Regulation system for protease production in Vibrio vulnificus

Vibrio vulnificus is a causative agent of serious food-borne diseases in humans related to consumption of raw seafoods. This human pathogen secretes a metalloprotease (VVP) that evokes enhancement of the vascular permeability and disruption of the capillaries. Production of microbial proteases is generally induced at early stationary phase of its growth. This cell density dependent regulation of VVP production in V. vulnificus known to be the quorum-sensing. When V. vulnificus was cultivated in Luria-Bertani (LB) medium, accumulation of the autoinducer, the signal molecule operating the quorum-sensing system, was detected. Moreover, expression of the vvp gene encoding VVP was found to be closely related with expression of the luxS gene that encode the synthase of the autoinducer precursor (luxS). These findings may indicate VVP production is controlled by the quorum-sensing system in LB medium. Furthermore, this system functioned more effectively at 26 degrees C than at 37 degrees C. When incubated at 37 degrees C in human serum supplemented with ferric chloride, production of VVP and expression of vvp increased in proportion to the concentration of ferric ion; whereas, expression of luxS was not increased. This suggests that VVP production in human serum containing ferric ion may be regulated mainly by the system other than the quorum-sensing system.     

Inhibition of nitric oxide production in lipopolysaccharide-stimulated RAW264.7 macrophage cells by lignans isolated from Euonymus alatus leaves and twigs

The 80% methanolic extract of Euonymus alatus leaves and twigs afforded three new lignans, (−)-threo-4,9,4′,9′-tetrahydroxy-3,7,3′,5′-tetramethoxy-8-O-8′-neolignan (1), (−)-threo-4,9,4′,9′-tetrahydroxy-3,5,7,3′-tetramethoxy-8-O-8′-neolignan (2), (7R,8R,7′R)-(+)-lyoniresinol (3), together with seventeen known lignans (4-20). The structures of 1-20 were elucidated by extensive 1D and 2D spectroscopic methods including ¹H NMR, ¹³C NMR, ¹H-¹H COSY, HMQC, HMBC and NOESY. All the isolated compounds except for dilignans (19 and 20) significantly inhibited nitric oxide production in lipopolysaccharide-stimulated RAW264.7 cells.     

Inhibition of nitric oxide production by natural oxyprenylated coumarins and alkaloids in RAW 264.7 cells

A series of naturally occurring 3,3-dimethylallyloxy- and geranyloxycoumarins and alkaloids were chemically synthesized and tested as anti-inflammatory agents for their inhibitory effects on nitric oxide production in LPS-stimulated RAW 264.7 cells. Results indicated that the alkaloid of fungal origin 3-methylbut-2-enyl-4-methoxy-8-[(3-methylbut-2-enyl)oxy]quinoline-2-carboxylate, commonly known as Ppc-1, and coumarins having an unsubstituted 2-benzopyrone ring exhibited the highest activity with IC₅₀ values from 23 to 34 μM without having poor or not detectable cytotoxicity. Indomethacine and L-NAME used as reference drugs provided by far less activities.     

Regulation systems of protease and hemolysin production in Vibrio vulnificus

Vibrio vulnificus, a gram‐negative halophilic estuarine bacterium, is an opportunistic human pathogen that causes rapidly progressive fatal septicemia and necrotizing wound infection. This species also causes hemorrhagic septicemia called vibriosis in cultured eels. It has been proposed that a range of virulence factors play roles in pathogenesis during human and/or eel infection. Among these factors, a metalloprotease (V. vulnificus protease [VVP]) and a cytolytic toxin (V. vulnificus hemolysin [VVH]) are of significant importance. VVP elicits the characteristic edematous and hemorrhagic skin damage, whereas VVH exhibits powerful hemolytic and cytolytic activities and contributes to bacterial invasion from the intestine to the blood stream. In addition, a few V. vulnificus strains isolated from diseased eels have recently been found to produce a serine protease designated as V. vulnificus serine protease (VvsA) instead of VVP. Similarly to VVP, VvsA may possess various toxic activities such as collagenolytic, cytotoxic and edema‐forming activity. In this review, regulation of V. vulnificus VVP, VVH and VvsA is clarified in terms of expression at the mRNA and protein levels. The explanation is given on the basis of the quorum sensing system, which is dependent on bacterial cell density. In addition, the roles of environmental factors and global regulators, such as histone‐like nucleoid structuring protein, cyclic adeno monophosphate receptor protein, RpoS, HlyU, Fur, ToxRS, AphB and LeuO, in this regulation are outlined. The cumulative impact of these regulatory systems on the pathogenicity of V. vulnificus is here delineated.     

Nystatin-induced nitric oxide production in mouse macrophage-like cell line RAW264.7

Nystatin is known to deplete lipid rafts from mammalian cell membranes. Lipid rafts have been reported to be necessary for lipopolysaccharide signaling. In this study, it was unexpectedly found that lipopolysaccharide-induced nitric oxide production was not inhibited, but rather increased in the presence of a non-cytotoxic concentration of nystatin. Surprisingly, treatment with nystatin induced only NO production and iNOS expression in RAW264.7 cells. At the concentration used, no changes in the expression of GM1 ganglioside, a lipid raft marker on RAW264.7 cells, was seen. From studies using several kinds of inhibitors for signaling molecules, nystatin-induced NO production seems to occur via the iκB/NF-κB and the PI3 K/Akt pathway. Furthermore, because nystatin is known to activate the Na-K pump, we examined whether the Na-K pump inhibitor amiloride suppresses nystatin-induced NO production. It was found that amiloride significantly inhibited nystatin-induced NO production. The results suggest that a moderate concentration of nystatin induces NO production by Na-pump activation through the PI3 kinase/Akt/NF-κB pathway without affecting the condition of lipid rafts.     

Identification and structure activity relationship of novel flavone derivatives that inhibit the production of nitric oxide and PGE2 in LPS-induced RAW 264.7 cells

In an effort to identify novel anti-inflammatory compounds, a series of flavone derivatives were synthesized and biologically evaluated for their inhibitory effects on the production of nitric oxide (NO) and prostaglandin E₂ (PGE₂), representative pro-inflammatory mediators, in LPS-induced RAW 264.7 cells. Their structure-activity relationship was also investigated. In particular, we found that compound 3g displayed more potent inhibitory activities on PGE₂ production, similar inhibitory activities on NO production and less weak cytotoxicity than luteolin, a natural flavone known as a potent anti-inflammatory agent.     

Anti-inflammatory phenolics isolated from Juniperus rigida leaves and twigs in lipopolysaccharide-stimulated RAW264.7 macrophage cells

Inflammation is an essential host defense system particularly in response to infection and injury; however, excessive or undesirable inflammatory responses contribute to acute and chronic human diseases. A high-throughput screening effort searching for anti-inflammatory compounds from medicinal plants deduced that the methanolic extract of Juniperus rigida S. et L. (Cupressaceae) inhibited significantly nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. Activity-guided fractionation and isolation yielded 13 phenolic compounds, including one new phenylpropanoid glycosides, 3,4-dimethoxycinnamyl 9-O-β-D-glucopyranoside (1). Among the isolated compounds, phenylpropanoid glycosides with p-hydroxy group (2, 4) and massoniaside A (7), (+)-catechin (10), amentoflavone (11) effectively inhibited LPS-induced NO production in RAW264.7 cells.     

Sentulic acid isolated from Sandoricum koetjape Merr attenuates lipopolysaccharide and interferon gamma co-stimulated nitric oxide production in murine macrophage RAW264 cells

A seco-triterpenoid, sentulic acid (SA) isolated from Sandoricum koetjape Merr attenuated nitric oxide (NO) production following co-stimulation with lipopolysaccharide (LPS) and interferon-gamma (IFNγ) in RAW264.7 macrophage cells. The mRNA expression levels of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), IFNγ, interleukin (IL)-6, and IL-12 in LPS/IFNγ co-stimulated RAW264.7 cells also decreased upon SA treatment. To determine the molecular mechanisms underlying the inhibitory effect of SA on LPS/IFNγ-induced NO production in RAW264.7 cells, we further analyzed Toll-like receptor (TLR) signaling by western blotting. The expression of TLR4 and IFN signaling molecules in cells treated with SA was significantly suppressed compared to that in cells not treated with SA. Additionally, SA inhibited the binding of LPS to the TLR4 receptor in RAW264.7 cells stimulated with Alexa Fluor 488-conjugated LPS. These results demonstrate that SA attenuates NO production after LPS/IFNγ co-stimulation in RAW264.7 cells by inhibiting the binding of LPS to TLR4. Our findings suggest that SA is beneficial for the treatment of inflammatory diseases.     

Peroxiredoxin-1, a possible target in modulating inflammatory cytokine production in macrophage like cell line RAW264.7

Peroxiredoxin (PRX), a scavenger of H₂O₂ and alkyl hydroperoxides in living organisms, protects cells from oxidative stress. Contrary to its known anti‐oxidant roles, the involvement of PRX‐1 in the regulation of lipopolysaccharide (LPS) signaling is poorly understood, possible immunological functions of PRX‐1 having been uncovered only recently. In the present study, it was discovered that the PRX‐1 deficient macrophage like cell line (RAW264.7) has anti‐inflammatory activity when stimulated by LPS. Treatment with LPS for 3 hrs resulted in increased gene expression of an anti‐inflammatory cytokine, interleukin‐10 (IL‐10), in PRX‐1 knock down RAW264.7 cells. Gene expression of pro‐inflammatory cytokines IL‐1β and tumor necrosis factor‐ α (TNF‐α) did not show notable changes under the same conditions. However, production of these cytokines significantly decreased in PRX‐1 knock down RAW264.7 cells with 12 hrs of stimulation. Production of IL‐10 was also increased in PRX‐1 knock down RAW264.7 cells with 12 hrs of stimulation. We predicted that higher concentrations of IL‐10 would result in decreased expression of IL‐1β and TNF‐α in PRX‐1 knock‐down cells. This was confirmed by blocking IL‐10, which reestablished IL‐1β and TNF‐α secretion. We also observed that increased concentrations of IL‐10 do not affect the NF‐κB pathway. Interestingly, STAT3 phosphorylation by LPS stimulation was significantly increased in PRX‐1 knockdown RAW264.7 cells. Up‐regulation of IL‐10 in PRX‐1 knockdown cells and the resulting downregulation of proinflammatory cytokine production seem to involve the STAT3 pathway in macrophages. Thus, down‐regulation of PRX‐1 may contribute to the suppression of adverse effects caused by excessive activation of macrophages through affecting the STAT3 signaling pathway.     

水产品中创伤弧菌和副溶血弧菌双重PCR检测方法的建立

目的 建立一种同步检测创伤弧菌和副溶血弧菌的双重PCR方法。方法 选择副溶血弧菌tlh基因和创伤弧菌vvhA基因作为靶序列各设计一对引物。用合成的引物对副溶血弧菌和创伤弧菌进行双重PCR扩增,确定特异性和最低检出限。然后用此方法对53株副溶血弧菌和7株创伤弧菌进行检测。结果 确定了双重PCR检测创伤弧菌和副溶血弧菌的最优反应条件,其中退火温度为60 ℃,方法具有较好的特异性。对副溶血弧菌的最低限为1.0×102 CFU/mL,创伤弧菌最低限为4.2×104 CFU/mL。双重PCR对分离株检测符合率达100%。结论 建立的双重PCR方法简便、快速、特异性好,可同时检测副溶血弧菌和创伤弧菌,为水产品中病原菌的基层检测提供解决方案。     

Fucoidan对巨噬细胞RAW264.7的免疫调节作用

目的 观察褐藻多糖硫酸酯（Fucoidan）对巨噬细胞RAW264.7体外吞噬活性、细胞因子TNF-α和IL-6分泌,以及Toll样受体4（TLR4）mRNA表达的影响。方法 实验分对照组,Fucoidan高、中、低剂量组（浓度分别是200、400和800 μg/mL）。药物处理6~48 h后,MTT法检测RAW264.7细胞活力;中性红比色法检测细胞吞噬活性;ELISA法检测培养上清中TNF-α和IL-6的分泌水平;实时定量PCR检测Toll样受体4（TLR4）mRNA表达量。结果 与对照组相比,Fucoidan显著增强RAW264.7细胞代谢活力和吞噬能力（P＜0.01）,增加TNF-α和IL-6的分泌,上调TLR4的表达,呈剂量依赖关系。结论 Fucoidan可上调TLR4表达,增强巨噬细胞代谢和吞噬活性,增加TNF-α和IL-6的分泌,具有潜在的调节免疫作用。     

Isolation and characterization of hemolysin mutants of Vibrio vulnificus

Abstract The importance of the cytolysin/hemolysin in the virulence of Vibrio vulnificus was investigated using both the naturally occuring virulent and avirulent colony variants and ethylmethane-sulfonate generated mutants. Both virulent and avirulent isogenic morphotypes produced similar amounts of hemolysin. Two mutants deficient in the production of hemolysin and negative for CHO cell activity were characterized and their virulence for mice was examined. Non-hemolytic mutants were found to be as virulent as their parent strain. It is concluded that the hemolysin produced by V. vulnificus is not required for the full virulence of this pathogen.     

Pyranocoumarins from Glehnia littoralis inhibit the LPS-induced NO production in macrophage RAW 264.7 cells

A new dihydropyranocoumarin, (+)-cis-(3′S,4′S)-diisobutyrylkhellactone (1), together with five known compounds, 3′-senecioyl-4′-acetylkhellactone (2), 3′-isovaleryl-4′-acetylkhellactone (3), 3′,4′-disenecioylkhellactone (4), 3′-isovaleryl-4′-senecioylkhellactone (5), and 3′,4′-diisovalerylkhellactone (6), was isolated from Glehnia littoralis. Their chemical structures were elucidated based on the spectroscopic data interpretation, particularly 1D and 2D NMR data including HMQC and HMBC. All the isolated compounds showed the potential to inhibit LPS-induced nitric oxide production in RAW 264.7 cells with IC₅₀ values ranging from 7.4 to 44.3 μM.     

变性高效液相色谱技术对创伤弧菌检测的研究

应用PCR结合变性高效液相色谱技术对创伤弧菌进行检测,建立创伤弧菌快速准确的检测新方法。经过DHPLC分析条件优化,在DHPLC非变性温度下分析创伤弧菌特异性PCR扩增产物。同时进行方法特异性、灵敏度、重复性实验。实验结果表明所建立的创伤弧菌PCR-DHPLC检测方法特异性强、灵敏度高、重现性好、结果稳定可靠、检测时间短,检测低限可达到124 CFU/mL,是创伤弧菌快速检测的新技术。     

A novel multiplex PCR for the identification of Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus

AIM: To establish a simple multiplex polymerase chain reaction (PCR) that will identify Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus. METHODS AND RESULTS: A total of 429 Vibrio spp. from various origins were tested with the novel primers targeting toxR. The reverse primers were all designed to be species specific, while the forward primer was universal. The primers correctly identified all the V. parahaemolyticus, V. cholerae and V. vulnificus isolates tested. CONCLUSIONS: The toxR multiplex PCR works well when the initial colony morphology is known. If not, Vibrio alginolyticus might represent a diagnostic obstacle. SIGNIFICANCE AND IMPACT OF THE STUDY: The method provides a fast and reliable way of identifying the main Vibrio spp. involved in food-borne disease. The method could prove very useful for laboratories working with identification of these Vibrio spp.     

Inhibition of nitric oxide production in LPS-stimulated RAW 264.7 cells by stem bark of Ulmus pumila L.

This study was designed to isolate and identify a potent inhibitory compound against nitric oxide (NO) production from the stem bark of Ulmus pumila L. Ethyl acetate fraction of hot water extract registered a higher level of total phenolics (756.93 mg GAE/g) and also showed strong DPPH (IC₅₀ at 5.6 μg/mL) and ABTS (TEAC value 0.9703) radical scavenging activities than other fractions. Crude extract and its fractions significantly decreased nitrite accumulation in LPS-stimulated RAW 264.7 cells indicating that they potentially inhibited the NO production in a concentration dependent manner. Based on higher inhibitory activity, the ethyl acetate fraction was subjected to Sephadex LH-20 column chromatography and yielded seven fractions and all these fractions registered appreciable levels of inhibitory activity on NO production. The most effective fraction F1 was further purified and subjected to ¹H, ¹³C-NMR and mass spectrometry analysis and the compound was identified as icariside E₄. The results suggest that the U. pumila extract and the isolated compound icariside E₄ effectively inhibited the NO production and may be useful in preventing inflammatory diseases mediated by excessive production of NO.     

Modification of the lipidome in RAW264.7 macrophage subjected to stable silencing of oxysterol-binding proteins

Oxysterol-binding protein homologs (ORPs) are implicated in lipid metabolism, vesicle transport and cell signaling. In this study we generated RAW264.7 cells with ORP1L, ORP3, or ORP8 silenced using shRNA lentiviruses. The lipidome of the cells under basal serum-free culture conditions or as treated with oxidized LDL (oxLDL), enzymatically modified LDL (E-LDL), or lipopolysaccharide (LPS) was analyzed by mass spectrometry. Reduction in each ORP resulted in distinct and complex effects on macrophage lipidome. Under basal conditions, ORP1L silencing had strongest effects on phosphatidylinositols (PI, increase), free cholesterol (FC, increase), and cholesteryl esters (CE, increase). ORP3 silencing affected most the glucosyl ceramides (GluCer, decrease) and PE-plasmalogens (PE-pl, decrease), while ORP8 silencing increased FC and CE, and decreased GluCer and PE-pl. Upon LPS treatment, the ORP effects were modified: under these conditions ORP1L silencing caused increase of Cer, ORP3 silencing decrease of PI, and ORP8 silencing decrease of PI and increase of PE, not detectable under basal conditions. The lipid species data were subjected to multivariate statistical analysis of principal components, revealing numerous specific alterations upon ORP silencing. The cells cultured in basal conditions or treated with LPS showed qualitatively different responses. However, in LPS-stimulated cells silencing of any of the three ORPs decreased the relative amount of arachidonic acid-containing PI species, increased the corresponding PE species, and favored 16-carbon sphingomyelin (SM) species at the expense of the 24-carbon ones. As a conclusion, the present study reveals the distinct and sophisticated roles of different ORP proteins as regulators of macrophage lipid composition, with implications for inflammatory signaling.