<b>OBJECTIVEb>To observe the binding power of polymyxin B (PMB) and its simulation peptide to lipopolysaccharide (LPS) and lipoid A.

<b>METHODSb>LPS and lipoid A were separately coated on biosensor. 5 microl of PMB (0.01 microg/L) 5 microl of its simulating peptide 1 (PMBSP1 0.01 microg/L) and 5 microl of its simulating peptide 2 (PMBSP2, 0.01 microg/L) were respectively added into the hydrophobic sample pool. The combining power of PMB and its simulating peptides PMBSP1 and PMBSP2 to LPS and lipoid A was compared. RESULTS (1) PMBSP1 almost did not bind LPS and lipoid A, while PMB and PMBSP2 possessed high affinity with LPS and lipoid A. (2) The peak value (98.41 +/- 7.31) rad/s of PMBSP2 binding LPS was much higher than that (83.58 +/- 5.42) rad/s of PMB in binding LPS (P < 0.05). While the peak value of PMB in binding lipoid A was similar to that of PMBSP2. (3) The peak value of PMB binding LPS was significantly lower than that of PMB in binding lipoid A (P < 0.05). But there was no difference between the peak value of PMBSP2 in binding LPS and that of PMBSP2 in binding lipoid A. (4) PMBSP2 could bind to LPS and lipoid A in a shorter time to reach peak levels.

<b>CONCLUSIONb>Compared with PMB, the PMBSP2 could bind to LPS and lipoid A in a shorter time. In addition, PMBSP2 exhibited similar affinity to LPS and lipoid A. This indicated that PMBSP might possess better anti-LPS activity due to its lack of space steric hindrance when PMBSP binding the lipoid A of LPS.

Cell Wall ; chemistry ; Endotoxins ; Gram-Negative Bacteria ; Lipopolysaccharides ; chemistry ; toxicity ; Peptides ; chemistry ; pharmacology ; Polymyxin B ; chemistry ; pharmacology

<b>OBJECTIVEb>To investigate the mechanism of polymyxin B (PMB) antagonizing the biological activity of lipopolysaccharide (LPS).

<b>METHODSb>The affinity of PMB for LPS and lipid A was assayed by biosensor, and the neutralization of PMB for LPS (2 ng/ml) was detected by kinetic turbidimetric limulus test. The releases of TNF-alpha and IL-6 in murine peritoneal macrophages a (PMphi) after exposure to LPS (100 ng/ml) were detected, and the expression levels of TLR4, TNF-alpha and IL-6 mRNA in PMphi induced by LPS (100 ng/ml) were measured by RT-PCR.

<b>RESULTSb>PMB had high-affinity to LPS and lipid A with dissociation equilibrium constants of 18.9 nmol/L and 11.1 nmol/L, respectively, and neutralized LPS in a dose-dependent manner. Furthermore, PMB could markedly inhibit the expressions of TLR4, TNF-alpha and IL-6 mRNA and the release of cycokines in LPS-stimulated murine peritoneal macrophages.

<b>CONCLUSIONSb>PMB neutralizes LPS and inhibites the expression and release of cycokines in macrophages, in which the affinity of PMB for lipid A plays an important role.

Animals ; Cytokines ; analysis ; Limulus Test ; Lipid A ; antagonists & inhibitors ; Lipopolysaccharides ; antagonists & inhibitors ; Macrophages ; chemistry ; Mice ; Polymyxin B ; pharmacology

Lipopolysaccharides (LPS) contamination in herbal crude polysaccharides is inevitable. The present study was performed to explore the effect of polymyxin B on abolishing the influence of LPS contamination in mononuclear cells. LPS was pretreated with polymyxin B sulfate (PB) at different concentrations for 1, 5 or 24 h, and then used to stimulate RAW264.7 and mouse peritoneal macrophages (MPMs). The nitric oxide (NO) and tumor necrosis factor-α (TNF-α) in cell culture supernatant, as the indications of cell response, were assayed. Bupleurum chinensis polysaccharides (BCPs) with trace amount contamination of LPS was treated with PB. 30 μg·mL of PB, treating LPS (10 and 1 000 ng·mL in stimulating RAW264.7 and MPMs respectively) at 37 °C for 24 h, successfully abolished the stimulating effect of LPS on the cells. When the cells were stimulated with LPS, BCPs further promoted NO production. However, pretreated with PB, BCPs showed a suppression of NO production in MPMs and no change in RAW264.7. In the in vitro experiments, LPS contamination in polysaccharide might bring a great interference in assessing the activity of drug. Pretreatment with PB (30 μg·mL) at 37 °C for 24 h was sufficient to abolish the effects of LPS contamination (10 and 1 000 ng·mL).
Animals ; Bupleurum ; chemistry ; Drug Contamination ; Drugs, Chinese Herbal ; analysis ; pharmacology ; Lipopolysaccharides ; analysis ; antagonists & inhibitors ; Macrophages ; drug effects ; metabolism ; Mice ; Nitric Oxide ; metabolism ; Polymyxin B ; analysis ; pharmacology ; Polysaccharides ; analysis ; pharmacology ; Tumor Necrosis Factor-alpha ; metabolism

Mouse spleen cells activated in a mixed lymphocyte reaction release a soluble factor, which induces a significant proliferative response in fresh mouse spleen cells. This proliferation inducing factor (PIF) was found to be heat stable (90 degrees C for 45 min) and also resistant to trypsin or chymotrypsin treatment. By using a sizing HPLC column, the molecular weight of PIF appears to be 25 kDa. Mouse spleen cells treated with anti-thy-1 + complement lost Con-A induced proliferative responses but responded well to PIF. B cell depleted spleen cells obtained by negative selection panning, did not respond to PIF. These results indicate that B cells proliferated in response to PIF. Polymixin-B, which blocks the B cell proliferative response to LPS, did not inhibit PIF induced proliferation.
Animal ; B-Lymphocytes/physiology* ; B-Lymphocytes/drug effects ; Bone Marrow/metabolism ; Cell Division/physiology ; Chromatography, High Pressure Liquid ; Chymotrypsin/pharmacology ; Dose-Response Relationship, Drug ; Growth Substances/pharmacology* ; Growth Substances/chemistry ; Heat ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Molecular Weight ; Polymyxin B/pharmacology ; Protein Denaturation ; Spleen/metabolism* ; Thymus Gland/metabolism ; Trypsin/pharmacology

<b>OBJECTIVEb>To study possible immunobiological potential of Osmunda japonica Thunb.

<b>METHODSb>Immunomodulatory effects of ethanol extracts prepared from rhizomes of O. japonica and phenolic compounds isolated from the extracts were investigated under the in vitro conditions using the rat peritoneal cells (2×10(6)/mL; 24 h culture). Biosynthesis of nitric oxide (NO) was assayed by Griess reagent, production of prostaglandin E2 (PGE2) and secretion of cytokines were determined by enzyme-linked immunoabsorbent assay.

<b>RESULTSb>The extracts activated dose dependently, with the onset at 2.5-5 μmol/L concentrations, the high output NO production, and secretion of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). Mild enhancement of NO was produced by the aldehyde-type phenolics 4-hydroxybenzaldehyde and 3,4-hydroxybenzaldehyde. In contrasts, the acetone-type phenolics 4-hydroxybenzalacetone and 3,4-hydroxybenzalacetone inhibited production of immune mediators including cytokines (TNF-α, IL-1β, IL-6), NO, and PGE2. The 3,4-hydroxybenzalacetone was more effective than 4-hydroxybenzaldehyde. The IC50s estimates ranged within the interval of 5-10 μmol/L. No signs of cytotoxicity were observed up to the 50 μmol/L concentration of the compounds.

<b>CONCLUSIONb>Phenolic compounds contained in medicinal herb Osmunda japonica possess distinct immunomodulatory activity.

Animals ; Cell Survival ; drug effects ; Cells, Cultured ; Dinoprostone ; biosynthesis ; Female ; Ferns ; chemistry ; Immunologic Factors ; pharmacology ; Interferon-gamma ; pharmacology ; Lipopolysaccharides ; pharmacology ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Peritoneum ; cytology ; drug effects ; Phenols ; chemistry ; isolation & purification ; pharmacology ; Plant Extracts ; chemistry ; isolation & purification ; pharmacology ; Polymyxin B ; pharmacology ; Proline ; analogs & derivatives ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Thiocarbamates ; pharmacology

<b>BACKGROUNDb>It has been identified that in patients with pre-eclampsia, several factors were released into the serum, which can regulate the proliferation of vascular smooth muscle cells and stimulate the synthesis of extracellular matrix (ECM). However, the signal transduction pathway of the growth factors and cytokines synthesized by endothelial cells leading to the proliferation and ECM synthesis of human umbilical arterial smooth muscle cell (HUASMC) is unknown. The aim of this study was to research the effects of protein kinase C (PKC) inhibitor, polymyxin B (PMB), on the proliferation, nuclear factor-kappa B (NF-kappaB) activation, and expression of procollagen I and III mRNA in HUASMC cultured with pre-eclamptic umbilical sera.

<b>METHODSb>Normal HUASMCs were treated with pre-eclamptic umbilical serum (pre-eclamptic group), pre- eclamptic umbilical serum plus PMB (PMB group), or normal umbilical serum (normal group). The expression of I-kappaB, NF-kappaB was detected by Western blotting after the HUASMC was incubated for 2 hours. The proliferation of HUASMC was evaluated by MTT, the cell cycle was analyzed by flow cytomerty, and the expression of procollagen I, III mRNA was measured by RT-PCR assay after HUASMC was incubated for 48 hours. ANOVA was used for statistical analysis.

<b>RESULTSb>Umbilical sera in pre-eclampsia could stimulate the proliferation, the DNA synthesis, the transition of G(0) + G(1) phase or G(2)/M phase to S and phase, the activation of NF-kappaB, and the expression of procollagen I mRNA of HUASMC as compared with normal umbilical sera (P < 0.01). PMB could inhibit the proliferation, the DNA synthesis, the transition of G(0) + G(1) or G(2)/M phase phase to S phase, the activation of NF-kappaB, and the expressions of procollagen I mRNA of HUASMC stimulated by umbilical sera in pre-eclampsia (P < 0.01).

<b>CONCLUSIONb>PKC-NF-kappaB signal transduction pathway may play a key role in SMC phenotype modulation, which is more important in the pathogenesis of placental blood vessel in pre-eclampsia.

Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen Type I ; genetics ; Collagen Type III ; genetics ; Female ; Humans ; Muscle, Smooth, Vascular ; cytology ; metabolism ; Myocytes, Smooth Muscle ; metabolism ; NF-kappa B ; physiology ; Polymyxin B ; pharmacology ; Pre-Eclampsia ; metabolism ; pathology ; Pregnancy ; Protein Kinase C ; antagonists & inhibitors ; physiology ; RNA, Messenger ; analysis ; Signal Transduction ; Umbilical Arteries ; metabolism

<b>AIMb>To elucidate the genetic basis for the pronounced resistance that the oral pathogen, Porphyromonas gingivalis (P. gingivalis), exhibits towards the cationic antimicrobial peptide, polymyxin B.

<b>METHODOLOGYb>A genetic screen of P. gingivalis clones generated by a Tn4400'-based random insertion mutagenesis strategy was performed to identify bacteria harboring novel genetic mutations that render P. gingivalis susceptible to killing by the cationic antimicrobial peptide, polymyxin B (PMB, 50 microg x mL(-1)).

<b>RESULTSb>P. gingivalis (ATCC 33277) is unusually resistant to the cationic antimicrobial peptide, PMB at relatively high concentrations (200 microg x mL(-1)). Approximately 2,700 independent Tn4400'-derived mutants of P. gingivalis were examined for increased sensitivity to PMB killing at a relatively low dose (50 microg x mL(-1)). A single PMB-sensitive mutant was obtained in this phenotypic screen. We determined that the Tn4400' transposon was integrated into the gene encoding the lipid A 4'-phosphatase, PGN_0524, demonstrating that this insertion event was responsible for its increased susceptibility of this clone to PMB-dependent killing. The resulting mutant strain, designated 0524-Tn4400', was highly sensitive to PMB killing relative to wild-type P. gingivalis, and exhibited the same sensitivity as the previously characterized strain, 0524KO, which bears a genetically engineered deletion in the PGN_0524 locus. Positive ion mass spectrometric structural (MALDI-TOF MS) analyses revealed that lipid A isolates from 0524-Tn4400' and 0524KO strains displayed strikingly similar MALDI-TOF MS spectra that were substantially different from the wildtype P. gingivalis lipid A spectrum. Finally, intact 0524-Tn4400' and 0524KO mutant bacteria, as well as their corresponding LPS isolates, were significantly more potent in stimulating Toll-like receptor 4 (TLR4)-dependent E-selectin expression in human endothelial cells relative to intact wild-type P. gingivalis or its corresponding LPS isolate.

<b>CONCLUSIONb>The combined molecular evidence provided in this report suggests that PGN_0524, a lipid A 4'-phosphatase, is the sole genetic element conferring the ability of the periodontopathogen, P. gingivalis, to evade the killing activity of cationic antimicrobial peptides, such as PMB. These data strongly implicate PGN_0524 as a critical virulence factor for the ability of P. gingivalis to evade front-line host innate defenses that are dependent upon cationic antimicrobial peptide activity and TLR 4 sensing.

Anti-Bacterial Agents ; pharmacology ; Chromosome Mapping ; DNA Transposable Elements ; genetics ; Drug Resistance, Bacterial ; genetics ; E-Selectin ; analysis ; immunology ; Endothelial Cells ; immunology ; microbiology ; Gene Deletion ; Humans ; Lipid A ; analysis ; immunology ; Lipopolysaccharides ; analysis ; immunology ; Mutagenesis, Insertional ; genetics ; Open Reading Frames ; genetics ; Phosphoric Monoester Hydrolases ; genetics ; physiology ; Polymyxin B ; pharmacology ; Porphyromonas gingivalis ; enzymology ; genetics ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Toll-Like Receptor 4 ; analysis ; immunology ; Virulence Factors ; physiology