheres (ECMs). It was confirmed that after coating thiolated Eudragit, the percentage of TECMs remained on the isolated porcine intestinal mucosa surface was significantly higher than those of CMs and ECMs. Likewise, gamma camera imaging of Tc-99m labeled microsphere distribution in rats after oral administration also suggested that TECMs had comparatively stronger mucoadhesive characters. Therefore, our results indicated that TECMs have potentials to be an oral protein drug carrier.

Lipolysis is stimulated by PEGylated conjugated linoleic acid through the cyclic adenosine monophosphate-independent signaling pathway in 3T3-L1 cells: activation of MEK/ERK MAPK signaling pathway and hyper-secretion of adipo-cytokines.

We previously reported that PEGylated conjugated linoleic acid (PCLA) as a pro-drug treatment of cultures of 3T3-L1 cells containing differentiated adipocytes caused de-differentiation by downregulation of PPARgamma2-induced adipogenesis, and cell apoptosis induced by PCLA was lower than that induced by conjugated linoleic acid (CLA) owing to the biocompatible and hydrophilic properties of poly(ethylene glycol) (PEG). To further investigate our previous observations, the present study is designed to evaluate the lipolytic action of PCLA and its role in biochemical signaling pathways of 3T3-L1 cells when compared to the CLA itself. Although both CLA and PCLA stimulated lipolysis, our results indicated a sensitivity difference between CLA and PCLA treatment: a time-dependent effect on lipolysis and p-extracellular signal-related kinases (ERK) expression was observed for PCLA-treated, but not for CLA-treated cultures. Also, the induction by PCLA of mitogen-activated protein kinase kinase (MEK)/ERK mitogen-activated protein kinase (MAPK) activation was linked to secretion of adipo-cytokines, interleukin-6 (IL-6), and interleukin-8 (IL-8), in time-dependent manners. Interestingly, adenylyl cyclase inhibitor, 2', 5'-dideoxyadenosine (DDA), pre-treatment did not prevent PCLA-stimulated lipolysis. In fact, isoproterenol, but not PCLA, caused a significant increase in cyclic adenosine monophosphate (cAMP) levels, suggesting that the PCLA-induced lipolysis was not mediated in the conventional cAMP-dependent pathway and the cAMP was the intracellular mediator for isoproterenol-induced lipolysis. Overall, our findings provide support for a role for PCLA as a pro-drug in the regulation of metabolism in adipose tissue.

Pluronic F127 enhances the effect as an adjuvant of chitosan microspheres in the intranasal delivery of Bordetella bronchiseptica antigens containing dermonecrotoxin.

We have studied a vaccine delivery system in vitro and in vivo based on chitosan microspheres (CMs) prepared in the presence of selected immunomodulators, Pluronic block copolymer F127 (F127). The Bordetella bronchiseptica multiple antigens containing dermonecrotoxin (BBD), a virulent factor leading to atrophic rhinitis (AR) in swine was loaded in CMs/F127 or CMs alone. The microspheres, prepared using an ionic gelation process with tripolyphosphate, demonstrated release profiles that showed a greater amount of BBD being released from BBD-loaded CMs/F127 (BBD-CMs/F127). In vitro experiments using mouse alveolar macrophage cells (RAW 264.7) demonstrated that BBD-CMs/F127 have significantly higher immune-stimulating activities than controls. The highest immune-stimulating activities by the BBD-CMs/F127 using RAW 264.7 cells were mirrored in the in vivo studies following nasal administration to mice. The mice immunized with BBD-CMs/F127 showed higher BBD specific IgA antibody responses in nasal wash, saliva and serum than mice immunized with BBD-CMs alone. Protective immunity was measured by survival rate after challenge with B. bronchiseptica via the nasal cavity. The survival rate of the group treated with BBD-CMs/F127 was higher than those of other groups. These results suggested that CMs/F127 represents a novel mucosal delivery system and that F127 could enhance the delivery of BBD-CMs in the vaccination scheme.

Enhanced anticancer effect of conjugated linoleic acid by conjugation with Pluronic F127 on MCF-7 breast cancer cells.

This study is designed to evaluate whether conjugated linoleic acid-coupled Pluronic F127 (Plu-CLA) enhances anticancer efficacy in MCF-7 breast cancer cells when compared to conjugated linoleic acid (CLA) itself. CLA was simply coupled to Pluronic F127 through ester linkage between carboxyl group of CLA and hydroxyl one of Pluronic at melting state without solvent or catalyst. Plu-CLA significantly enhanced apoptosis with increasing concentration compared with CLA itself. Moreover, it was found that p53, p21, and Bax were up-regulated, whereas Bcl-2 and procaspase 9 were down-regulated with increasing concentration of Plu-CLA. These results were attributed to the sensitization activity of Pluronic F127.

Leptin-induced matrix metalloproteinase-2 secretion is suppressed by trans-10,cis-12 conjugated linoleic acid.

It has long been recognized that leptin, a hormone made by adipocytes, is an important circulating signal for the regulation of body weight. In addition, matrix metalloproteinase (MMP), especially MMP-2, an adipocyte-secreted protein which promotes multi-cellular adipose clusters, is up-regulated in obesity. The present study is designed to evaluate whether trans-10,cis-12 conjugated linoleic acid (t-CLA) can suppress leptin-induced MMP-2 secretion in 3T3-L1 cells. The result showed that expressions of adipocyte marker proteins were significantly reduced by t-CLA-treated cultures, but not by linoleic acid (LA)-treated ones. Interestingly, MMP-2 secretion was significantly increased by leptin-treated cultures, thereby leading to accelerate adipocyte differentiation, indicating that MMP-2 was a necessary mediator of adipogenesis. However, increasing concentration of t-CLA significantly reduced leptin-induced MMP-2 secretion and triglyceride (TG) content. These findings provide support for a role for t-CLA in the regulation of metabolism in leptin-induced adipose tissue development.