Aim： To investigate the effects of advanced glycation end products （AGEs） on calcification in human aortic smooth muscle cells （HASMCs） in vitro and the underlying mechanisms. Methods： AGEs were ...artificially prepared. Calcification of HASMCs was induced by adding inorganic phosphate （Pi, 2 mmol/L） in the media, and observed with Alizarin red staining. The calcium content in the supernatant was measured using QuantiChrome Calcium Assay Kit. Expression of the related mRNAs and proteins was analyzed using real-time PCR and Western blot, respectively. Chromatin immunoprecipitation （CHIP） assay was used to detect the binding of NF-KB to the putative IGFIR promoter. Results： AGEs （100 pg/mL） significantly enhanced Pi-induced calcification and the levels of osteocalcin and Cbfal in HASMCs. Further- more, the treatment decreased the expression of insulin-like growth factor I receptor （IGFIR）. Over-expression of IGFIR in HASMCs suppressed the AGEs-induced increase in calcium deposition. When IGFIR expression was knocked down in HASMCs, AGEs did not enhance the calcium deposition. Meanwhile, AGEs time-dependently decreased the amounts of IκBa and Flag-tagged p65 in the cytoplasmic extracts, and increased the amount of nuclear p65 in HASMCs. In the presence of NF-κB inhibitor PDTC （50 pmol/L）, the AGEs-induced increase in calcium deposition was blocked. Over-expression of p65 significantly enhanced Pi-induced mineralization, but suppressed IGFIR mRNA level. Knockdown of p65 suppressed the AGEs-induced increase in calcium deposition, and rescued the IGFIR expression. The ChIP analysis revealed that NF-κB bound the putative IGFIR promoter at position -230 to -219 bp. The inhibition of IGF1R by NF-κB was abolished when IGFIR reporter plasmid contained mutated binding sequence for NF-κB or an NF-κB reporter vector. Conclusion： The results demonstrate that AGEs promote calcification of human aortic smooth muscle cells in vitro via activation of NF-κB and down-regulation of IGFIR expression.
Mycoplasma pneumoniae (M. pneumoniae) is one of the common pathogens causing atypical pneumonia. In recent years, resistance to macrolides has become more common, especially in China. Previous ...studies have confirmed that the mutation at position 2063 in domain V of the 23S rRNA is the most prevalent, followed by the mutation at position 2064. Reported molecular detection methods for the identification of these mutations include direct sequencing, restriction fragment length polymorphism analysis, real-time polymerase chain reaction (PCR) with high-resolution melt analysis, and nested PCR-linked with capillary electrophoresis, etc. The most commonly used method for monitoring resistance-conferring mutations in M. pneumoniae is direct DNA sequencing of PCR or nested PCR products. However, these methods are time-consuming, labor-intensive or need expensive equipments. Therefore the development of rapid and sensitive methods is very important for monitoring the resistance globally.
In this study, we reported a fast and cost-effective method for detecting 2063 and/or 2064 macrolide resistant mutations from specimens using a modified allele-specific PCR analysis, and all results were compared with the sequencing data. We also analyzed the clinical courses of these samples to confirm the modified allele-specific PCR results.
Among 97 M. pneumoniae specimens, 88 were found to possess mutations by this method, and all modified allele-specific PCR analysis results were consistent with the sequencing data. The data of the clinical courses of these 97 cases showed that they suffered from severe pneumonia. Erythromycin showed better efficacy on cases from which no macrolide resistance mutation was found on their specimens. However, in some cases from which mutations were detected, erythromycin monotherapy had poor efficacy, and on these patients severe symptoms improved only when azithromycin was added to the treatment.
The drug-resistant M. pneumoniae is very common in Beijing, China. Our modified allele-specific PCR analysis can identify erythromycin resistant mutations more rapidly from specimens than any other method currently available. Erythromycin is still effective for treating patients infected with the mutation negative M. pneumoniae, but this treatment fails to work on mutant organisms. This method can facilitate clinicians in selecting appropriate therapy within short timescales.
Serving as an important second messenger, calcium ion has unique properties and universal ability to transmit diverse signals that trigger primary physiological actions in cells in response to ...hormones, pathogens, light, gravity, and stress factors. Being a second messenger of paramount significance, calcium is required at almost all stages of plant growth and development, playing a fundamental role in regulating polar growth of cells and tissues and participating in plant adaptation to various stress factors. Many researches showed that calcium signals decoding elements are involved in ABA-induced stomatal closure and plant adaptation to drought, cold, salt and other abiotic stresses. Calcium channel proteins like AtTPC1 and TaTPC1 can regulate stomatal closure. Recently some new studies show that Ca(2+) is dissolved in water in the apoplast and transported primarily from root to shoot through the transpiration stream. The oscillating amplitudes of Ca(2+)(o) and Ca(2+)(i) are controlled by soil Ca(2+) concentrations and transpiration rates. Because leaf water use efficiency (WUE) is determined by stomatal closure and transpiration rate, so there may be a close relationship between Ca(2+) transporters and stomatal closure as well as WUE, which needs to be studied. The selection of varieties with better drought resistance and high WUE plays an increasing role in bio-watersaving in arid and semi-arid areas on the globe. The current paper reviews the relationship between calcium signals decoding elements and plant drought resistance as well as other abiotic stresses for further study.
Objective To investigate acrylamide （ACR）-induced subacute neurotoxic effects on the central nervous system （CNS） at the synapse level in rats. Methods Thirty-six Sprague Dawley （SD） rats were ...randomized into three groups, （1） a 30 mg/kg ACR-treated group, （2） a 50 mg/kg ACR-treated group, and （3） a normal saline （NS）-treated control group. Body weight and neurological changes were recorded each day. At the end of the test, cerebral cortex and cerebellum tissues were harvested and viewed using light and electron microscopy. Additionally, the expression of Synapsin I and P-Synapsin I in the cerebral cortex and cerebellum were investigated. Results The 50 mg/kg ACR-treated rats showed a significant reduction in body weight compared with untreated individuals （P 〈 0.05）. Rats exposed to ACR showed a significant increase in gait scores compared with the NS control group （P 〈 0.05）. Histological examination indicated neuronal structural damage in the 50 mg/kg ACR treatment group. The active zone distance （AZD） and the nearest neighbor distance （NND） of synaptic vesicles in the cerebral cortex and cerebellum were increased in both the 30 mg/kg and 50 mg/kg ACR treatment groups. The ratio of the distribution of synaptic vesicles in the readily releasable pool （RRP） was decreased. Furthermore, the expression levels of Synapsin I and P-Synapsin I in the cerebral cortex and cerebellum were decreased in both the 30 mg/kg and 50 mg/kg ACR treatment groups. Conclusion Subacute ACR exposure contributes to neuropathy in the rat CNS. Functional damage of synaptic proteins and vesicles may be a mechanism of ACR neurotoxicity.
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