Killing rate curve and combination effects of surfactin C produced from Bacillus subtilis complex BC1212 against pathogenic Mycoplasma hyopneumoniae

This study evaluated the killing rate as well as the antimycoplasmal effect of surfactins isolated from Bacillus subtilis complex BC1212, either alone or in combination with various antibacterials against Mycoplasma hyopneumoniae. Prior to the killing rate and the combination effect studies of surfactins, the minimal inhibitory concentrations (MICs) of various antibacterials and surfactins (consisting of surfactins A, B, C, and D) against M. hyopneumoniae were investigated. The MIC of all surfactins was found to be 64 μg/ml. The MICs of colistin (COL), norfloxacin (NFX), oxytetracycline (OTC), streptomycin (SM) and tiamulin (TIA) were >256, 0.063, 0.025, 4, and 0.015 μg/ml, respectively. In the killing rate curve studies, surfactin C at 2× MIC and 4× MIC concentrations was found to reduce viability by >3 log₁₀ c.c.u./ml within 2–4 h of incubation. Combination of surfactin C with other antibacterials showed additive interaction from the viewpoint of fractional inhibitory concentration (FIC) index of >0.5 but ≤2 as a borderline. Taking all results into consideration, surfactin C may be useful as a preventive or therapeutic adjuvant in the pathogenesis of mycoplasmal infection.     

Nitric oxide production by a murine macrophage cell line (RAW264.7 cells) stimulated with Aggregatibacter actinomycetemcomitans surface-associated material

Nitric oxide (NO) may play a crucial role in the pathogenesis of periodontal disease and, hence, the aim of the present study was to test the hypothesis that Aggregatibacter actinomycetemcomitans surface-associated material (SAM) stimulates inducible nitric oxide synthase (iNOS) activity and NO production by the murine macrophage cell line RAW264.7. Cells were stimulated with untreated or heat-treated A. actinomycetemcomitans SAM and with or without pre-treatment with l-N⁶-(1-Iminoethyl)-lysine (L-NIL) (an iNOS inhibitor), polymyxin B, interferon-gamma (IFN-γ) and Interleukin-4 (IL-4), IL-10, genistein [a protein tyrosine kinase (PTK) inhibitor], bisindolylmaleimide [a protein kinase C (PKC) inhibitor], bromophenacyl bromide (BPB) [a phospholipase A₂ (PLA2) inhibitor] or wortmannin [phosphatidylinositol 3-kinase (PI-3K) inhibitor]. The iNOS activity and nitrite production in the cell cultures were determined. Untreated but not heat-treated A. actinomycetemcomitans SAM-stimulated both iNOS activity and nitrite production in RAW264.7 cells. l-NIL, IL-4, IL-10, genistein, bisindolylmaleimide, or BPB, suppressed but IFN-γ enhanced both iNOS activity and nitrite production by A. actinomycetemcomitans SAM-stimulated cells. Wortmannin and polymyxin B failed to alter both iNOS activity or nitrite production by A. actinomycetemcomitans SAM treated cells. Therefore, the present study suggests that a heat-sensitive protein constituent(s) of A. actinomycetemcomitans SAM stimulates both iNOS activity and nitrite production by RAW264.7 cells in a cytokine, PTK, PKC, and PLA₂ but not PI-3K-dependent fashion.     

Pyrrolidinone diterpenoid from Isodon excisus and inhibition of nitric oxide production in lipopolysaccharide-induced macrophage RAW264.7 cells

A new pyrrolidinone diterpenoid, excisusin F (1), was isolated from the aerial parts of Isodon excisus (Lamiaceae), together with four known compounds, and their structures were determined mainly by NMR (1D and 2D) and mass spectrometry. Excisusin F (1) and inflexarabdonin E (3) showed potent inhibitory effects of LPS-induced nitric oxide production in RAW264.7 cells with the IC₅₀ value of 10.4 and 3.8 μM, respectively.     

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.     

Organization of the ribosomal RNA genes in Mycoplasma hyopneumoniae: the 5S rRNA gene is separated from the 16S and 23S rRNA genes

Summary In order to study the organization of the ribosomal RNA genes of Mycoplasma hyopneumoniae the rRNA genes were cloned in phage vectors EMBL3 and EMBL4. By subcloning the restriction fragments into various plasmids and analysing the resulting clones by Southern and Northern blot hybridization, a restriction map of the rRNA genes was generated and the organization of the rRNA genes was determined. The results show that the genes for the 16S and 23S rRNAs are closely spaced and occur only once in the genome, whereas the 5S rRNA gene is separated from the other two genes by more than 4 kb.     

Toxoplasma gondii partially inhibits nitric oxide production of activated murine macrophages

Activated macrophages produce nitric oxide (NO) and as such are able to control the multiplication of Toxoplasma gondii. Until now, no reports have described a possible modulation of NO production of macrophages after T. gondii infection. To investigate this possibility, murine blood monocyte-derived and peritoneal macrophages were activated in vitro with interferon-gamma and lipopolysaccharide and infected with T. gondii and Trypanosoma cruzi, and NO production was evaluated. NO was produced by monocyte-derived macrophages only if cultured in the presence of macrophage-colony-stimulating factor. Monocyte-derived or peritoneal macrophages infected with T. gondii presented a significant reduction in NO production. NO production inhibition was not detected after T. cruzi infection. Macrophages infected with higher T. gondii/macrophage ratios presented lower NO production. Furthermore, only viable T. gondii could cause partial inhibition of NO production. In macrophages activated 24 h before the interaction, partial inhibition was detected after 3 h of infection and continued for 48 h. In macrophages activated immediately after the interaction, partial inhibition was not detected at 12 h, but was observed at 24 h. T. gondii-infected macrophages present lower inducible nitric oxide synthase expression as assayed by immunofluorescence. T. gondii did not develop in monocyte-derived macrophages producing NO, but were not totally eliminated. These results demonstrate that T. gondii infection partially inhibits NO production by murine macrophages, suggesting that a deactivating macrophage mechanism may be used for better survival into phagocytic cells.     

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.     

Involvement of protein kinase C in the inhibition of lipopolysaccharide-induced nitric oxide production by thapsigargin in RAW 264.7 macrophages

This study explored the effects of inhibition of endoplasmic reticulum (ER) Ca²⁺-ATPase on lipopolysaccharide (LPS)-induced protein kinase C (PKC) activation, nuclear factor-κB (NF-κB) translocation, inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in RAW 264.7 macrophages. Thapsigargin (TG) irreversibly inhibits ER Ca²⁺-ATPase and LPS-induced NO production is reduced even after washout. TG also attenuated LPS-stimulated iNOS expression by using immunoblot analysis. However, another distinct fully reversible ER Ca²⁺-ATPase inhibitor, 2,5-di-tert-butylhydroquinone (DBHQ), ionophore A23187 and ionomycin could exert a similar effect to TG in increasing intracellular calcium concentration; however, these agents could not mimic TG in reducing iNOS expression and NO production. LPS increased PKC- and -β activation, and TG pretreatment attenuated LPS-stimulated PKC activation. Not did pretreatment with DBHQ, A23187 and ionomycin reduce LPS-stimulated PKC activation. Furthermore, NF-κB-specific DNA–protein-binding activity in the nuclear extracts was enhanced by treatment with LPS, and TG pretreatment attenuated LPS-stimulated NF-κB activation. None of DBHQ, A23187 and ionomycin pretreatment reduced LPS-stimulated NF-κB activation. These data suggest that persistent inhibition of ER Ca²⁺-ATPase by TG would influence calcium release from ER Ca²⁺ pools that was stimulated by the LPS activated signal processes, and might be the main mechanism for attenuating PKC and NF-κB activation that induces iNOS expression and NO production.     

Inhibitory effects of Irigenin from the rhizomes of Belamcanda chinensis on nitric oxide and prostaglandin E(2) production in murine macrophage RAW 264.7 cells

In the present study, we investigated antiinflammatory effects of six flavonoids isolated from the rhizomes of Belamcanda chinensis (Iridaceae) in RAW 264.7 macrophages. The results indicated that irigenin concentration dependently inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin (PG) E(2) production. Furthermore, this compound inhibited the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 proteins and mRNAs without an appreciable cytotoxic effect. Treatment of the transfectant RAW 264.7 cells with irigenin reduced the level of nuclear factor-kappaB (NF-kappaB) activity, also effectively lowered NF-kappaB binding measured by electrophoretic mobility shift assay (EMSA), which was associated with decreased p65 protein levels in the nucleus. On the basis of the above data, we suggest that the effect of irigenin in decreasing LPS-induced NO and PGE(2) synthesis is due to diminish the mRNA and protein expression of iNOS and COX-2, respectively, also may be due to under the suppression of NF-kappaB activation. Therefore, irigenin isolated from the rhizomes of Belamcanda chinensis could be offered as a leading compound for anti-inflammation.     

Polysaccharides isolated from <Emphasis Type="Italic">Phellinus baumii</Emphasis> stimulate murine splenocyte proliferation and inhibit the lipopolysaccharide-induced nitric oxide production in RAW264.7 murine macrophages

Polysaccharides isolated from Phellinus baumii (PBP) significantly enhanced both lipopolysaccharide (LPS)-induced B lymphocyte proliferation and concanavalin A (Con A)-induced T lymphocyte proliferation. However, PBP (12.5–100 μg ml⁻¹) significantly suppressed the LPS-induced nitric oxide (NO) production in RAW264.7 cells in a concentration-dependent manner. The maximal inhibition of PBP on NO production was 37.5% at 100 μg ml⁻¹. These results provide useful in vitro information to explain the immunostimulating activity and anti-inflammatory activity of PBP.     

Proteomic comparative analysis of pathogenic strain 232 and avirulent strain J of <Emphasis Type="Italic">Mycoplasma hyopneumoniae</Emphasis>

Mycoplasma hyopneumoniae is an important pathogen of pigs causing enzootic pneumonia of swine. The pathogen remains largely enigmatic as far as the host-pathogen interactions are concerned. In the present study, the protein profiles of two strains of M. hyopneumoniae were compared by two-dimensional gel electrophoresis and mass spectrometry. The results indicate that the major adhesin P97, the 50-kDa protein derived from P159 adhesin, and the 43-kDa cleavage product of P102 are expressed at much higher levels in the pathogenic strain 232. In contrast, the avirulent strain J switches its focus to metabolism and expresses more glyceraldehyde 3-phosphate dehydrogenase in gluconeogenesis and lactate dehydrogenase, pyruvate dehydrogenase, and phosphate acetyltransferase in the pyruvate metabolism pathway. We speculate that the avirulent strain may have developed better capabilities to cope with the rich environment during repeated inoculations. Simultaneously, the capability to infect host cells may become less important so that the adhesion-related protein genes are down-regulated. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 2, pp. 264–271.     

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.     

The enhancing action of D-galactosamine on lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophage cells

The effect of D-galactosamine (D-GalN) on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells was examined. D-GalN augmented the production of NO, but not tumor necrosis factor (TNF)-alpha in LPS-stimulated RAW 264.7 cells. Pretreatment of D-GalN augmented the NO production whereas its post-treatment did not. D-GalN augmented the NO production in RAW 264.7 cells stimulated with either TNF-alpha and interferon-gamma. The augmentation of LPS-induced NO production by D-GalN was due to enhanced expressions of an inducible type of NO synthase mRNA and proteins. Intracellular reactive oxygen species (ROS) were exclusively generated in RAW 264.7 cells stimulated with D-GalN and LPS. Scavenging of intracellular ROS abrogated the augmentation of NO production. It was therefore suggested that D-GalN might augment LPS-induced NO production through the generation of intracellular ROS.     

Inhibitory effect of resveratrol dimerized derivatives on nitric oxide production in lipopolysaccharide-induced RAW 264.7 cells

Four types of resveratrol dimerized analogues were synthesized and evaluated in vitro on LPS-induced NO production in RAW 264.7 cells. The results showed that several compounds, especially those containing 1,2-diphenyl-2,3-dihydro-1H-indene core (type I), exhibited good inhibitory activities. Among 25 analogues, 12b showed a significant inhibitory activity (49% NO production at 10 μM, IC₅₀ = 3.38 μM). Further study revealed that compound 12b could suppress LPS-induced iNOS expression, NO production, and IL-1β release in a concentration-dependently manner. The mechanism of action (MOA) involved for its anti-inflammatory responses was through signaling pathways of p38 MAPK and JNK1/2, but not ERK1/2.     

8-Hydroxyquinoline inhibits iNOS expression and nitric oxide production by down-regulating LPS-induced activity of NF-kappaB and C/EBPbeta in Raw 264.7 cells

In activated macrophage, large amounts of nitric oxide (NO) are generated by inducible nitric oxide synthase (iNOS), resulting in acute or chronic inflammatory disorders. In Raw 264.7 cells stimulated with lipopolysaccharide (LPS) to mimic inflammation, 8-hydroxyquinoline (8HQ) inhibited the LPS-induced expression of both iNOS protein and mRNA in a parallel dose-dependent manner. 8HQ did not enhance the degradation of iNOS mRNA. To investigate the mechanism by which 8HQ inhibits iNOS gene expression, we examined the activation of MAP kinases in Raw 264.7 cells. We did not observe any significant change in the phosphorylation of MAPKs between LPS alone and LPS plus 8HQ-treated cells. Moreover, 8HQ significantly inhibited the DNA-binding activity of nuclear factor-kappaB (NF-kappaB) and CCAAT/enhancer-binding protein beta (C/EBPbeta), but not activator protein-1 and cAMP response element-binding protein. Taken together, these results suggest that 8HQ acts to inhibit inflammation through inhibition of NO production and iNOS expression through blockade of C/EBPbeta DNA-binding activity and NF-kappaB activation.     

Nitric oxide production: a mechanism of Chlamydia trachomatis inhibition in interferon-γ-treated RAW264.7 cells

Abstract IFN-γ and/or LPS induced nitrite production and inhibition of Chlamydia trachomatis (CT) replication in the murine macrophage cell line, RAW264.7. Linear regression analysis demonstrated a strong correlation between nitrite production and inhibition of CT replication (correlation coefficients: −0.93, P < 0.001). l -NMMA specifically inhibited nitrite production and restored CT replication (55–71%). Inducible nitric oxide synthase (iNOS) mRNA was analyzed by Northern and dot blot hybridization with an iNOS cDNA probe. A strong correlation between iNOS mRNA expression and inhibition of CT replication also was observed (correlation coefficient: −0.97, P < 0.05). Furthermore, anti-TNF-α antibody, which completely neutralized biological activity of the secreted TNF-α, neither inhibited nitrite production nor restored CT replication in the LPS- and/or IFN-γ-treated RAW264.7 cells. In mouse peritoneal macrophages treated with IFN-γ, both l -NMMA and anti-TNF-α antibody inhibited nitrite production and restored CT replication. However, l -NMMA and the antibody had no effect upon nitrite production and CT inhibition in LPS-treated peritoneal macrophages. These data indicate that NO production is one mechanism for inhibition of CT replication in IFN-γ-activated murine macrophages.     

Synthesis of aurones and their inhibitory effects on nitric oxide and PGE2 productions in LPS-induced RAW 264.7 cells

Sulfuretin is one of major constituents of Rhus verniciflua that exerts anti-inflammatory activities. Some of aurones were synthesized as sulfuretin derivatives and evaluated for their abilities to inhibit NO and PGE₂ production in LPS-induced RAW 264.7 cells in order to reveal the relationship. Of the aurones synthesized in the present study, 2h and 2i, which possess C-6 hydroxyl group in A-ring and methoxy substituents in B-ring, more potently inhibited NO and PGE₂ production and were less cytotoxic than sulfuretin.