Analysis of bacterial community using pyrosequencing in semen from patients with chronic pelvic pain syndrome: a pilot study.

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.

Vibrio oceanisediminis sp. nov., a nitrogen-fixing bacterium isolated from an artificial oil-spill marine sediment.

A Gram-staining-negative, halophilic, facultatively anaerobic, motile, rod-shaped and nitrogen-fixing bacterium, designated strain S37T, was isolated from an artificial oil-spill sediment sample from the coast of Taean, South Korea. Cells grew at 10-37 °C and pH 5.0-9.0, with optimal growth at 28 °C and pH 6.0-8.0. Growth was observed with 1-9 % (w/v) NaCl in marine broth, with optimal growth with 3-5 % NaCl, but no growth was observed in the absence of NaCl. According to the results of 16S rRNA gene sequence analysis, strain S37T represents a member of the genus Vibrio of the class Gammaproteobacteria and forms a clade with Vibrio plantisponsor MSSRF60T (97.38 %), Vibrio diazotrophicus ATCC 33466T (97.31 %), Vibrio aestuarianus ATCC 35048T (97.07 %) Vibrio areninigrae J74T (96.76 %) and Vibrio hispanicus LMG 13240T (96.76 %). The major fatty acids were C16 : 0, C16 : 1ω7c/C16 : 1ω6c and C18 : 1ω7c/C18 : 1ω6c. The DNA G+C content was 41.9 %. The DNA-DNA hybridization analysis results showed a 30.2 % association value with the closely related type strain V. plantisponsor DSM 21026T. On the basis of phenotypic and chemotaxonomic characteristics, strain S37T represents a novel species of the genus Vibrio, for which the name Vibrio oceanisediminis sp. nov., is proposed with the type strain S37T ( = KEMB 2255-005T = JCM 30409T).

Burkholderia eburnea sp. nov., isolated from peat soil.

A novel aerobic bacterium, designated strain RR11(T), was isolated from peat soil and was characterized by using a polyphasic taxonomic approach and identified in order to determine its taxonomic position. Strain RR11(T) is a Gram-negative, non-sporulating, motile, short-rod-shaped bacterium. 16S rRNA gene sequence analysis identified this strain as a member of the genus Burkholderia of the class Betaproteobacteria. The highest degrees of gene sequence similarity were found with Burkholderia tropica Ppe8(T) (98.0 %), B. bannensis E25(T) (97.3 %), B. ferrariae FeGI01(T) (97.1 %), B. unamae MTI-641(T) (97.1 %) and B. heleia SA41(T) (97.1 %). Strain RR11(T) had the following chemotaxonomic characteristics: the major ubiquinone was Q-8, the DNA G+C content was 60.8 mol%, the major fatty acids were C16 : 0, C19 : 0 cyclo ω8c and C17 : 0 cyclo and the polar lipid profile contained phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and an unknown aminophospholipid. Based on its morphological, physiological and chemotaxonomic characteristics, together with 16S rRNA gene sequence comparison results, strain RR11(T) represents a novel species, for which the name Burkholderia eburnea sp. nov. is proposed. The type strain is strain RR11(T) ( = KEMC 7302-065(T) = JCM 18070(T)).

Proteomic analysis of differentially expressed proteins in vitamin C-treated AGS cells.

BACKGROUND: Vitamin C (ascorbic acid) is an essential nutrient of most living tissues that readily acts as a strong reducing agent, which is abundant in fruits and vegetables. Although, it inhibits cell growth in many human cancer cells in vitro, treatment in cancer is still controversial. Hence, the purpose of this study was to investigate the molecular mechanism of the inhibitory effect of vitamin C on AGS cell growth, and protein profiles in AGS cells after exposure to vitamin C treatment, by using proteomic tools. RESULTS: Vitamin C showed a cytotoxic effect on AGS cells (IC50 300 µg/mL) and, 20 differentially expressed proteins (spot intensities which show ≥2 fold change and statistically significant, p<0.05 between the control and vitamin-C treated group) were successfully identified by assisted laser desorption/ ionization-time of flight/mass spectrometry (MALDI-TOF/MS). Of the 20 proteins, six were up-regulated and fourteen were down-regulated. Specifically, 14-3-3σ, 14-3-3ϵ, 14-3-3δ, tropomyosin alpha-3 chain and tropomyosin alpha-4 chain were down-regulated and peroxiredoxin-4 and thioredoxin domain-containing proteins 5 were up-regulated. The identified proteins are mainly involved in cell mobility, antioxidant and detoxification, signal transduction and protein metabolism. Further, the expressions of 14-3-3 isoforms were verified with immuno-blotting analysis. CONCLUSIONS: Our proteome results suggest that the apoptosis related proteins were involved in promoting and regulating cell death of AGS cells, and might be helpful to understand the molecular mechanism of vitamin C on AGS cell growth inhibition.

Burkholderia humi sp. nov., isolated from peat soil.

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.

Vitamin C induces apoptosis in AGS cells by down-regulation of 14-3-3σ via a mitochondrial dependent pathway.

Ascorbic acid (vitamin C) is an essential component of most living cells. Apart from antioxidant activity, it has been reported to inhibit cancer cell growth in vitro in human cancer cells. However, the cellular mechanism underlying anticancer activity has not been fully elucidated. In this study, vitamin C showed a cytotoxic effect on human gastric cancer cell line AGS (LD50 300µg/ml). Further, flow cytometry analysis showed that vitamin C increased the sub-G1 (apoptosis) population and apoptosis confirmed by fluorescein isothiocyanate-Annexin V double staining in AGS cells. Moreover, specific immuno-blotting revealed the expression of the phosphorylated form of Bad (S136), 14-3-3σ, pro-caspases-3, -6, -8, and-9 protein levels were significantly decreased and Bax/Bcl-xL ratio was increased in a dose-dependent manner. Also, wound healing assay results showed that vitamin C inhibited AGS cell proliferation. These findings suggest that vitamin C induces apoptosis and might be a potential therapeutic agent for gastric cancer.

Induction of the cell cycle arrest and apoptosis by flavonoids isolated from Korean Citrus aurantium L. in non-small-cell lung cancer cells.

This study investigated the anti-proliferative and apoptotic effect of flavonoids isolated from Korean Citrus aurantium L. using A549 lung cancer cells. Flavonoids potently inhibited of A549 cells in a dose-dependent manner, whereas flavonoids had a weak inhibitory effect on proliferation of WI-38 cells. Flow cytometry and Western blot analysis showed that flavonoids induced cell cycle arrest at the G2/M checkpoint by controlling the proteins expression level of cyclin B1, cdc2, cdc25c and p21(WAF1/CIP1). Also, flavonoids induced apoptosis through the regulation of the expression of caspases, cleaved PARP and Bax/Bcl-xL ratio. The activity of caspase-3 on A549 cells increased in a dose-dependent manner. These results clearly indicated that the anti-cancer effect of flavonoids on A549 cells follows multiple cellular pathways through G2/M arrest and the induction of apoptosis.

Regulation of Proinflammatory Mediators via NF-κB and p38 MAPK-Dependent Mechanisms in RAW 264.7 Macrophages by Polyphenol Components Isolated from Korea Lonicera japonica THUNB.

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.

Polyphenolic extract isolated from Korean Lonicera japonica Thunb. induce G2/M cell cycle arrest and apoptosis in HepG2 cells: involvements of PI3K/Akt and MAPKs.

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.

Suppressive effect of flavonoids from Korean Citrus aurantium L. on the expression of inflammatory mediators in L6 skeletal muscle cells.

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.

Comparative root protein profiles of Korean ginseng (Panax ginseng) and Indian ginseng (Withania somnifera).

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.

Flavonoids Isolated from Korea Citrus aurantium L. Induce G2/M Phase Arrest and Apoptosis in Human Gastric Cancer AGS Cells.

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.

Proteomic analysis of effects on natural herb additive containing immunoglobulin Yolksac (IgY) in pigs.

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.

Suppressive Effect on Lipopolysaccharide-Induced Proinflammatory Mediators by Citrus aurantium L. in Macrophage RAW 264.7 Cells via NF-κB Signal Pathway.

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.

Korean Scutellaria baicalensis water extract inhibits cell cycle G1/S transition by suppressing cyclin D1 expression and matrix-metalloproteinase-2 activity in human lung cancer cells.

AIM OF THE STUDY: Scutellaria baicalensis Georgi is a widely used medicinal herb in several Asian countries including Korea. The various medicinal properties attributed to Scutellaria baicalensis include anti-bacterial, anti-viral, anti-inflammatory and anti-cancer effects. The present study investigated the cytotoxicity of Scutellaria baicalensis water extract (SBWE) on A549 non-small-cell-lung cancer cells and the A549 expression of cyclin D1, cyclin-dependent kinase 4 (CDK4) and matrix metalloproteinase-2 (MMP-2), and the effects of SBWE on cell cycle progression, especially the G1/S phase, and on cell motility. MATERIALS AND METHODS: SBWE cytotoxicity was assessed by a standard colorimetric assay utilizing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and expression of cyclin D1 and CDK4 protein in SBWE-treated A549 cells was assessed by Western blot analysis. Flow cytometry analysis was performed to determine the effect of SBWE on A549 cell cycle progression. A549 cell MMP-2 activity was examined by zymography. Cell motility and migration was assessed by a scratch wound healing assay. RESULTS: SBWE was not cytotoxic. The production of Cyclin D1, CDK4 and MMP-2 activity were significantly decreased in a SBWE dose-dependent manner, with maximum inhibition occurring at SBWE concentrations of 250 µg/ml and 500 µg/ml. SBWE inhibited cell cycle progression in the G1/S phase and significantly inhibited the motility of A549 cells. CONCLUSIONS: Cyclin D1 protein may be associated with MMP-2 activity and cell motility. Thus, SBWE promotes a strong protective effect against MMP-2 mediated metastasis and cell proliferation through the down-regulation of cyclin D1. SBWE may be a useful chemotherapeutic agent for lung cancer.

Polychlorinated biphenyls have inhibitory effect on testicular steroidogenesis by downregulation of P450(17alpha) and P450(scc).

Polychlorinated biphenyls (PCBs) are environmental pollutants that are quite toxic to biological systems. This study examined the inhibitory effect of PCB126 and PCB114 on testicular steroidogenesis in male rats. Male Sprague Dawley rats received weekly intraperitoneal injections of PCB126 (0.2 mg/kg) or PCB114 (20 mg/kg) or vehicle (corn oil). Animals from each group were sacrificed at 2, 5 and 8 weeks after the injections. Blood and testis tissue samples were collected for the hormone assay, Western blotting and reverse transcriptase polymerase chain reaction (RT-PCR). The testosterone, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were assayed, and the expression levels of the mRNA and proteins associated with the testosterone biosynthesis pathway were measured to determine the effect of PCB126 and PCB114 on testicular steroidogenesis. The results showed that the testis weight was significantly higher in the PCB126-treated rats given eight shots. Moreover, the serum testosterone levels were significantly lower in the PCB126 and PCB114-treated groups than the control. The transcription and translation levels of P450(17alpha) and P450(scc) were significantly lower in the PCB126-treated groups than the control. These results suggest that PCB126 may affect testicular steroidogenesis by downregulating P450(17alpha), P450(scc) and have inhibitory effect on the testicular functions.

Hypothyroidism induced by polychlorinated biphenyls and up-regulation of transthyretin.

Polychlorinated biphenyls are environmental pollutants that are toxic to many biological systems. This study examined whether or not PCB126 and PCB114 have adverse effects on the serum thyroxine level and the serum proteome in rats. The results showed a lower serum total thyroxine level in the PCB126 and PCB114-treated groups than the control. Western blotting showed that the levels of transthyretin expression were significantly higher in the PCB-treated group than the control group. These results suggest that the PCB-mediated hypothyroidism is caused by the displacement of thyroxine from transthyretin.