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Find video protocols related to scientific articles indexed in Pubmed.
Ethanol-induced alcohol dehydrogenase E (AdhE) potentiates pneumolysin in Streptococcus pneumoniae.
Infect. Immun.
PUBLISHED: 10-15-2014
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Alcohol impairs the host immune system, rendering hosts more vulnerable to infection. Therefore, alcoholics are at increased risk of acquiring serious bacterial infections caused by Streptococcus pneumoniae, including pneumonia. Nevertheless, how alcohol affects pneumococcal virulence remains unclear. Here we showed that S. pneumoniae type 2 D39 is ethanol tolerant, and that alcohol up-regulates alcohol dehydrogenase E (AdhE) and potentiates pneumolysin (Ply). Hemolytic activity, colonization, and virulence of S. pneumoniae, as well as host cell myeloperoxidase activity, pro-inflammatory cytokine secretion, and inflammation, were significantly attenuated in adhE mutant bacteria (?adhE) compared to D39 wild-type bacteria. Therefore, AdhE might act as a pneumococcal virulence factor. Moreover, in the presence of ethanol, S. pneumoniae AdhE produced acetaldehyde and NADH, which subsequently led Rex (redox-sensing transcriptional repressor) to dissociate from the adhE promoter. An increase in AdhE in the ethanol condition conferred an increase of Ply and H2O2 levels. Consistently, S. pneumoniae D39 caused higher cytotoxicity to RAW 264.7 cells than ?adhE during the ethanol stress condition, and alcoholic mice were more susceptible to infection with the D39 wild-type bacteria than the ?adhE. Taken together, these data indicate that AdhE increases Ply in the ethanol stress condition, thus potentiating pneumococcal virulence.
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Preparation and testing of a Vi conjugate vaccine using pneumococcal surface protein A (PspA) from Streptococcus pneumoniae as the carrier protein.
Vaccine
PUBLISHED: 08-27-2014
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In the current study pneumococcal surface protein A (PspA) was conjugated to Vi capsular polysaccharide from Salmonella Typhi to make available a vaccine against typhoid fever that has the potential to also provide broad protection from Streptococcus pneumoniae. High yielding production processes were developed for the purification of PspAs from families 1 and 2. The purified PspAs were conjugated to Vi with high recovery of both Vi and PspA. The processes developed especially for PspA family 2 could readily be adapted for large scale production under cGMP conditions. Previously we have shown that conjugation of diphtheria toxoid (DT) to Vi polysaccharide improves the immune response to Vi but can also enhance the response to DT. In this study it was shown that conjugation of PspA to Vi enhanced the anti-PspA response and that PspA was a suitable carrier protein as demonstrated by the characteristics of a T-cell dependent response to the Vi. We propose that a bivalent vaccine consisting of PspA from families 1 and 2 bound to Vi polysaccharide would protect against typhoid fever and has the potential to also protect against pneumococcal disease and should be considered for use in developing countries.
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Adenylate kinase from Streptococcus pneumoniae is essential for growth through its catalytic activity.
FEBS Open Bio
PUBLISHED: 07-08-2014
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Streptococcus pneumoniae (pneumococcus) infection causes more than 1.6 million deaths worldwide. Pneumococcal growth is a prerequisite for its virulence and requires an appropriate supply of cellular energy. Adenylate kinases constitute a major family of enzymes that regulate cellular ATP levels. Some bacterial adenylate kinases (AdKs) are known to be critical for growth, but the physiological effects of AdKs in pneumococci have been poorly understood at the molecular level. Here, by crystallographic and functional studies, we report that the catalytic activity of adenylate kinase from S . pneumoniae (SpAdK) serotype 2 D39 is essential for growth. We determined the crystal structure of SpAdK in two conformations: ligand-free open form and closed in complex with a two-substrate mimic inhibitor adenosine pentaphosphate (Ap5A). Crystallographic analysis of SpAdK reveals Arg-89 as a key active site residue. We generated a conditional expression mutant of pneumococcus in which the expression of the adk gene is tightly regulated by fucose. The expression level of adk correlates with growth rate. Expression of the wild-type adk gene in fucose-inducible strains rescued a growth defect, but expression of the Arg-89 mutation did not. SpAdK increased total cellular ATP levels. Furthermore, lack of functional SpAdK caused a growth defect in vivo. Taken together, our results demonstrate that SpAdK is essential for pneumococcal growth in vitro and in vivo.
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Streptococcus pneumoniae ClpL modulates adherence to A549 human lung cells through Rap1/Rac1 activation.
Infect. Immun.
PUBLISHED: 06-30-2014
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Caseinolytic protease L (ClpL) is a member of the HSP100/Clp chaperone family, which is found mainly in Gram-positive bacteria. ClpL is highly expressed during infection for refolding of stress-induced denatured proteins, some of which are important for adherence. However, the role of ClpL in modulating pneumococcal virulence is poorly understood. Here, we show that ClpL impairs pneumococcal adherence to A549 lung cells by inducing and activating Rap1 and Rac1, thus increasing phosphorylation of cofilin (inactive form). Moreover, infection with a clpL mutant (?clpL) causes a greater degree of filopodium formation than D39 wild-type (WT) infection. Inhibition of Rap1 and Rac1 impairs filopodium formation and pneumococcal adherence. Therefore, ClpL can reduce pneumococcal adherence to A549 cells, likely via modulation of Rap1- and Rac1-mediated filopodium formation. These results demonstrate a potential role for ClpL in pneumococcal resistance to host cell adherence during infection. This study provides insight into further understanding the interactions between hosts and pathogens.
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ATF3 Confers Resistance to Pneumococcal Infection Through Positive Regulation of Cytokine Production.
J. Infect. Dis.
PUBLISHED: 06-20-2014
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Activating transcription factor-3 (ATF3) is known as a suppressor of cytokine production after exposure to lipopolysaccharide or during gram-negative bacterial infection. However, the mechanism by which ATF3 regulates innate immunity against gram-positive bacterial infection, particularly Streptococcus pneumoniae, remains unknown.
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Chinese yam extracts containing ?-sitosterol and ethyl linoleate protect against atherosclerosis in apolipoprotein E-deficient mice and inhibit muscular expression of VCAM-1 in vitro.
J. Food Sci.
PUBLISHED: 01-11-2014
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Atherosclerosis is a chronic inflammatory disease, which is associated with increased expression of adhesion molecules and monocyte recruitment into the arterial wall. This study evaluated whether hexane extracts from the edible part (DB-H1) or bark region (DB-H2) of Dioscorea. batatas Decne have anti-atherosclerotic properties in vivo and in vitro experiments. We also identified bioactive components in the hexane extracts. Thirty-six apolipoprotein E (ApoE(-/-) ) mice and 12 control (C57BL/6J) mice were given a Western-type diet for 11 or 21 wk. To examine the effects of yam extracts on lesion development, ApoE(-/-) mice were orally administered DB-H1 or DB-H2 for the duration of the study (200 mg/kg b.w./day, 3 times per wk). Both DB-H1 and DB-H2 significantly reduced the total atherosclerotic lesion area in the aortic root. In addition, plasma concentrations of total cholesterol, oxidized-low-density lipoprotein, and c-reactive protein were decreased by administration of DB-H1 and DB-H2. Consistent with the in vivo observations, DB-H1 and DB-H2 inhibited tumor necrosis factor (TNF)-?-induced vascular cell adhesion molecule-1 expression and adhesion of THP-1 monocytes to TNF-?-activated vascular smooth muscle cells. It was also found that treatment with DB-H1 or DB-H2 resulted in the inhibition nitric oxide (NO) and reactive oxygen species production and iNOS expression in macrophages. Thus, DB-H1 and DB-H2 seem to influence atherosclerosis by affecting the production of inflammatory mediators in vivo. Our results suggest that yam extracts have the potential to be used in the prevention of atherosclerosis.
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Modified opsonization, phagocytosis, and killing assays to measure potentially protective antibodies against pneumococcal surface protein A.
Clin. Vaccine Immunol.
PUBLISHED: 08-07-2013
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The standard opsonophagocytosis killing assay (OPKA) for antibodies to pneumococcal capsular polysaccharide was modified to permit an evaluation of the protection-mediating antibodies to pneumococcal surface protein A (PspA). We found that by increasing the incubation time with the complement and phagocytes from 45 min to 75 min, the protective activity was readily detected. In another modification, we used a capsule type 2 target strain that expressed PspA but not pneumococcal surface protein C (PspC). With these modifications separately or in combination, rabbit antisera to the recombinant ?-helical or proline-rich domains of PspA mediated >50% killing of the target strain. The ability of normal human sera to mediate the killing of pneumococci in this modified OPKA correlated with their levels of antibodies to PspA and their ability to protect mice against fatal infection with a type 3 strain. Passive protection of mice against pneumococci and killing in the modified OPKA were lost when normal human sera were adsorbed with recombinant PspA (rPspA) on Sepharose, thus supporting the potential utility of the modified OPKA to detect protective antibodies to PspA. In the standard OPKA, monoclonal antibodies to PspA were strongly protective in the presence of subprotective amounts of anti-capsule. Thus, the currently established high-throughput OPKA for antibodies to capsule could be modified in one of two ways to permit an evaluation of the opsonic efficacy of antibodies to PspA.
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Kobophenol A enhances proliferation of human osteoblast-like cells with activation of the p38 pathway.
Int. Immunopharmacol.
PUBLISHED: 06-19-2013
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Bone cell proliferation, bone formation, and bone resorption are the main factors involved in the homeostasis of the bone mass. Osteoblast death is a problem experienced by postmenopause women. Herbal medicines have attracted considerable attention for use as a drug or a drug substitute in the treatment of bone-related diseases, such as osteoporosis. This study investigated the effects of kobophenol A on the proliferation in human osteoblast cells. Kobophenol A stimulated the proliferation of osteoblast cells by the increases in DNA synthesis and the enhancement of cell cycle progression. Kobophenol A stimulation induced the expression of the cyclin B1 and cyclin-dependent kinase 1 (CDK1). Treatment of osteoblast cells with p38 MAPK inhibitor SB203580 significantly inhibited kobophenol A-enhanced proliferation. In addition, kobophenol A induced phosphorylation of p38 MAPK. Treatment of osteoblast cells with kobophenol A resulted in improvement of ROS scavenging activity. Moreover, kobophenol A treatment up-regulated the Bcl-2 level, but down-regulated the level of Bax expression. We also demonstrate that kobophenol A increased alkaline phosphatase (ALP) activity after 2 days. Taken together, the results of this study reveal that kobophenol A has proliferative effects and enhances ALP activity in osteoblast cells and these findings provide insights into the development of a therapeutic approach of kobophenol A in the prevention of osteoporosis and other bone disorders.
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Ethanol induces cell cycle arrest and triggers apoptosis via Sp1-dependent p75NTR expression in human neuroblastoma cells.
Cell Biol. Toxicol.
PUBLISHED: 03-26-2013
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Ethanol exposure has deleterious effects on the central nervous system. Although several mechanisms for ethanol-induced damage have been suggested, the precise mechanism underlying ethanol-induced neuronal cell death remains unclear. Recent studies indicate that the p75 neurotrophin receptor (p75NTR) has a critical role in the regulation of neuronal survival. This study was designed to examine the role of p75NTR in ethanol-induced apoptotic signaling in neuroblastoma cells. Ethanol caused highly increased level of p75NTR expression. The use of small interfering RNA to inhibit p75NTR expression markedly attenuated ethanol-induced cell cycle arrest and apoptosis. DNA binding activity of Sp1 was increased by ethanol, whereas inhibition of Sp1 activity by mithramycin, a Sp1 inhibitor, or short hairpin RNA suppressed ethanol-induced p75NTR expression. In addition, inhibitors of casein kinase 2 (CK2) and extracellular signal-regulated kinase (ERK) augmented ethanol-induced p75NTR expression. Our results also demonstrate that inhibition of ERK and CK2 caused a further increase in the activation of the p75NTR proximal promoter induced by ethanol. This increased activation was partially suppressed by the deletion of the Sp1 binding sites. These results suggest that Sp1-mediated p75NTR expression is regulated at least in part by ERK and CK2 pathways. The present study also showed that treatment with ethanol resulted in significant increases in the expression of p21, but not the levels of p53 and p53 target genes such as Bax, Puma, and Bcl-2. Furthermore, the inhibition of p75NTR expression or Sp1 activity suppressed ethanol-induced p21 expression, cell cycle arrest, and apoptosis. These data suggest that ethanol increases p75NTR expression, and CK2 and ERK signaling inversely regulate Sp1-mediated p75NTR expression in ethanol-treated neuroblastoma cells. Thus, our study provides more insight into the mechanisms underlying ethanol actions.
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Streptococcus pneumoniae ClpP protease induces apoptosis via caspase-independent pathway in human neuroblastoma cells: cytoplasmic relocalization of p53.
Toxicon
PUBLISHED: 02-11-2013
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Streptococcus pneumoniae causes the most severe form of the bacterial meningitis which is the major cause of bacterial meningitis. Virulence factors produced by S. pneumoniae have been known to contribute significantly to the disease process. ClpP protease (ClpP) which is essential for virulence and survival under stress conditions in S. pneumoniae was examined for the ability to induce apoptosis and the mechanism of the induction of apoptosis in human neuron-like cells, SK-N-SH neuroblastoma cells. ClpP inhibited cell growth and induced apoptosis in SK-N-SH cells. Treatment with ClpP resulted in hypodiploid DNA contents, increased Bax/Bcl-2 ratio and induction of reactive oxygen species (ROS) production. The release of cytochrome c from mitochondria into the cytosol, which is an initiator of the activation of caspase cascades, was not observed in ClpP-treated cells. In addition, pretreatment with Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), a broad spectrum caspase inhibitor, could not rescue apoptotic cells from ClpP toxicity. Coincidently, caspase-3 and -8 activation and cleavage of PARP were not detected. Moreover, caspase independent apoptosis-inducing factor (AIF) was released from mitochondria and translocated to the nucleus in response to ClpP. We also found that ClpP treatment resulted in the increase of p53 activity and cytoplasmic p53 levels were increased by ClpP, suggesting that functional activation of p53 is intact despite increased cytoplasmic accumulation. Taken together, these data suggest that ClpP contributes to neuronal damage in meningitis and provide further insight into the mechanisms underlying action of pneumococcal virulence factors during bacterial pathogenesis.
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Anti-oxidative stress effect of red ginseng in the brain is mediated by peptidyl arginine deiminase type IV (PADI4) repression via estrogen receptor (ER) ? up-regulation.
J Ethnopharmacol
PUBLISHED: 02-04-2013
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Ginseng has been used as an anti-stress agent, and its active ingredient, ginsenoside, is similar in structure to estrogen. However, the effect of ginseng on the stressed brain is not completely understood. The aim of this study is to understand systematically how red ginseng (RG) affects gene expressions in the brain of immobilization (IMO) stressed mice to elucidate its underlying mechanism.
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Antistress effect of red ginseng in brain cells is mediated by TACE repression via PADI4.
J Ginseng Res
PUBLISHED: 02-01-2013
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Ginseng is known to have antistress effects. Previously, red ginseng (RG) was shown to repress stress-induced peptidyl arginine deiminase type IV (PADI4) via estrogen receptor ? (ER?) in the brain, thus inhibiting brain cell apoptosis. Moreover, tumor necrosis factor (TNF)-? plays a critical role in immobilization (IMO) stress. However, the signaling pathway of RG-mediated repressesion of inflammation is not completely understood. In this study, we determined how RG modulated gene expression in stressed brain cells. Since secretion of TNF-? is modulated via TNF-? converting enzyme (TACE) and nuclear factor (NF)-?B, we examined the inflammatory pathway in stressed brain cells. Immunohistochemistry revealed that TACE was induced by IMO stress, but RG repressed TACE induction. Moreover, PADI4 siRNA repressed TACE expression compared to the mock transfected control suggesting that PADI4 was required for TACE expression. A reporter assay also revealed that H2O2 oxidative stress induced NF-?B in neuroblastoma SK-N-SH cells, however, RG pretreatment repressed NF-?B induction. These findings were supported by significant induction of nitric oxide and reactive oxygen species (ROS) by oxidative stress, which could be repressed by RG administration. Taken together, RG appeared to repress stress-induced PADI4 via TACE and NF-?B in brain cells thus preventing production of ROS and subsequently protecting brain cells from apoptosis.
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Inactivated pep27 mutant as an effective mucosal vaccine against a secondary lethal pneumococcal challenge in mice.
Clin Exp Vaccine Res
PUBLISHED: 01-15-2013
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A pep27 mutant may be able to elicit mucosal immunity against pneumococcal diseases, and could be employed as an inexpensive attenuated vaccine. However, this particular mutant contains an erythromycin-resistance marker. The purpose of the current study is to develop a markerless pep27 mutant and assess whether this inactivated mutant is able to induce mucosal immunity.
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Heat-shock protein ClpL/HSP100 increases penicillin tolerance in Streptococcus pneumoniae.
Adv. Otorhinolaryngol.
PUBLISHED: 08-18-2011
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Penicillin resistance and tolerance has been an increasing threat to the treatment of pneumococcal pneumoniae. However, no penicillin tolerance-related genes have been claimed. Here we show that a major heat shock protein ClpL/HSP100 could modulate the expression of a cell wall synthesis enzyme PBP2x, and subsequently increase cell wall thickness and penicillin tolerance in Streptococus pneumoniae.
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Stereocalpin A inhibits the expression of adhesion molecules in activated vascular smooth muscle cells.
Int. Immunopharmacol.
PUBLISHED: 07-26-2011
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Up-regulation of cell adhesion molecules on vascular smooth muscle cells (VSMCs) and leukocyte recruitment to the vascular wall contribute to vascular inflammation and atherosclerosis. Stereocalpin A, a chemical compound of the Antarctic lichen Ramalina terebarata, displays tumoricidal activity against several different tumor cell types. However, other biological activities of stereocalpin A and its molecular mechanisms remain unknown. In this study, our work is directed toward studying the in vitro effects of stereocalpin A on the ability to suppress the expression of adhesion molecules induced by TNF-? in vascular smooth muscle cells. Pretreatment of VSMCs for 2h with stereocalpin A at nontoxic concentrations of 0.1-10 ?g/ml inhibited TNF-?-induced adhesion of THP-1 monocytic cells and expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). Stereocalpin A reduced TNF-?-induced production of intracellular reactive oxygen species (ROS) and phosphorylation of p38, ERK, JNK and Akt. Stereocalpin A also inhibited NK-?B activation induced by TNF-?. Moreover, stereocalpin A inhibited TNF-?-induced ??? kinase activation, subsequent degradation of ????, and nuclear translocation of NF-?B. Hence, we describe a new anti-inflammatory activity and mechanism of stereocalpin A, owing to the negative regulation of TNF-?-induced adhesion molecule and MCP-1 expression, monocyte adhesion and ROS production in vascular smooth muscle cells. These results suggest that stereocalpin A has the potential to exert a protective effect by modulating inflammation within the atherosclerotic lesion.
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Sulforaphane suppresses vascular adhesion molecule-1 expression in TNF-?-stimulated mouse vascular smooth muscle cells: involvement of the MAPK, NF-?B and AP-1 signaling pathways.
Vascul. Pharmacol.
PUBLISHED: 07-04-2011
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Atherosclerosis is a long-term inflammatory disease of the arterial wall. Increased expression of the cell adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) is associated with increased proliferation of vascular smooth muscle cells (VSMCs), leading to increased neointima or atherosclerotic lesion formation. Therefore, the functional inhibition of adhesion molecules could be a critical therapeutic target of inflammatory disease. In the present study, we investigate the effect of sulforaphane on the expression of VCAM-1 induced by TNF-? in cultured mouse vascular smooth muscle cell lines. Pretreatment of VSMCs for 2h with sulforaphane (1-5?g/ml) dose-dependently inhibited TNF-?-induced adhesion of THP-1 monocytic cells and protein expression of VCAM-1. Sulforaphane also suppressed TNF-?-induced production of intracellular reactive oxygen species (ROS) and activation of p38, ERK and JNK. Furthermore, sulforaphane inhibited NK-?B and AP-1 activation induced by TNF-?. Sulforaphane inhibited TNF-?-induced ??? kinase activation, subsequent degradation of ???? and nuclear translocation of p65 NF-?B and decreased c-Jun and c-Fos protein level. This study suggests that sulforaphane inhibits the adhesive capacity of VSMC and downregulates the TNF-?-mediated induction of VCAM-1 in VSMC by inhibiting the MAPK, NF-?B and AP-1 signaling pathways and intracellular ROS production. Thus, sulforaphane may have beneficial effects to suppress inflammation within the atherosclerotic lesion.
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Streptococcus pneumoniae pep27 mutant as a live vaccine for serotype-independent protection in mice.
Vaccine
PUBLISHED: 04-04-2011
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Streptococcus pneumoniae (pneumococcus) is responsible for significant morbidity and mortality in worldwide. After introduction of current pneumococcal vaccines, a marked decrease in the incidence of pneumococcal disease was observed. Unfortunately, serotype shifts in carriage and disease, including capsular switch and presence of antimicrobial resistance, have been found. Here we report live attenuated vaccine strain which is avirulent and can protect from systemic and mucosal pneumococcal diseases. Pep27, an autolysis-inducing factor of S. pneumoniae is known to mediate LytA-dependent and -independent lysis and it was thus expected to effect virulence. The loss of Pep27 had a much larger than expected decrease in virulence and has made the Pep27 mutant strain sufficiently avirulent to be used as a live vaccine. The pep27 mutation unexpectedly had lower level of capsular polysaccharide than the wild type (type 2, D39) strain. Moreover, the pep27 mutant showed rapid clearance by 24 h post intranasal infection, and was not detected in lung and blood suggesting that mutant could not invade into the tissue. Even when 2×10(8)CFU were injected intravenously the mutant was not detected in the blood or brain after 4 h. Whereas 4 h after injection of 6×10(6) CFU of the wild type parent D39 strain, bacteremia was readily detected. Two dose intranasal immunizations with the live pep27 mutant in the absence of adjuvant elicited IgG antibody and serotype-independent protection against lethal intranasal challenge. Thus Pep27 was essential for virulence, and intranasal immunization with the pep27 mutant could provide protective immunity.
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Decrease in penicillin susceptibility due to heat shock protein ClpL in Streptococcus pneumoniae.
Antimicrob. Agents Chemother.
PUBLISHED: 03-21-2011
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Antibiotic resistance and tolerance are increasing threats to global health as antibiotic-resistant bacteria can cause severe morbidity and mortality and can increase treatment cost 10-fold. Although several genes contributing to antibiotic tolerance among pneumococci have been identified, we report here that ClpL, a major heat shock protein, could modulate cell wall biosynthetic enzymes and lead to decreased penicillin susceptibility. On capsular type 1, 2, and 19 genetic backgrounds, mutants lacking ClpL were more susceptible to penicillin and had thinner cell walls than the parental strains, whereas a ClpL-overexpressing strain showed a higher resistance to penicillin and a thicker cell wall. Although exposure of Streptococcus pneumoniae D39 to penicillin inhibited expression of the major cell wall synthesis gene pbp2x, heat shock induced a ClpL-dependent increase in the mRNA levels and protein synthesized by pbp2x. Inducible ClpL expression correlated with PBP2x expression and penicillin susceptibility. Fractionation and electron micrograph data revealed that ClpL induced by heat shock is localized at the cell wall, and the ?clpL showed significantly reduced net translocation of PBP2x into the cell wall. Moreover, coimmunoprecipitation with either ClpL or PBP2x antibody followed by reprobing with ClpL or PBP2x antibody showed an interaction between ClpL and PBP2x after heat stress. This interaction was confirmed by His tag pulldown assay with either ClpLHis? or PBP2xHis?. Thus, ClpL stabilized pbp2x expression, interacted with PBP2x, and facilitated translocation of PBP2x, a key protein of cell wall synthesis process, contributing to the decrease of antibiotic susceptibility in S. pneumoniae.
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Korean Red Ginseng Up-regulates C21-Steroid Hormone Metabolism via Cyp11a1 Gene in Senescent Rat Testes.
J Ginseng Res
PUBLISHED: 01-19-2011
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Ginseng (Panax ginseng Meyer) has been shown to have anti-aging effects in animal and clinical studies. However, the molecular mechanisms by which ginseng exerts these effects remain unknown. Here, the anti-aging effect of Korean red ginseng (KRG) in rat testes was examined by system biology analysis. KRG water extract prepared in feed pellets was administered orally into 12 month old rats for 4 months, and gene expression in testes was determined by microarray analysis. Microarray analysis identified 33 genes that significantly changed. Compared to the 2 month old young rats, 13 genes (Rps9, Cyp11a1, RT1-A2, LOC365778, Sv2b, RGD1565959, RGD1304748, etc.) were up-regulated and 20 genes (RT1-Db1, Cldn5, Svs5, Degs1, Vdac3, Hbb, LOC684355, Svs5, Tmem97, Orai1, Insl3, LOC497959, etc.) were down-regulated by KRG in the older rats. Ingenuity Pathway Analysis of untreated aged rats versus aged rats treated with KRG showed that the affected most was Cyp11a1, responsible for C21-steroid hormone metabolism, and the top molecular and cellular functions are organ morphology and reproductive system development and function. When genes in young rat were compared with those in the aged rat, sperm capacitation related genes were down-regulated in the old rat. However, when genes in the old rat were compared with those in the old rat treated with KRG, KRG treatment up-regulated C21-steroid hormone metabolism. Taken together, Cyp11a1 expression is decreased in the aged rat, however, it is up-regulated by KRG suggesting that KRG seems enhance testes function via Cyp11a1.
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Comparative in vitro activities of torezolid (DA-7157) against clinical isolates of aerobic and anaerobic bacteria in South Korea.
Antimicrob. Agents Chemother.
PUBLISHED: 09-13-2010
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Resistance of Gram-positive pathogens to first-line antimicrobial agents has been increasing in many parts of the world. We compared the in vitro activities of torezolid with those of other antimicrobial agents, including linezolid, against clinical isolates of major aerobic and anaerobic bacteria. Torezolid had an MIC(90) of ?0.5 ?g/ml for the Gram-positive bacterial isolates tested and was more potent than either linezolid or vancomycin.
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Inhibition of TNF-?-induced adhesion molecule expression by diosgenin in mouse vascular smooth muscle cells via downregulation of the MAPK, Akt and NF-?B signaling pathways.
Vascul. Pharmacol.
PUBLISHED: 07-06-2010
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Atherosclerosis is a chronic inflammatory disease and the expression of adhesion molecules on vascular smooth muscle cells (VSMCs) contributes to the progress of the disease. Diosgenin, a precursor of steroid hormones, has been shown to have a variety of biological activities including anti-inflammatory activity; however, its molecular mechanisms are poorly understood. This study examined the effect of diosgenin on the expression of adhesion molecules induced by TNF-? in cultured mouse VSMC cell line, MOVAS-1. Preincubation of VSMCs for 2h with diosgenin (0.1-10 ?M) dose-dependently inhibited TNF-?-induced adhesion of THP-1 monocytic cells and mRNA and protein expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). Diosgenin abrogated TNF-? induced production of intracellular reactive oxygen species (ROS) and phosphorylation of p38, ERK, JNK and Akt. Diosgenin was also shown to inhibit NK-?B activation induced by TNF-?. Furthermore, diosgenin inhibited TNF-?-induced I?B kinase activation, subsequent degradation of I?B?, and nuclear translocation of NF-?B. Our results indicate that diosgenin inhibits the adhesive capacity of VSMC and the TNF-?-mediated induction of ICAM-1 and VCAM-1 in VSMC by inhibiting the MAPK/Akt/NF-?B signaling pathway and ROS production, which may explain the ability of diosgenin to suppress inflammation within the atherosclerotic lesion and modulate immune response.
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ATF3 plays a key role in Kdo2-lipid A-induced TLR4-dependent gene expression via NF-?B activation.
PLoS ONE
PUBLISHED: 06-28-2010
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Activating transcription factor 3 (ATF3) is a negative regulator of proinflammatory cytokine expression in macrophages, and ATF3 deficient mice are more susceptible to endotoxic shock. This study addresses the role of ATF3 in the Kdo(2)-Lipid A-induced Toll-like receptor 4 (TLR4) signaling pathway in mouse embryonic fibroblasts (MEF). Kdo(2)-Lipid A upregulates ATF3 expression in wild type MEF cells and induces both nuclear factor kappa B (NF-?B) and c-Jun N-terminal kinase (JNK) activation via the TLR4 signaling pathway, while neither of these pathways is activated in ATF3-/- MEF cells. Interestingly, in contrast to Kdo(2)-Lipid A, the activation of both NF-?B and JNK by TNF-? was normal in ATF3-/- MEF cells.
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Virulence attenuation of Streptococcus pneumoniae clpP mutant by sensitivity to oxidative stress in macrophages via an NO-mediated pathway.
J. Microbiol.
PUBLISHED: 05-01-2010
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ClpP protease is essential for virulence and survival under stress conditions in several pathogenic bacteria. The clpP mutation in a murine infection model has demonstrated both attenuation of virulence and a sensitivity to hydrogen peroxide. However, the underlying mechanisms for these changes have not been resolved. Because macrophages play a major role in immune response and activated macrophages can kill microbes via oxygen-dependant mechanisms, we investigated the effect of the clpP mutation on its sensitivity to macrophage-mediated oxygen-dependant mechanisms. The clpP mutant derived from D39 (serotype 2) exhibited a higher sensitivity to oxidative stresses such as reactive oxygen intermediates, reactive nitrogen intermediates, and H(2)O(2), but no sensitivity to osmotic stress (NaCl) and pH. Moreover, viability of the clpP mutant was significantly increased in murine macrophage cells by treatment with S-methylisothiourea sulfate, which inhibits inducible nitric oxide synthase (iNOS) activity and subsequently elicits lower level secretions of nitric oxide (NO). However, viability of wild type was unchanged. Taken together, these results indicate that ClpP is involved in the resistance to oxidative stresses after entrapment by macrophages and subsequently contributes to virulence via NO mediated pathway.
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The inhibitory effect of acrylamide on NCAM expression in human neuroblastoma cells: involvement of CK2/Ikaros signaling pathway.
Toxicol In Vitro
PUBLISHED: 03-31-2010
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Acrylamide has been known to have a neurotoxic effect which is associated with nerve damage in both the central and peripheral nervous systems. Since neural cell adhesion molecule (NCAM) plays an important role in the processes of neuronal development and synaptic plasticity, the down-regulation of NCAM may lead impaired spatial memory and reduced long-term potentiation. We examined the effect of acrylamide on NCAM expression and the mechanisms of its effect in human neuroblastoma cells. Treatment with acrylamide resulted in the decrease of NCAM expression, which was reversed by CK2 inhibitor, 4,5,6,7-tetrabromobenzotriazole (TBB). Moreover, Western blot analysis showed that acrylamide induced the expression of CK2. Acrylamide dose-dependently decreased the DNA binding affinity of the Ikaros transcription factor, which is a bifunctional differentiation factor. In addition, the cells treated with acrylamide and CK2 inhibitor showed increased Ikaros activity compared with the acrylamide treatment only. Small interfering RNA-mediated depletion of CK2-? also increased Ikaros activity in acrylamide-treated cells. Overall, these data suggest that acrylamide decreases the Ikaros DNA binding activity via the CK2 pathway, resulting in a decrease of NCAM expression and provide further insight into the mechanisms underlying acrylamide actions.
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Characterization of ASC-2 as an antiatherogenic transcriptional coactivator of liver X receptors in macrophages.
Mol. Endocrinol.
PUBLISHED: 04-02-2009
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Activating signal cointegrator-2 (ASC-2) functions as a transcriptional coactivator of many nuclear receptors and also plays important roles in the physiology of the liver and pancreas by interacting with liver X receptors (LXRs), which antagonize the development of atherosclerosis. This study was undertaken to establish the specific function of ASC-2 in macrophages and atherogenesis. Intriguingly, ASC-2 was more highly expressed in macrophages than in the liver and pancreas. To inhibit LXR-specific activity of ASC-2, we used DN2, which contains the C-terminal LXXLL motif of ASC-2 and thereby acts as an LXR-specific, dominant-negative mutant of ASC-2. In DN2-overexpressing transgenic macrophages, cellular cholesterol content was higher and cholesterol efflux lower than in control macrophages. DN2 reduced LXR ligand-dependent increases in the levels of ABCA1, ABCG1, and apolipoprotein E (apoE) transcripts as well as the activity of luciferase reporters driven by the LXR response elements (LXREs) of ABCA1, ABCG1, and apoE genes. These inhibitory effects of DN2 were reversed by overexpression of ASC-2. Chromatin immunoprecipitation analysis demonstrated that ASC-2 was recruited to the LXREs of the ABCA1, ABCG1, and apoE genes in a ligand-dependent manner and that DN2 interfered with the recruitment of ASC-2 to these LXREs. Furthermore, low-density lipoprotein receptor (LDLR)-null mice receiving bone marrow transplantation from DN2-transgenic mice showed accelerated atherogenesis when administered a high-fat diet. Taken together, these results indicate that suppression of the LXR-specific activity of ASC-2 results in both defective cholesterol metabolism in macrophages and accelerated atherogenesis, suggesting that ASC-2 is an antiatherogenic coactivator of LXRs in macrophages.
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Characterization of the Streptococcus pneumoniae BgaC protein as a novel surface beta-galactosidase with specific hydrolysis activity for the Galbeta1-3GlcNAc moiety of oligosaccharides.
J. Bacteriol.
PUBLISHED: 03-06-2009
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Streptococcus pneumoniae is a causative agent of high morbidity and mortality. Although sugar moieties have been recognized as ligands for initial contact with the host, only a few exoglycosidases have been reported to occur in S. pneumoniae. In this study, a putative beta-galactosidase, encoded by the bgaC gene of S. pneumoniae, was characterized for its enzymatic activity and virulence. The recombinant BgaC protein, expressed and purified from Escherichia coli, was found to have a highly regiospecific and sugar-specific hydrolysis activity for the Galbeta1-3-GlcNAc moiety of oligosaccharides. Interestingly, the BgaC hydrolysis activity was localized at the cell surface of S. pneumoniae, indicating that BgaC is expressed as a surface protein although it does not have a typical signal sequence or membrane anchorage motif. The surface localization of BgaC was further supported by immunofluorescence microscopy analysis using an antibody raised against BgaC and by a reassociation assay with fluorescein isothiocyanate-labeled BgaC. Although the bgaC deletion mutation did not significantly attenuate the virulence of S. pneumoniae in vivo, the bgaC mutant strain showed relatively low numbers of viable cells compared to the wild type after 24 h of infection in vivo, whereas the mutant showed higher colonization levels at 6 and 24 h postinfection in vivo. Our data strongly indicate for the first time that S. pneumoniae bgaC encodes a surface beta-galactosidase with high substrate specificity that is significantly associated with the infection activity of pneumococci.
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Reduction-sensitive and cysteine residue-mediated Streptococcus pneumoniae HrcA oligomerization in vitro.
Mol. Cells
PUBLISHED: 02-20-2009
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In both gram-positive and several gram-negative bacteria, the transcription of dnaK and groE operons is negatively regulated by HrcA; however, the mechanism modulating HrcA protein activity upon thermal stress remains elusive. Here, we demonstrate that HrcA is modulated via reduction and oligomerization in vitro. Native-PAGE analysis was used to reveal the oligomeric structure of HrcA. The oligomeric HrcA structure became monomeric following treatment with the reducing agent dithothreitol, and this process was reversed by treatment with hydrogen peroxide. Moreover, the mutant HrcA C118S exhibited reduced binding to CIRCE elements and became less oligomerized, suggesting that cysteine residue 118 is important for CIRCE element binding as well as oligomerization. Conversely, HrcA mutant C280S exhibited increased oligomerization. An HrcA double mutant (C118S, C280S) was monomeric and exhibited a level of oligomerization and CIRCE binding similar to wild type HrcA, suggesting that cysteine residues 118 and 280 may function as checks to one another during oligomer formation. Biochemical fractionation of E. coli cells overexpressing HrcA revealed the presence of HrcA in the membrane fraction. Together, these results suggest that the two HrcA cysteine residues at positions 118 and 280 function as reduction sensors in the membrane and mediate oligomerization upon stress.
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Repeated intracerebral hemorrhage from developmental venous anomaly alone.
J Korean Neurosurg Soc
PUBLISHED: 01-31-2009
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Developmental venous anomalies (DVAs) are hemodynamically low flow, low resistance vascular malformations without clinical significance. Although most DVAs are asymptomatic and are found incidentally, sometimes they can be symptomatic with intracerebral hemorrhage, many of which are usually caused by associated cavernous malformations (CMs) rather than the DVAs themselves. Only a few cases have been reported in the literature where an intracerebral hemorrhage has been caused by a DVA alone. This report describes a case of an intracerebral hemorrhage due to DVA alone with review of the literature.
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Pneumococcal surface protein A inhibits complement deposition on the pneumococcal surface by competing with the binding of C-reactive protein to cell-surface phosphocholine.
J. Immunol.
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In the presence of normal serum, complement component C3 is deposited on pneumococci primarily via the classical pathway. Pneumococcal surface protein A (PspA), a major virulence factor of pneumococci, effectively inhibits C3 deposition. PspAs C terminus has a choline-binding domain that anchors PspA to the phosphocholine (PC) moieties on the pneumococcal surface. C-reactive protein (CRP), another important host defense molecule, also binds to PC, and CRP binding to pneumococci enhances complement C3 deposition through the classical pathway. Using flow cytometry of PspA(+) and PspA(-) strains, we observed that the absence of PspA led to exposure of PC, enhanced the surface binding of CRP, and increased the deposition of C3. Moreover, when the PspA(-) mutant was incubated with a pneumococcal eluate containing native PspA, there was decreased deposition of CRP and C3 on the pneumococcal surface compared with incubation with an eluate from a PspA(-) strain. This inhibition was not observed when a recombinant PspA fragment, which lacks the choline-binding region of PspA, was added to the PspA(-) mutant. Also, there was much greater C3 deposition onto the PspA(-) pneumococcus when exposed to normal mouse serum from wild-type mice as compared with that from CRP knockout mice. Furthermore, when CRP knockout mouse serum was replenished with CRP, there was a dose-dependent increase in C3 deposition. The combined data reveal a novel mechanism of complement inhibition by a bacterial protein: inhibition of CRP surface binding and, thus, diminution of CRP-mediated complement deposition.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.