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One of the recent advances in medical nanotechnology has been the development of nanoformulations to overcome drug-resistant bacterial infections. Herein, we disclose a new nano-antibiotic formulation based on sceptrin-Au nano-aggregates (SANA), which are drug-metal ion multiple complexes. Sceptrin is a natural compound from a marine organism (sponge) and was reported as a potential compound with drug activities. SANA consists of a sceptrin-Au ion and is a self-assembled nano-formation with electrostatic interaction. Interestingly, SANA showed superior antibiotic/antibiofilm activity toward carbapenem-resistant Gram-negative bacteria with low toxicity to red blood cells and endothelial cells. The working mechanism of SANA was identified with analysis of the extracellular reactive oxygen species level and membrane depolarization of bacteria. The feasibility of SANA as a new nano-antibiotic was demonstrated in CRPA-contaminated medical supplies where SANA inhibited the formation of biofilms as well as the growth of CRPA. This work presents a new concept for the development of next-generation nano-antibiotics and a more feasible clinical translational pathway.
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Células Endoteliales , Bacterias Gramnegativas , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Oligopéptidos , Pirroles/farmacologíaRESUMEN
Multidrug-resistant (MDR) bacteria have become a severe problem for public health. Developing new antibiotics for MDR bacteria is difficult, from inception to the clinically approved stage. Here, we have used a new approach, modification of an antibiotic, ciprofloxacin (CFX), with triphenylphosphonium (TPP, PPh3) moiety via ester- (CFX-ester-PPh3) and amide-coupling (CFX-amide-PPh3) to target bacterial membranes. In this study, we have evaluated the antibacterial activities of CFX and its derivatives against 16 species of bacteria, including MDR bacteria, using minimum inhibitory concentration (MIC) assay, morphological monitoring, and expression of resistance-related genes. TPP-conjugated CFX, CFX-ester-PPh3, and CFX-amide-PPh3 showed significantly improved antibacterial activity against Gram-positive bacteria, Staphylococcus aureus, including MDR S. aureus (methicillin-resistant S. aureus (MRSA)) strains. The MRSA ST5 5016 strain showed high antibacterial activity, with MIC values of 11.12 µg/mL for CFX-ester-PPh3 and 2.78 µg/mL for CFX-amide-PPh3. The CFX derivatives inhibited biofilm formation in MRSA by more than 74.9% of CFX-amide-PPh3. In the sub-MIC, CFX derivatives induced significant morphological changes in MRSA, including irregular deformation and membrane disruption, accompanied by a decrease in the level of resistance-related gene expression. With these promising results, this method is very likely to combat MDR bacteria through a simple TPP moiety modification of known antibiotics, which can be readily prepared at clinical sites.
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Two of the most critical factors for the survival of glioblastoma (GBM) patients are precision diagnosis and the tracking of treatment progress. At the moment, various sophisticated and specific diagnostic procedures are being used, but there are relatively few simple diagnosis methods. This work introduces a sensing probe based on a turn-on type fluorescence response that can measure the cysteine (Cys) level, which is recognized as a new biomarker of GBM, in human-derived cells and within on-site human clinical biopsy samples. The Cys-initiated chemical reactions of the probe cause a significant fluorescence response with high selectivity, high sensitivity, a fast response time, and a two-photon excitable excitation pathway, which allows the imaging of GBM in both mouse models and human tissue samples. The probe can distinguish the GBM cells and disease sites in clinical samples from individual patients. Besides, the probe has no short or long-term toxicity and immune response. The present findings hold promise for application of the probe to a relatively simple and straightforward following of GBM at clinical sites.
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We have explored a new research field of fluorophores through the manipulation of fluorophore-binding proteins. The development of a new imaging agent for tracing a specific organelle or a particular site within a living organism has been of great interest in the field of basic science as well as translational medicine. In this work and for the first time, we will disclose a new naphthalene-based dipolar dye and its complex, with serum albumin (SA), and show their applicability for the selective imaging of mitochondria in cells and the intestine in a mouse. The SA-binding dipolar dye, IPNHC, was synthesized straightforwardly, and we identified its photophysical properties and binding mode with SA. IPNHC-SA complex showed a bright emission in the blue wavelength range with a high quantum yield and stability. In the fluorescence imaging study, bright fluorescence images of mouse intestines were observed under a UV light, as well as two-photon (TP) deep tissue imaging after intravenous injection of IPNHC and IPNHC-SA complex. The present findings hold great promise for the application of the fluorescent complex for use in the tracing and tracking of intestine-related diseases at clinical sites.
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Colorantes Fluorescentes/química , Intestinos/citología , Mitocondrias/química , Mitocondrias/metabolismo , Naftalenos/química , Imagen Óptica/métodos , Albúmina Sérica/química , Animales , RatonesRESUMEN
BACKGROUND: Although empirical antibacterial treatments are currently recommended for inflammatory chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), physicians cannot verify infections in most cases. Therefore, in this study, the microbiota of semen was investigated via pyrosequencing to obtain evidence underlying infectious disease. METHODS: Patients diagnosed with CP/CPPS (n=17) and healthy volunteers (n=4) participated in the study. Whole DNA was purified from the participants' semen. The DNA was amplified by polymerase chain reaction (PCR) using universal bacterial primers. All semen samples were also cultured using conventional methods. Pyrosequencing analysis of the PCR-amplified DNA was performed. RESULTS: None of the semen samples showed colony formation in conventional bacterial cultures. However, pyrosequencing revealed multiple bacterial genera in all samples, including an abundance of fastidious bacteria. Corynebacterium, Pseudomonas, Sphingomonas, Staphylococcus, and Streptococcus were frequently detected nonspecifically in both the patient and control groups. However, Achromobacter, Stenotrophomonas, and Brevibacillus were more frequently found in the CP/CPPS patients. CONCLUSIONS: The identification of various dominant species in the CP/CPPS group other than those reported in previous studies might be helpful for future etiological analysis of CP/CPPS.
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Effective biological treatment of marine wastewater is not well-known. Accumulation of nitrogen and phosphorus from land-based effluent is a crucial cause of red-tide in marine systems. The purpose of the study is to reduce nitrogen and phosphorus in marine wastewater with a pilot plant-scale sequencing batch reactor (SBR) system by using marine sediment as eco-friendly and effective biological materials, and elucidate which bacterial strains in sludge from marine sediment influence the performance of SBR. By applying eco-friendly high efficiency marine sludge (eco-HEMS), the treatment performance was 15 m3 d-1 of treatment amount in 4.5 m3 of the reactor with the average removal efficiency of 89.3% for total nitrogen and 94.9% for total phosphorus at the optimal operation condition in summer. Moreover, the average removal efficiency was 84.0% for total nitrogen and 88.3% for total phosphorus in winter although biological treatment efficiency in winter is generally lower due to bacterial lower activity. These results were revealed by the DNA barcoding analysis of 16s rRNA amplicon sequencing of samples from the sludge in winter. The comparative analysis of the bacterial community composition in sludge at the high efficiency of the system showed the predominant genera Psychromonas (significantly increased to 45.6% relative abundance), Vibrio (13.3%), Gaetbulibacter (5.7%), and Psychroserpens (4.3%) in the 4 week adaptation after adding marine sediment, suggesting that those predominant bacteria influenced the treatment performance in winter.
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Bacterias/clasificación , Sedimentos Geológicos/microbiología , Nitrógeno/análisis , Fósforo/análisis , Análisis de Secuencia de ADN/métodos , Aguas Residuales/análisis , Bacterias/genética , Biodegradación Ambiental , Reactores Biológicos/microbiología , ADN Bacteriano/genética , ADN Ribosómico/genética , Filogenia , Proyectos Piloto , ARN Ribosómico 16S/genética , Aguas del Alcantarillado/microbiologíaRESUMEN
A latent turn-on fluorescent probe for the detection of malononitrile (NCCH2CN), a precursor of hydrogen cyanide (HCN) in the mammalian tissue metabolism, is developed based on reaction-based fluorophore generation for the first time. Malononitrile is utilized within a wide spectrum of academic and industrial applications, and it is a key reagent to make o-chlorobenzylidene malononitrile (CS gas; tear gas), which is used for riot control. Due to its extensive use as well as potential health risks and the environmental pollution, malononitrile monitoring method has been required. In this paper, we discovered that our key sensing platform, 6-(dimethylamino)-3-hydroxy-2-naphthaldehyde (named Mal-P1), responds sensitively and selectively towards malononitrile. The Knoevenagel condensation induced benzo [g]coumarin formation of Mal-P1 with malononitrile showed significant fluorescence turn-on response. In addition, Mal-P1 showed the malononitrile sensing ability in environmental samples (real water, CS gas) and imaging ability in biological sample (HeLa cell line) using fluorescence microscopy with low cytotoxicity. The successful demonstrations will facilitate further applications in a variety of fields.
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Aldehídos/química , Colorantes Fluorescentes/química , Naftalenos/química , Nitrilos/análisis , Contaminantes Químicos del Agua/análisis , Aldehídos/síntesis química , Aldehídos/toxicidad , Agua Potable/análisis , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/toxicidad , Células HeLa , Humanos , Lagos/análisis , Límite de Detección , Microscopía Confocal , Microscopía Fluorescente , Naftalenos/síntesis química , Naftalenos/toxicidad , Nitrilos/química , Ríos/química , Agua de Mar/análisis , Espectrometría de Fluorescencia , Contaminantes Químicos del Agua/química , o-Clorobencilidenomalonitrila/síntesis químicaRESUMEN
Fluorescence-based in vivo imaging is one of the most important tools for monitoring of biological processes in cells and tissues of live animal models. Fluorescence imaging agents have also been used to monitor the microcirculation. Tracking microcirculation of the blood is vital to gain further insight into various vascular disease-related anomalies within the human body. As monitoring of vascular circulation is performed with visualization of both immune cells and pathogens, which are mainly labelled with red and green, the favorable color option for blood vessels could be blue. However, currently available blueish color-labeled agents for vascular monitoring is generally confronted with quick bleaching, because of its short excitation and emission wavelengths. Hereby, what we propose in this report is a newly generated bright blue fluorescent dextran, named HCD-70K that monitors the blood vessels using blue and inter-compatible typical fluorescent materials. DBCO-functionalized dextran-70K was fabricated with hydroxy-coumarin dye via metal-free bioorthogonal click chemistry, and generated HCD-70K, which can flow within the blood vessel and decipher the whole structure of the blood vessel successfully. The synthesis, spectroscopic analysis, and quantum chemical calculations were conducted. Using two-photon microscopy, efficient deep in vivo blood vessel imaging of a mouse model revealed exceptional bio-imaging capabilities of the HCD-70K and consequently it provided a promising opportunity for efficient vascular visualization in various research areas.
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Vasos Sanguíneos/diagnóstico por imagen , Dextranos/química , Colorantes Fluorescentes/química , Imagen Óptica , Fotones , Animales , Teoría Funcional de la Densidad , Dextranos/administración & dosificación , Dextranos/síntesis química , Colorantes Fluorescentes/administración & dosificación , Colorantes Fluorescentes/síntesis química , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Estructura MolecularRESUMEN
Porous silicon nanoparticles (pSiNPs) have been utilized within a wide spectrum of biological studies, as well as in chemistry, chemical biology, and biomedical fields. Recently, pSiNPs have been constantly coming under the spotlight, mostly in biomedical applications, due to their advantages, such as controlled-release drug delivery in vivo by hydrolysis-induced degradation, self-reporting property through long life-time photoluminescence, high loading efficiency of substrate into pore, and the homing to specific cells/organ/bacteria by surface functionalization. However, the systematic degradation rate analysis of surface-functionalized pSiNPs in different biological media has not been conducted yet. In this paper, we prepared four different surface-functionalized pSiNPs samples and analyzed the degradation rate in six different media (DI H2O (deionized water), PBS (phosphate-buffered saline), HS (human serum), DMEM (Dulbecco's modified Eagle's medium), LB (lysogeny broth), and BHI (brain heart infusion)). The obtained results will now contribute to understanding the correlation between surface functionalization in the pSiNPs and the degradation rate in different biological media. The characterized data with the author's suggestions will provide useful insights in designing the new pSiNPs formulation for biomedical applications.
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A keloid is a type of unusually raised scar. Unlike other raised scars, keloids form larger sizes than the wound site due to overgrowth, generally related to various biological factors. To date, only a few diagnostic and therapeutic methods for keloids have been reported. The high recurrence rates and undesirable side effects of keloids, at the end stage, encourage the invention of novel diagnostic tools, in order to cure keloids at an earlier stage. In this review, we summarize the general information about keloid diagnosis, keloid biomarkers, and recently reported fluorescent probes that can sense the key biomarkers of keloids. The focused description of fluorescent probes for keloid biomarkers and the author's perspective give useful insights in order to design the next-generation diagnostic sensing system for keloids.
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Biomarcadores/metabolismo , Colorantes Fluorescentes/metabolismo , Queloide/diagnóstico , Queloide/metabolismo , HumanosRESUMEN
The aggregation of amyloid beta (Aß) proteins in senile plaques is a critical event during the development of Alzheimer's disease, and the postmortem detection of Aß-rich proteinaceous deposits through fluorescent staining remains one of the most robust diagnostic tools. In animal models, fluorescence imaging can be employed to follow the progression of the disease, and among the different imaging methods, two-photon microscopy (TPM) has emerged as one of the most powerful. To date, several near-infrared-emissive two-photon dyes with a high affinity for Aß fibrils have been developed, but there has often been a tradeoff between excellent two-photon cross-sections and large fluorescence signal-to-background ratios. In the current work, we introduced a twisted intramolecular charge state (TICT)-based de-excitation pathway, which results in a remarkable fluorescence increase of around 167-fold in the presence of Aß fibrils, while maintaining an excellent two-photon cross section, thereby enabling high-contrast exâ vivo and inâ vivo TPM imaging. Overall, the results suggest that adopting TICT de-excitation in two-photon fluorophores may represent a general method to overcome the tradeoff between probe brightness and signal-to-background ratio.
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Colorantes Fluorescentes/metabolismo , Imagen Óptica/métodos , Placa Amiloide/metabolismo , HumanosRESUMEN
Urinary tract infections (UTIs) are clinically important problems that lead to serious morbidity and mortality, and indwelling urinary catheters are a major factor of UTIs. In this study, we applied clustered regularly interspaced short palindromic repeats (CRISPR) genome editing to generate ΔluxS mutant strains from clinical isolates of Escherichiacoli SE15, which is one of major pathogens and can cause colonization and biofilm formation in the catheter. A major regulatory pathway of such biofilm formation on medical devices is the quorum sensing mechanism via small molecule autoinducer-2 synthesized by LuxS enzyme. Here, we used the CRISPR-Cas9 system for precise deletion of luxS gene in clinical isolate E. coli SE15. To this end, we constructed a donor DNA for homologous recombination to delete 93 bases in the chromosomal target (luxS) and observed the success rate of luxS deletion to be 22.7 %. We conducted biofilm assay to observe decreased biofilm formation in the E. coil SE15 ΔluxS mutants compared to wild-type E. coil SE15. Quantitative real-time PCR analysis of E. coil SE15 ΔluxS mutants showed that the expression of luxS was below detection level. We also observed that the relative mRNA levels of biofilm-formation-related genes, such as mqsR, pgaBC and csgEF, were significantly decreased in E. coil SE15 ΔluxS mutants compared to wild-type. We conclude that genome editing by CRISPR-Cas9 system is an effective tool to dissect the molecular mechanism of biofilm formation in medically important strains, and the study may serve as a basis for developing novel medical intervention against UTIs caused by biofilm.
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Proteínas Bacterianas/genética , Sistemas CRISPR-Cas , Escherichia coli/genética , Edición Génica , Catéteres Urinarios/microbiología , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Biopelículas , Liasas de Carbono-Azufre/genética , Liasas de Carbono-Azufre/metabolismo , Catéteres de Permanencia/microbiología , Cromosomas Bacterianos/genética , Cromosomas Bacterianos/metabolismo , ADN Bacteriano/genética , Escherichia coli/clasificación , Escherichia coli/aislamiento & purificación , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Humanos , Percepción de Quorum , ARN Mensajero/genética , ARN Mensajero/metabolismo , Recombinación GenéticaRESUMEN
A Gram-negative, aerobic, short-rod-shaped, non-motile bacterium designated Rs7(T), was isolated from peat soil collected from Russia and was characterized to determine its taxonomic position. 16S rRNA gene sequence analysis revealed that the strain Rs7(T) belongs to the class Betaproteobacteria. The highest degree of sequence similarities were determined to be with Burkholderia tropica Ppe8(T) (98.4 %), Burkholderia unamae MTI-641(T) (97.8 %), Burkholderia bannensis E25(T) (97.7 %), Burkholderia heleia SA41(T) (97.0 %), and Burkholderia sacchari IPT101(T) (97.0 %). Chemotaxonomic data revealed that the strain Rs7(T) possesses ubiquinone Q-8. The polar lipid profile of strain Rs7(T) contained phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, and an unknown amino phospholipid. The predominant fatty acids were C(16:0), C(19:0) cyclo ω8c, and C(17:0) cyclo, all of which corroborated the assignment of the strain to the genus Burkholderia. The DNA G+C content was 63.2 mol%. DNA-DNA hybridization experiments showed less than 37.8 % DNA relatedness with closely related type strains, thus confirming separate species status. The results of physiological and biochemical tests allowed phenotypic differentiation of strain Rs7(T) from the members of the genus Burkholderia. Based on these data, Rs7(T) (=KEMC 7302-068(T) = JCM 18069(T)) should be classified as the type strain for a novel Burkholderia species, for which the name Burkholderia humi sp. nov. is proposed.
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Burkholderia/aislamiento & purificación , Microbiología del Suelo , Composición de Base , Burkholderia/clasificación , Burkholderia/fisiología , Ácidos Grasos/química , Datos de Secuencia Molecular , Fenotipo , Filogenia , ARN Ribosómico 16S/genéticaRESUMEN
Lonicera japonica THUNB., which abundantly contains polyphenols, has been used as a traditional medicine for thousands of years in East Asian countries because of the anti-inflammation properties. This study aimed to investigate the anti-inflammatory mechanism of polyphenol components isolated from Korea L. japonica T. by nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinases (MAPKs) pathway. Polyphenols significantly decreased lipopolysaccharide- (LPS-) induced mRNA and protein expression of inducible nitric oxide synthase and cyclooxygenase-2, as well as mRNA expression of tumor necrosis factor-alpha, interleukin- (IL-) 1ß, and IL-6. Moreover, polyphenols inhibited nuclear translocation of NF-κB p65, phosphorylation/degradation of the inhibitor of κB, and phosphorylation of p38 MAPK, whereas the extracellular signal-regulated kinase and Janus N-terminal kinase were not affected. These results indicate that polyphenol components isolated from Korea L. japonica T. should have anti-inflammatory effect on LPS-stimulated RAW 264.7 cells through the decrease of proinflammatory mediators expression by suppressing NF-κB and p38 MAPK activity.
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Lonicera japonica Thunb. (L. japonica T.) has been used in Korean traditional medicine for long time because of its anti-cancer and hepatic protective effect. In this study, we investigated polyphenolic extract in L. japonica T. using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) and its anti-cancer effect on hepatocarcinoma cells. Human HepG2 cell line was treated with various concentrations of polyphenolic extract. Apoptosis was detective by cell morphology, cell cycle analysis and immunoblot analysis. Polyphenolic extract inhibited cell proliferation at 48h in a dose-dependent manner. Polyphenolic extract affected HepG2 cell viability by inhibiting cell cycle progression at the G2/M transition and inducing apoptosis. Polyphenolic extract also decreased the expression of CDK1, CDC25C, cyclin B1, pro-caspases-3 and -9 and poly ADP ribose polymerase, and affected the levels of mitochondrial apoptotic-related proteins. The phosphorylation of extracellular signal-related kinase ½ (ERK 1/2), c-Jun N-terminal kinase (JNK), and p-38 mitogen-activated protein kinases (MAPKs) were increased in HepG2 cells treated with polyphenolic extract, whereas Akt was dephosphorylated. These results indicate that inhibition of PI3K/Akt and activation of MAPKs are pivotal in G2/M cell cycle arrest and apoptosis of human hepatocarcinoma cells mediated by polyphenolic extract.
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Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Lonicera/química , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Extractos Vegetales/farmacología , Polifenoles/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Línea Celular Tumoral , Humanos , Fosforilación , Polifenoles/aislamiento & purificaciónRESUMEN
Citrus fruits (Citrus aurantium L.) have long been used as a traditional herbal medicine. The benefits of the flavonoids found in Citrus aurantium L. include anti-inflammation, anti-cancer, anti-viral and anti-bacterial activities, and enhancement of the immune response. The study investigated the effect of the flavonoids isolated from Citrus aurantium L. native to Korea on the production of pro-inflammatory mediators by blocking signal transduction mediated by nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) in lipopolysaccharide (LPS)-induced L6 skeletal muscle cells. The flavonoids decreased the production of inducible nitric oxide synthase, cyclooxygenase-2, interleukin-6 and tumor necrosis factor-alpha by suppressing NF-κB and MAPKs signal pathways in LPS-induced L6 skeletal muscle cells. These findings suggest that the flavonoids isolated from Korea Citrus aurantium L. might have anti-inflammatory effects that regulate the expression of inflammatory mediators in L6 skeletal muscle cells.
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Citrus/química , Flavonoides/farmacología , Mediadores de Inflamación/metabolismo , Células Musculares/efectos de los fármacos , Antiinflamatorios/farmacología , Línea Celular , Ciclooxigenasa 2/metabolismo , Humanos , Interleucina-6/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Células Musculares/metabolismo , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Ginsenosides and withanolides are the secondary metabolites from Panax ginseng and Withania somnifera, respectively. These compounds have similar biological properties. Two-dimensional electrophoresis (2-DE) analysis was utilized to reveal the protein profile in the roots of both plants, with the aim of clarifying similarly- and differentially-expressed proteins. Total proteins of Korea ginseng (P. ginseng) and Indian ginseng (W. somnifera) roots were separated by 2-DE using a pH 4-7 immobilized pH gradient strip in the first dimension and 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis in the second dimension. The protein spots were visualized by silver staining. Twenty-one P. ginseng proteins and 35 W. somnifera proteins were chosen for identification by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry; of these, functions were ascribed to 14 and 22 of the P. ginseng and W. somnifera proteins, respectively. Functions mainly included general cell metabolism, defense and secondary metabolism. ATPase and alcohol dehydrogenase proteins were expressed in both plants. The results of this study, to our knowledge, are the first to provide a reference 2-DE map for the W. somnifera root proteome, and will aid in the understanding of the expression and functions of proteins in the roots of Korean ginseng and Indian ginseng.
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Panax/química , Proteínas de Plantas/análisis , Raíces de Plantas/química , Withania/química , Adenosina Trifosfatasas/análisis , Adenosina Trifosfatasas/metabolismo , Alcohol Deshidrogenasa/análisis , Alcohol Deshidrogenasa/metabolismo , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Panax/metabolismo , Proteínas de Plantas/metabolismo , Raíces de Plantas/metabolismo , Proteoma , Proteómica , Tinción con Nitrato de Plata , Espectrometría de Masas en Tándem , Withania/metabolismoRESUMEN
Aim of the Study. Citrus species is used in traditional medicine as medicinal herb in several Asian countries including Korea. Flavonioids became known as various properties, such as anti-oxidants, anti-inflammation and anti-cancer, and so forth. The present study, the anti-cancer effect of flavonioids isolated from Citrus aurantium L. in human gastric cancer AGS cells has been investigated. Materials and Methods. The anti-proliferative activity was assayed using MTT assay. Cell cycle analysis was done using flow cytometry and apoptosis detection was done using by hoechst fluorescent staining and Annexin V-propidium iodide double staining. Western blot was used to detect the expression of protein related with cell cycle and apoptosis. Results. Flavonoids isolated from Citrus aurantium L. have the effect of anti proliferation on AGS cells with IC50 value of 99 µg/mL. Flavonoids inhibited cell cycle progression in the G2/M phase and decrease expression level of cyclin B1, cdc 2, cdc 25c. Flavonoids induced apoptosis through activate caspase and inactivate PARP. Conclusions. Flavonoids isolated from Citrus aurantium L. induced G2/M phase arrest through the modulation of cell cycle related proteins and apoptosis through activation caspase. These finding suggest flavonoids isolated from Citrus aurantium L. were useful agent for the chemoprevention of gastric cancer.
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Thirty male pigs were infected orally with E. coli and Salmonella typhimurium, and divided into a control group and two additive groups to determine the effect of an additive mixture on the changes in protein expression. The pigs were given a food supplemented with a natural herbal additive containing immunoglobulin yolksac (IgY) at concentrations of 0.5% or 1%. On the 1st day and after eight weeks of feeding, the body weight gain, food intake and serum GOT/GPT levels were examined. The GOT/GPT levels on the 1st day were similar in the three groups. However, after eight weeks of feeding, the GOT level was significantly lower in the additive treatment groups (0.5% and 1.0%). In addition, the changes in the spleen proteome as a response to the herbal additive were examined using two-dimensional polyacrylamide gel electrophoresis. A total of 31 differentially expressed protein spots were identified by comparing the protein profiles of the control and additive treated porcine spleens. Finally, 19 proteins were detected by MALDI-TOF/MS. Overall, the proteins detected are involved in a range of biological process, such as metabolic processes, biological processes, transport, carbohydrate metabolic processes, generation of precursors and energy. In conclusion, these results support of the hypothesis that a natural herbal additive containing IgY can affect the immune regulation system and reduce the stress of microbial infections.
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Aspartato Aminotransferasas/sangre , Inmunoglobulinas , Factores Inmunológicos/farmacología , Magnoliopsida , Extractos Vegetales/farmacología , Proteoma/efectos de los fármacos , Bazo/efectos de los fármacos , Alanina Transaminasa/sangre , Animales , Suplementos Dietéticos , Yema de Huevo , Electroforesis en Gel Bidimensional , Masculino , Proteómica , Bazo/metabolismo , PorcinosRESUMEN
Citrus fruits have been used as an edible fruit and a traditional medicine since ancient times. In particular, the peels of immature citrus fruits are used widely in traditional herbal medicine in Korea, as they are believed to contain bioactive components exerting anti-inflammatory activity. This study examined whether the crude methanol extract of Citrus aurantium L. (CME) has a suppressive effect on inducible enzymes and proinflammatory cytokines by inhibiting the NF-κB pathway in LPS-stimulated macrophage RAW 264.7 cells. The cells were pretreated with the indicated concentrations of CME (5, 10, 20, and 50 µg/mL) and then treated with LPS (1 µg/mL). The results showed that CME (10, 20, and 50 µg/mL) inhibited the LPS- (1 µg/mL) induced mRNA and protein expression of iNOS in macrophage Raw 264.7 cells. In addition, the expression of COX-2 was inhibited at the mRNA and protein levels by CME in a dose-dependent manner. The mRNA expression of proinflammatory cytokines, such as TNF-α and IL-6, were markedly reduced by CME (10, 20, and 50 µg/mL). Moreover, CME clearly suppressed the nuclear translocation of the NF-κB p65 subunits, which was correlated with its inhibitory effect on I-κB phosphorylation. These results suggest that CME has anti-inflammatory properties by modulating the expression of COX-2, iNOS, and proinflammatory cytokines, such as TNF-α and IL-6, in macrophage RAW 264.7 cells via the NF-κB pathway.