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The effects of the soluble forms of the endotoxin receptor molecules sMD-2 and sCD14 on bacterial growth were studied. When Escherichia coli and Bacillus subtilis were incubated at 37 degrees C for 18 hr with either sMD-2 or sCD14, growth of these bacteria was significantly inhibited as evaluated by viable cell counts and NADPH/NADH activity. A mutant of sCD14 (sCD14d57-64) lacking a region essential for LPS binding did not inhibit the growth of E. coli, whereas this mutant did inhibit the growth of B. subtilis. Addition of excess PG to the bacterial culture reversed the inhibitory effect of sMD-2 on the growth of B. subtilis, but not on the growth of E. coli. Furthermore, when evaluated by ELISA, both sMD-2 and sCD14 bound specifically to PG. Taken together, these results indicate that sMD-2 and sCD14 inhibit the growth of both Gram-positive and Gram-negative bacteria and further suggest that binding to PG and LPS is involved in the inhibitory effect of sMD-2 on Gram-positive bacteria and of sCD14 on Gram-negative bacteria, respectively.

Cepaic acid was isolated as a novel xanthylium yellow pigment from the dried outer scales of the yellow onion Allium cepa Linne. Its structure was elucidated on the basis of ESI-MS and 2D NMR spectroscopy as a 9-carboxy-1,3,6,8-tetrahydroxyxanthylium, which suggests that cepaic acid and other yellow pigments in the dried outer skin of onion were formed by the nucleophilic reaction of phloroglucinol derived from quercetin, a flavonol in onion scales, by autoxidation to glyoxylic acid. To our knowledge, this is the first report of such a pigment in yellow onion.

A highly sensitive analytical method for 25 kinds of primary aromatic amines and azo-dyes in paper products was developed. Free amines and total amines of the samples were analyzed. The amount of each azo-dye was calculated from the amount of total amines by subtracting the amount of free amines. Amines and azo-dyes were migrated into water at 23 degrees C for 24 hours. Free amines were extracted into dichloromethane after alkalization with sodium hydroxide solution. Azo-dyes were reduced to amines with sodium dithionite, alkalized, and then extracted into dichloromethane as total amines. They were determined by GC/MS. The recoveries of 100 microg/kg amines spiked into migration solution were in the range of 69-122%, except for 4,4'-oxydianiline and 4,4'-diaminodiphenylmethane which gave recovery rates of approximately 40%. The determination limits of amines were 4-20 microg/kg in paper. Amines and azo-dyes were surveyed in 17 kinds of base paper and 16 kinds of paper products for food contact use. Aniline was detected at levels of 4-20 microg/kg from most recycled papers, whereas the other amines were not detected in any sample.

A simple and rapid method using electron capture detector gas chromatography (GC-ECD) was developed for the determination of hexachlorobenzene (HCB) in Food Red Nos. 104 and 105. The sample was dissolved in water and HCB was extracted with hexane. The GC-ECD operating conditions were as follows: column, InertCap 5 MS/NP (30 m x 0.25 mm i. d. with 0.25 microm film thickness); oven temperature, 60 degrees C(1 min)--20 degrees C/min--150 degrees C(10 min)--20 degrees C/min--280 degrees C(5 min); inlet temperature, 260 degrees C; detector temperature, 300 degrees C; carrier gas, nitrogen (constant press 25 psi); inlet mode, splitless. For the evaluation of the method, HCB spiked into R104 and R105 at the levels of 2 microg/g and 5 microg/g was determined in replicate in five laboratories. Mean recoveries of HCB from R104 and R105 were 98.2-103.7%. Repeatability relative standard deviation values were 2.9-6.0% and reproducibility relative standard deviation values were 4.2-9.3%. HORRAT value was less than one. From these results, the validity of this method was confirmed.

IRAK-4 plays an essential role in Toll-like receptor (TLR)/IL-1 receptor signaling. However, its signaling and regulation mechanisms have remained elusive. We have reported previously that stimulation of TLR2, TLR4 or TLR9, but not TLR3, leads to downregulation of IRAK-4 protein. Here, we show that expression of MyD88 leads to downregulation of endogenous as well as exogenously expressed IRAK-4 protein in HEK293 cells. Expression of TRIF did not cause IRAK-4 downregulation although it induced NF-kappaB activation. Expression of either a deletion mutant of MyD88 lacking its death domain or MyD88s, neither of which induced NF-kappaB activation, did not lead to IRAK-4 downregulation. MyD88-induced downregulation was observed in an IRAK-4 mutant lacking the kinase domain, but not in another mutant lacking the death domain. These results demonstrate that downregulation of IRAK-4 requires activation of the MyD88-dependent pathway and that the death domains of both MyD88 and IRAK-4 are important for this downregulation.

The human prion protein (PrP) is a glycoprotein with a glycosylphosphatidylinositol (GPI) anchor at its C-terminus. Here we report alternative splicing within exon 2 of the PrP gene (PRNP) in the human glioblastoma cell line T98G. The open reading frame of the alternatively spliced mRNA lacked the GPI anchor signal sequence and encoded a 230 amino acid polypeptide. Its product, GPI-anchorless PrP (GPI(-) PrPSV), was unglycosylated and soluble in non-ionic detergent, and was found in the cytosolic fraction. We also detected low levels of alternatively spliced mRNA in human brain and non-neuronal tissues. When long-term passaged T98G cells were placed in a low-oxygen environment, alternatively spliced mRNA expression increased and expression of normally spliced PrP mRNA decreased. These findings imply that oxygen tension regulates GPI(-) PrPSV expression in T98G cells.

A novel crocetin glycosyl ester, neocrocin A (2), was isolated from gardenia yellow. The structure of 2 was elucidated as that of an all-trans-crocetin β-D-gentiobiosyl β-D-glucopyranosyl-(1→6)-D-2-deoxy-glucopyranos-2-yl diester based on chemical and spectral data. The findings provide evidence that the binding system of crocetin glycosides is not limited to the anomeric position.

Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria. It is a ligand for Toll-like receptor 4 (TLR4), which plays an essential role in innate immunity. Macrophages and dendritic cells exposed to LPS overproduce proinflammatory mediators, leading to septic shock. In this study, we screened for peptides that associate with TLR4 with a yeast two-hybrid screen using the human TLR4 extracellular domain as bait. A peptide (STM28) isolated from the screen inhibited LPS-induced nuclear factor-kappaB (NF-kappaB) activation in human and mouse macrophage cells and interacted with TLR4 in yeast and mammalian cells. STM28 showed no inhibitory effects against NF-kappaB activation induced by TLR1/2, TLR3 and TLR9 ligands in a mouse macrophage cell line, RAW 264. In addition, STM28 suppressed LPS-induced tumor necrosis factor-alpha production by differentiated THP-1 cells. Moreover, LPS-induced lethality in d-galactosamine-sensitized mice was significantly repressed by STM28 in a dose-dependent manner. These results demonstrate that STM28 selectivity inhibits TLR4-induced macrophage activation, and suggest that STM28 may have utility as a novel therapeutic agent for Gram-negative bacterial sepsis.

The differences in the constituents of ten ester-type gum bases used as natural food additives in Japan (urushi wax, carnauba wax, candelilla wax, rice bran wax, shellac wax, jojoba wax, bees wax, Japan wax, montan wax, and lanolin) were investigated. Several kinds of gum bases showed characteristic TLC patterns of lipids. In addition, compositions of fatty acid and alcohol moieties of esters in the gum bases were analyzed by GC/MS after methanolysis and hydrolysis, respectively. The results indicated that the varieties of fatty acids and alcohols and their compositions were characteristic for each gum base. These results will be useful for identification and discrimination of the ester-type gum bases.

MyD88 and IL-1R-associated kinase 1 (IRAK-1) play crucial roles as adaptor molecules in signal transduction of the TLR/IL-1R superfamily, and it is known that expression of these proteins leads to the activation of NF-κB in a TNFR-associated factor 6 (TRAF6)-dependent manner. We found in this study, however, that a dominant-negative mutant of TRAF6, lacking the N-terminal RING and zinc-finger domain, did not inhibit IRAK-1-induced activation of NF-κB in human embryonic kidney 293 cells, although the TRAF6 mutant strongly suppressed the MyD88-induced activation. The dominant-negative mutant of TRAF6 did not affect the IRAK-1-induced activation, regardless of the expression level of IRAK-1. In contrast, small interfering RNA silencing of TRAF6 expression inhibited MyD88-induced and IRAK-1-induced activation, and supplementation with the TRAF6 dominant-negative mutant did not restore the IRAK-1-induced activation. Expression of IRAK-1, but not MyD88, induced the oligomerization of TRAF6, and IRAK-1 and the TRAF6 dominant-negative mutant were associated with TRAF6. These results indicate that TRAF6 is involved but with different mechanisms in MyD88-induced and IRAK-induced activation of NF-κB and suggest that TRAF6 uses a distinctive mechanism to activate NF-κB depending on signals.

Rumput roman extract is used as a natural food preservative. Its antimicrobial activity and constituents were investigated as part of an ongoing study to evaluate its quality and safety as a food additive. The constituents were analyzed by GC/MS, and 5 major constituents were isolated and identified as capillin, capillene, caryophyllene oxide, alpha-curcumene and methyleugenol using NMR analysis. The antimicrobial activities against E. coli, S. cerevisiae and A. niger were measured by means of the halo test. Based on the results, we confirmed that capillin was the major active constituent. The concentrations of capillin and capillene were determined to 17.9 mg/mL and 36.1 mg/mL, respectively, from standard curves of authentic compounds on HPLC.

Helicobacter pylori is recognized as an etiologic agent of gastroduodenal diseases. Among toxic substances produced by H. pylori, LPS exhibits extremely low endotoxic activity as compared to the typical LPSs, such as that produced by Escherichia coli. We found that the LPS-low-responder stomach cancer cell line MKN28, which expresses Toll-like receptor 4 (TLR4) at extremely low levels, showed similar levels of interleukin-8 (IL-8) induction by H. pylori or E. coli LPS preparations. Weak IL-8 induction by H. pylori LPS preparations was suppressed by expression of a dominant negative mutant of TLR2 but not of TLR4. Data from luciferase reporter analysis indicated that cotransfection of TLR2-TLR1 or TLR2-TLR6 was required for the activation induced by H. pylori LPS preparations. In conclusion, the H. pylori LPS preparations significantly induce an inflammatory reaction via the receptor complex containing TLR2-TLR1 or TLR2-TLR6 but not that containing TLR4. The TLR2-TLR1 complex was preferentially recognized by the H. pylori LPS preparations over the TLR2-TLR6 complex. Whereas the magnitude of response to H. pylori LPS preparation was markedly less than that to E. coli LPS preparation in LPS-high-responder cells strongly expressing TLR4, it was comparable to that of E. coli LPS in low-responder cells expressing negligible amount of TLR4.

Novel fatty acyl and phospholipid derivatives of pyrrole polyamide were synthesized. Their cytotoxicity against a cancer cell line of MT-4 cells and those infected by human immunodeficiency virus (HIV) was examined. Although no anti-HIV activity was found, their cytotoxicitty against the cancer cells was significantly enhanced by introducing a lipophilic group into the pyrrole polyamide.

A determination method for epoxidized soybean oil (ESBO) in bottled foods was developed and used to survey bottled foods on the Japanese market. The amount of sample required was decreased to 20 g and the standard addition method was adopted for the quantification, because lipid in foods interrupted the hydrolysis of ESBO. The recoveries were 87.1 and 98.9% and the determination limit was 5.0 microg/g for a 20 g sample, be cause lipid in foods interupted the hydrolysis of ESBO. The recoveries using the internal standard method varied widely, because hydrolysis of the internal standard, cis-11,14-eicosadienoic acid ethyl ester, was affected more than that of ESBO by coexisting lipid in the sample. ESBO was not detected in any of the bottled baby food samples examined (14 samples), though it had been frequently detected in previous European surveys. This difference may be related to the low fat content and low fluidity of the bottled baby foods retailed in Japan. On the other hand, ESBO was detected at levels of 25.7-494.0 microg/g in liver paste, pasta sauce, Sungan in spicy oil, and spicy oil. These foods had higher fat content and higher fluidity. However, ESBO intake from these foods appears unlikely to exceed the TDI in the EU (1 mg/kg bw/day).

The application of liquid chromatography-nuclear magnetic resonance spectroscopy (LC-NMR) for the direct identification of ethyldimethylpyrazine, a food flavouring agent, has been studied. The commercial product is a mixture of two regio-isomers, 2-ethyl-3,5-dimethylpyrazine (1) and 2-ethyl-3,6-dimethylpyrazine (2); however, the exact composition of the mixture is unknown. Structural characterization by LC-MS and GC-MS was not possible because both regio-isomers yield the same molecular related ion and ion fragmentation. To rapidly identify the two regio-isomers, the product was analyzed by LC-NMR with on-flow and fraction loop modes. From the results, the structure elucidations of the two regio-isomers could be carried out without the need to isolate the isomers by the usual procedures.

Urushi wax is a natural gum base used as a food additive. In order to evaluate the quality of urushi wax as a food additive and to obtain information useful for setting official standards, we investigated the constituents and their concentrations in urushi wax, using the same sample as scheduled for toxicity testing. After methanolysis of urushi wax, the composition of fatty acids was analyzed by GC/MS. The results indicated that the main fatty acids were palmitic acid, oleic acid and stearic acid. LC/MS analysis of urushi wax provided molecular-related ions of the main constituents. The main constituents were identified as triglycerides, namely glyceryl tripalmitate (30.7%), glyceryl dipalmitate monooleate (21.2%), glyceryl dioleate monopalmitate (2.1%), glyceryl monooleate monopalmitate monostearate (2.6%), glyceryl dipalmitate monostearate (5.6%), glyceryl distearate monopalmitate (1.4%). Glyceryl dipalmitate monooleate isomers differing in the binding sites of each constituent fatty acid could be separately determined by LC/MS/MS.

Levels of eight harmful elements, i.e., antimony, arsenic, barium, cadmium, chromium, lead, mercury and selenium, were investigated in 45 baby toys and 10 paints, which were mainly made of polyvinyl chloride. All samples contained barium at levels of 0.3-3,700 mg/kg, several samples contained cadmium (0.2-26 mg/kg), chromium (0.5-280 mg/kg) and lead (1.5-1,300 mg/kg), and one sample contained antimony (5.3 mg/kg). They might have been used as colorants of the toy materials and paints. They were then evaluated using the migration test of ISO 8124-3, in which samples were ground up, and then soaked in 0.07 mol/L HCl at 37 degrees C for two hours. Barium, cadmium, chromium and lead migrated from some of the samples, but at levels lower than the migration limits required by ISO 8124-3. Compared with the Japanese official method, the ISO method resulted in higher migration, but there are significant differences in the migration limits, test method, and so on between them. Further investigation is needed in this area.

Sandarac resin, a natural gum base, is described as "a substance composed mainly of sandaracopimaric acid obtained from the secretion of sandarac trees" in the List of Existing Food Additives in Japan. To evaluate its quality as a food additive, the main constituents in a sandarac resin product were investigated. Three constituents were isolated and identified as sandaracopimaric acid, sandaracopimarinol and 4-epidehydroabietic acid by MS and 2D-NMR. Quantification of the main constituent, sandaracopimaric acid, was performed by HPLC and its content in the product was determined to be 11.6%.

Food contact plastics and rubbers possibly contain many kinds of chemicals such as monomers, oligomers, additives, degradation products of polymers and additives, and impurities. Among them, bisphenol A, nonylphenol, benzylbutyl phthalate, styrene oligomers and hydroxylated benzophenones have been reported to possess estrogenic activities. In this study, other chemicals related to food contact plastics and rubbers, and their metabolites induced by the S9-mixture were tested for their estrogenic activities using the yeast two-hybrid assay. Among the 150 chemicals, 10 chemicals such as bis(4-hydroxyphenyl) methane, 4-cyclohexylphenol, 4-phenylphenol, 4,4'-isopropylidenediphenol alkylphosphite, two type of styrenated phenol (including mono type), tris(nonylphenyl) phosphite, 2,2'-dihydroxy-4-methoxybenzophenone, 2-hydroxy-4-methoxybenzophenone and 2,4-diphenyl-4-methyl-1-pentene, their metabolites and the metabolites of 6 other chemicals, such as 2-(phenylmethyl) phenol, styrenated phenol (di and tri type), 1-(N-phenylamino)naphthalene, 4-tert-butylphenylsalicylate, nonylphenol ethoxylates and 2-methyl-6-tert-butylphenol, displayed estrogenic activities. All of them contained a phenol group in their chemical structures or formed one easily by hydrolysis or metabolism. However, most of the chemicals related to food contact plastics and rubbers, and their metabolites did not show any estrogenicity.

Jojoba wax is a natural gum base used as a food additive in Japan, and is obtained from jojoba oil with a characteristically high melting point. Although the constituents of jojoba oil have been reported, the quality of jojoba wax used as a food additive has not yet been clarified. In order to evaluate its quality as a food additive and to obtain basic information useful for setting official standards, we investigated the constituents and their concentrations in jojoba wax. LC/MS analysis of the jojoba wax showed six peaks with [M+H]+ ions in the range from m/z 533.6 to 673.7 at intervals of m/z 28. After isolation of the components of the four main peaks by preparative LC/MS, the fatty acid and long chain alcohol moieties of the wax esters were analyzed by methanolysis and hydrolysis, followed by GC/MS. The results indicated that the main constituents in jojoba wax were various kinds of wax esters, namely eicosenyl octadecenoate (C20:1-C18:1) (1), eicosenyl eicosenoate (C20:1-C20:1) (II), docosenyl eicosenoate (C22:1-C20:1) (III), eicosenyl docosenoate (C20:1-C22:1) (IV) and tetracosenyl eiosenoate (C24:1-C20:1) (V). To confirm and quantify the wax esters in jojoba wax directly, LC/MS/MS analysis was performed. The product ions corresponding to the fatty acid moieties of the wax esters were observed, and by using the product ions derived from the protonated molecular ions of wax esters the fatty acid moieties were identified by MRM analysis. The concentrations of the wax esters I, II and III, in jojoba wax were 5.5, 21.4 and 37.8%, respectively. In summary, we clarified the main constituents of jojoba wax and quantified the molecular species of the wax esters without hydrolysis by monitoring their product ions, using a LC/MS/MS system.

An analytical method for residual sodium chlorite in several kinds of processed herring roe treated with sodium chlorite was studied. Sodium chlorite was extracted with 9 mmol/L sodium carbonate. After centrifugation, the supernatant was filtered through a 0.2 microm nylon filter. The filtrate was deproteinized by ultrafiltration and chloride ion was removed with an On-Guard Ag cartridge column. The eluate was subjected to conductivity detector-ion chromatography. Recoveries of sodium chlorite from herring roe spiked at the level of 5 mg/kg were 88 +/- 3.7% (n = 5, CV 4.2%). The method had a quantitation limit of 5 mg/kg for processed herring roes.

Nitric oxide (NO) produced by macrophages plays an important role in host defense and inflammation. We found that two agrochemicals, alachlor and carbaryl, inhibit lipopolysaccharide (LPS)-induced NO production by macrophages. In the present study, we investigated this inhibitory mechanism in RAW 264 cells. Both chemicals inhibited LPS-induced iNOS protein and mRNA expression as well as murine iNOS promoter activity. When treating these chemicals with reducing agents, the inhibition by carbaryl was reversed, but not the inhibition by alachlor. These chemicals also inhibited LPS-induced interferon-{beta} (IFN-{beta}) expression, an indispensable factor for LPS-induced iNOS expression. The inhibited iNOS expression, however, was not restored by exogenous IFN-{beta} supplementation. LPS-induced nuclear translocation of NF-{kappa}B, which is necessary for the expression of IFN-{beta} and iNOS, was inhibited by these chemicals: however, the LPS-induced degradation of I{kappa}B-{alpha} and I{kappa}B-{beta} was inhibited only by alachlor. These results indicate that alachlor and carbaryl differentially impair the LPS-induced NF-{kappa}B activation, leading to the inhibition of NO production.

Endotoxin tolerance provides protection against mortality under various conditions of stress. However, the induction of endotoxin tolerance thus far has no clinical application because of endotoxin toxicity and the excessive immune suppression that follows the tolerance induction. In this study, we examined whether a novel, synthetic lipopolysaccharide (LPS) receptor agonist, ER-803058 (ER) can induce endotoxin tolerance with accompanying low toxicity. The stimulative effects of ER on tumor necrosis factor (TNF)-alpha production from RAW264 cells were 50% to 70% lower than those of the corresponding quantities of LPS. ER pretreatment also diminished TNF-alpha secretion induced by a subsequent LPS shock. However, the degree of desensitization with ER pretreatment (10 ng/mL, 55.5% +/- 6.7%; 100 ng/mL, 42.3 +/- 4.9%) was modest in contrast with that measured for the corresponding LPS pretreatment (10 ng/mL, 36.7% +/- 3.7%; 100 ng/mL, 20.0% +/- 3.6%). The minimum in vivo dose (0.02 mg/kg/body weight) of ER-induced negligible production of TNF-alpha and interleukin (IL)-6 in rats, and resulted in a modest endotoxin tolerance with respect to TNF-alpha secretion. Although the plasma TNF-alpha level after ER pretreatment was decreased (48.2% +/- 1.1%), the suppression was not statistically significant. Interestingly, even this minimal quantity of ER pretreatment evoked a dramatic improvement in survival (90% survival) against administration of a lethal dose of LPS, which is inconsistent with the modest TNF-alpha suppression. Furthermore, ER pretreatment preserved normal plasma albumin levels and prevented the increase of plasma blood urea nitrogen levels seen with LPS. These results indicate that pretreatment with ER can effectively induce endotoxin tolerance, with a consequent improvement in mortality without toxicity and without subsequent excessive immunosuppression.

HIV-1 has a huge genetic diversity. So far, nine subtypes have been isolated, namely, subtypes A, B, C, D, F, G, H, J, and K. Epidemiological study provides information which may help in the development of HIV-1 prevention programs or health policies. In the future, subtyping may also be critical for vaccine development, and an effective anti-viral drug will need to be effective for different subtypes of HIV virus. The analysis of the nucleotide sequence of the v3 region is considered the most reliable method for determining the HIV-1 subtype. However, the procedures for determining the v3 sequences are complicated and time consuming, requiring expensive reagents, equipment, and well-trained personnel. The polymerase chain reaction (PCR) method using subtype-specific primers for HIV-1 subtyping is easier and faster. The objective of this study was to develop subtype-specific primers for subtyping PCR. The specific primers were designed for subtypes A, B, C, D, F, G, and CRF01_AE, and these primers could be applied to assay for various HIV-1 subtypes in the clinical samples. The specific primers were designed for each subtypes in the gp41 region. The result of PCR was compared with the subtypes which was determined by the v3 sequence. The results of subtyping by PCR using the newly designed primers could detect 29 of 33 patients tested, and all matched those obtained by nucleotide sequencing of the env v3 region except for three subjects, which were differentiated as CRF02_AG. The newly designed primers functioned accurately and conclusively. In comparison with PCR as a method for the determination of subtypes, sequence analysis requires better-trained personnel, more expensive reagents, and more equipment and time.

Interleukin-1 receptor-associated kinase (IRAK)-4 is a key mediator in the Toll-like receptor (TLR) signaling. We found that stimulation of TLR2, TLR4, or TLR9, but not TLR3, caused a decrease in IRAK-4 protein without affecting its mRNA level in a mouse macrophage cell line, RAW 264. The decrease in IRAK-4 was accompanied by the appearance of a smaller molecular weight protein (32 kD), which was recognized by an anti-IRAK-4 antibody raised against the C-terminal region. The decrease in IRAK-4 and the appearance of the 32-kD protein occurred with slower kinetics than the activation of IRAK-1 and were suppressed by inhibitors of the proteasome, inducible inhibitor of κBα phosphorylation or protein synthesis, but not by caspase inhibitors. These results indicate that prolonged stimulation of TLR2, TLR4, or TLR9 causes a down-regulation of IRAK-4 protein, which may be mediated through cleavage of IRAK-4 by a protease induced by the activation of nuclear factor-κB.

An HPLC method on C18 column using a gradient mobile phase is proposed for the separate determination of residual benzoyl peroxide (BP) and benzoic acid (BA) in flour and wheat products. The recoveries obtained were quite excellent, from 96.0 to 99.3% for BP added to the flour, and 91.3% for BA added to the flour. Analysis of 10 samples of commercial foods such as flour and wheat products, detected 0.7 microg/g of BP in imported noodles. Furthermore, we successfully verified the existence of BP by LC-MS. These methods are simple and reliable for determination and verifying the amount of BP and BA in foods since now the use of BP as a food additive is permitted in many countries.

In a previous study, a chemically synthesized disaccharide precursor of lipid A (406; identical to lipid IVA) was shown to have dramatically reduced lethality, B-cell mitogenicity, and tumor necrosis factor induction in macrophages when its hydroxyl groups were replaced with either succinyl or acetyl residues (K. Tanamoto, FEBS Lett. 351:325-329, 1994). Succinylated 406 was found to lose Limulus amoebocyte lysate gelation activity completely as a result of the modification (about 10(5)-fold), too, as expected. However, acetyl 406, surprisingly, exhibited activity comparable to that of the original 406. Both succinylated and acetylated 406 lost pyrogenicity completely. These results indicate that one of the typical endotoxic activities was dissociated from the others and that the ability to induce Limulus amoebocyte lysate gelation is not always representative of endotoxin activity.

Carthamin is a well-known major pigment in carthamus red colourant. When analysed by HPLC on an ODS column, the colorant separated into two distinct reddish pigments, with both components having almost identical photodiode array spectra. LC/MS analysis suggested one of the compounds was carthamin, whilst the other was an unknown minor pigment. The minor pigment was purified and isolated from the colorant by preparative LC/MS collecting the faction based on monitoring the deprotonated molecule [M-H](-) m/z 953 in electrospray negative-ion mode. The structure was elucidated as a hydroxyethyl ether of carthamin, a novel compound, by means of NMR and HR-FAB-MS analyses.

Bacterial lipopolysaccharide (LPS), the major structural component of the outer wall of Gram-negative bacteria, is a potent activator of macrophages. Activated macrophages produce a variety of inflammatory cytokines. Excessive production of cytokines in response to LPS is regarded as the cause of septic shock. On the other hand, macrophages exposed to suboptimal doses of LPS are rendered tolerant to subsequent exposure to LPS and manifest a profoundly altered response to LPS. Increasing evidence suggests that monocytic cells from patients with sepsis and septic shock survivors have characteristics of LPS tolerance. Thus, an understanding of the molecular mechanisms underlying activation and deactivation of macrophages in response to LPS is important for the development of therapeutics for septic shock and the treatment of septic shock survivors. Over the past several years, significant progress has been made in identifying and characterizing several key molecules and signal pathways involved in the regulation of macrophage functions by LPS. In this paper, we summarize the current findings of the functions of the LPS receptor complex, which is composed of CD14, Toll-like receptor 4 (TLR4), and myeloid differentiation protein-2 (MD-2), and the signal pathways of this LPS receptor complex with regard to both activation and deactivation of macrophages by LPS. In addition, recent therapeutic approaches for septic shock targeting the LPS receptor complex are described.

We investigated the labeling and content of plasticizers of soft plastic toys. First, we investigated material labeling in 96 products purchased in fiscal years 2000 and 2001. Among these products, 43% of those purchased in fiscal 2000 and 68% of those purchased in fiscal 2001 were labeled. We then investigated the kinds and amounts of plasticizers in 73 soft polyvinyl chloride (PVC) toys. Three kinds of phthalates and six other kinds of plasticizers were detected in the soft PVC toys. Diisononyl phthalate, di(2-ethylhexyl) phthalate, diisononyl adipate, and O-acetyl tributyl citrate were detected at high frequency, and in large amounts. The average total content of all plasticizers was 280 mg/g for the products purchased in fiscal 2000 and 227 mg/g for those in fiscal 2001. In fiscal 2001, antioxidants, 4-nonylphenol, and bisphenol A were investigated in addition to plasticizers.

Lipopolysaccharide (LPS) preparations are known to often contain substances which activate cells through Toll-like receptor 2 (TLR2), and it is suspected that bacterial lipoproteins are responsible for this activation. We compared the mode of action of the TLR2-stimulatory substances with that of a synthetic bacterial lipopeptide (tripalmitoyl-Cys-Ser-Ser-Asn-Ala [Pam3CSSNA]), as well as with that of peptidoglycan. Six out of eight LPS preparations tested induced NF-κB-dependent reporter activity in 293 cells expressing CD14 and TLR2. Phenol extract (PEX) prepared fromEscherichia coliLPS by modified phenol extraction induced reporter activity in 293 cells expressing TLR2, and this activity was enhanced by coexpression of CD14, whereas the activity of Pam3CSSNA was not dependent on CD14. The activity of PEX, but not that of Pam3CSSNA or peptidoglycan, was also enhanced by LPS binding protein or serum and blocked by polymyxin B. In addition, the activity of PEX was inhibited by a lipid A precursor (compound 406) in 293 cells expressing CD14 and TLR2. These results indicate thatE. coliLPS preparations contain LPS-mimetic TLR2-stimulatory substances which differ from bacterial lipopeptides or peptidoglycan.

MD-2 has been reported to be required for the translocation of the Toll-like receptor 4 (TLR4) to the cell surface. However, the mechanism by which MD-2 promotes TLR4 translocation is unknown. We identified the presence of two forms of TLR4 with different molecular masses (approximately 110 and 130 kDa) when TLR4 was expressed together with MD-2. Expressing TLR4 alone produced only the 110-kDa form. Using a membrane-impermeable biotinylation reagent, we found that only the 130-kDa form of TLR4 was expressed on the cell surface. When a cellular extract prepared from cells expressing TLR4 and MD-2 was treated with N -glycosidase, the two forms of TLR4 converged into a single band whose size was smaller than the 110-kDa form of TLR4. Mutation of TLR4 at Asn 526 or Asn 575 resulted in the disappearance of the 130-kDa form and prevented TLR4 from being expressed on the cell surface without affecting the ability of TLR4 to associate with MD-2. These results indicate that TLR4 is able to undergo multiple glycosylations without MD-2 but that the specific glycosylation essential for cell surface expression requires the presence of MD-2.

We studied altered gene expressions in BALB/3T3 cells treated by different tumor promoters in the promotion phase of a transformation assay, an in vitro model of a two-stage carcinogenicity test, using fluorescent mRNA differential display analysis. Expression of the NP95 gene, which was previously found to be the gene of a murine nuclear protein associated with cell proliferation, was increased in the cultures treated by 12-O-tetradecanoylphorbol-13-acetate (TPA), okadaic acid, and orthovanadate. The upregulation of NP95 mRNA was confirmed by reverse transcription-PCR, and Northern blot. TPA, okadaic acid, and orthovanadate enhanced cell proliferation as measured by a 5-bromo-2′-deoxyuridine incorporation assay. The expression level of NP95 mRNA was not affected by the treatment with typical carcinogens benzo[a]pyrene and 3-methylcholanthrene at concentrations at which they act as initiators of cell transformation. These facts may imply that the enhancement of cell transformation by these tumor promoters is due, at least in part, to the acceleration of cell proliferation. NP95 mRNA was also increased in the transformed BALB/3T3 cells. Overexpression of NP95 may also participate in the maintenance of the transformed phenotype.

The lipid A portion has been identified as the active center responsible for lipopolysaccharide (LPS)-induced macrophage activation. However, we found thatSalmonella(Salmonella entericaserovars Abortusequi, Minnesota, and Typhimurium) lipid A is inactive in human macrophages, despite its LPS being highly active. Thus we investigated the critical role of polysaccharide inSalmonellaLPS-induced activation of NF-κB. In human monocytic cell line THP-1,Salmonellalipid A and syntheticSalmonella-type lipid A (516) did not induce NF-κB-dependent reporter activity up to 1 μg/ml, whereas strong activation was observed in response toSalmonellaLPS. The difference in activity between this lipid A and LPS was further examined by using 293 cells expressing human CD14/Toll-like receptor 4 (TLR4)/MD-2, and similar results were obtained in these cells as well. A polysaccharide preparation obtained fromSalmonellaLPS was inactive in 293 cells expressing human CD14/TLR4/MD-2 even in combination with 516.Salmonella entericaserovar Minnesota Re LPS, whose structure consists of lipid A and two molecules of 2-keto-3-deoxyoctonic acid, but not its lipid A exhibited strong activity in THP-1 cells and 293 cells expressing human CD14/TLR4/MD-2. These results indicate that the polysaccharide portion covalently bound to lipid A plays the principal role inSalmonellaLPS-induced activation of NF-κB through human CD14/TLR4/MD-2.

We developed a competitive enzyme-linked immunosorbent assay (ELISA) to detect prion protein contained in materials derived from cattle, aiming at establishing a method to detect abnormal prion protein (PrPSc) in food products. Rabbit polyclonal antibodies were raised against bovine prion peptides. Using these antibodies, we have established a competitive ELISA that is capable of detecting recombinant bovine prion protein (rBoPrP) in the range of 12 to 1,200 ng and we used it to determine prion protein contents in bovine cerebral cortex. This assay system was evaluated by spiking food products with various amounts of rBoPrP. The determination gave 2-fold higher values in minced meat homogenates and lower values in large intestine homogenates than the values expected from the spiked amounts. This assay provides a simple determination method of spiked rBoPrP, and therefore is expected to be useful for investigating sample pretreatment methods.

Human glioblastoma cell line T98G produced a cellular form of prion protein (PrP(C)), and we confirmed expression of PrP mRNA by RT-PCR. Immunoblot analysis of whole cell lysate revealed one major (35 kDa) and two faint bands (31, 25 kDa) that reacted with monoclonal anti-human PrP antibody 3F4. Cells treated with tunicamycin produced only a 25 kDa band, representing a deglycosylated form of PrP. Similarly, peptide: N-glycosidase F treatment of whole cell lysate altered the Asn-linked form to the deglycosylated form. When T98G cells were cultured for a longer period, the amount of PrP(C) per cell increased on Day 4 to 16 in a time-dependent manner. When the cells were cultured at high cell-density, the cells on Day 4 produced the same amount of PrP(C) as those on Day 16 of the usual culture. Moreover, in a serum-free medium, cells cultured at a low cell-density produced the same amount of PrP(C) as those cultured at the high cell-density. These results demonstrate that PrP(C) production in T98G cells was dependent on the phase of the cell cycle, probably the G1 phase.

Salmonellalipid A is inactive in human macrophages despite being potently active in murine macrophages. We investigated the molecular basis for this species-specific action ofSalmonellalipid A. When murine CD14 (mCD14), mTLR4, and mMD-2 were all expressed in human monocytic THP-1 cells, these cells were capable of responding toSalmonellalipid A. Expressing each of these proteins separately did not impart such responsiveness. Expression of mTLR4 plus mMD-2, but not mCD14 plus mTLR4 or mCD14 plus mMD-2, conferred this responsiveness. In THP-1 cells expressing mCD14, mTLR4, and mMD-2, replacing mCD14 with human CD14 had no effect on responsiveness toSalmonellalipid A or syntheticSalmonella-type lipid A (compound 516). When mTLR4 was replaced with human TLR4, the responses to these lipid A preparations were decreased to half, and the replacement of mMD-2 decreased responsiveness to one-third, although the responses toEscherichia colilipid A or syntheticE. coli-type lipid A (compound 506) were not affected. These results indicate that both TLR4 and MD-2 participate in the species-specific action ofSalmonellalipid A.

The biological properties of the lipid A fromFlavobacterium meningosepticum, which we recently isolated and whose complete chemical structure has been determined (H. Kato, T. Iida, Y. Haishima, A. Tanaka, and K. Tanamoto. J. Bacteriol. 180:3891–3899, 1998), were studied. The lipid A exhibited generally moderate activity compared toSalmonella entericasubsp.entericaserovar abortus equi lipopolysaccharide (LPS) used as a control in the assay systems tested; lethal toxicity in galactosamine-sensitized mice, mitogenicity in mouse spleen cells, induction of tumor necrosis factor alpha (TNF-α) release from mouse peritoneal macrophages and J774-1 mouse macrophage-like and human THP-1 line cells, nitric oxide induction activity from J774-1 cells, andLimulusgelation activity. The moderate activity of theF. meningosepticumlipid A may be explained by its unique fatty acid composition and the lack of a phosphate group in position 4′. It is noteworthy that the lipid A apparently induced TNF-α release from peritoneal macrophages in LPS-unresponsive C3H/HeJ mice and that the activation was suppressed by the LPS-specific antagonist, succinylated lipid A precursor. Significant splenocyte mitogenicity in C3H/HeJ mice was also observed with the lipid A. Taken together with the previous results concerningPorphyromonas gingivalislipid A, which has a high level of structural similarity to the lipid A ofF. meningosepticum, and the induction of TNF-α release in macrophages from C3H/HeJ mice, the lipid A ofF. meningosepticum, which has novel fatty acids, may possibly play an role for the activation of C3H/HeJ macrophages.

The lipid A from Comamonas testosteroni has been isolated and its complete chemical structure determined [Iida, T., Haishima, Y., Tanaka, A., Nishijima, K., Saito, S. & Tanamoto, K. (1996). Eur J Biochem 237, 468–475]. In this work, the relationship between its chemical structure and biological activity was studied. The lipid A was highly homogeneous chemically and was characterized by the relatively short chain length (C10) of the 3-hydroxy fatty acid components directly bound to the glucosamine disaccharide backbone by either amide or ester linkages. The lipid A exhibited endotoxic activity in all of the assay systems tested (mitogenicity in mouse spleen cells; induction of tumour necrosis factor alpha release from both mouse peritoneal macrophages and mouse macrophage-like cell line J774-1, as well as from the human monocytic cell line THP-1; induction of nitric oxide release from J774-1 cells; Limulus gelation activity and lethal toxicity in galactosamine-sensitized mice) to the same extent as did ‘Salmonella minnesota’ lipid A or Escherichia coli LPS used as controls. The strong endotoxic activity of the C. testosteroni lipid A indicates that the composition of 3-hydroxydecanoic acid is not responsible for the low endotoxicity of the lipid A observed in members of the genus Rhodopseudomonas, as has previously been suggested. Furthermore, both the lack of a second acylation of the 3-hydroxy fatty acid attached at the 3′ position, and the substitution of the hydroxyl group of the 3-hydroxy fatty acid attached at position 2, do not affect the manifestation of endotoxic activity or species specificity.

Lipopolysaccharide (LPS) obtained from Porphyromonas gingivalis was found to exhibit marked lethal toxicity in galactosamine-sensitized C3H/HeJ mice. Although no lethality was observed in mice intraperitoneally challenged with 1 mg of P. gingivalis LPS without galactosamine, when they were sensitized with 30 mg of galactosamine, challenge with 1 and 10 μg of LPS resulted in 67 and 100% lethality, respectively. The lethal dose of LPS was almost the same in LPS-responsive C57BL/6 mice and non-LPS-responsive C3H/HeJ mice. Furthermore, when 1 μg of P. gingivalis LPS was administered to each mouse 90 min before the challenge with the same LPS with galactosamine, tolerance to the lethal action of LPS was induced, and the mice were completely protected from death, even at a dose 100-fold greater than the lethal dose of LPS. Neither a lethal effect nor induction of tolerance to the lethality of P. gingivalis LPS was exhibited by Salmonella LPS in galactosamine-sensitized C3H/HeJ mice. A protein-LPS complex derived from Pseudomonas aeruginosa , which exhibited strong lethality and induced tolerance to a subsequent challenge with a lethal dose of LPS in galactosamine-sensitized LPS-responsive mice, did not exhibit lethal toxicity in galactosamine-sensitized C3H/HeJ mice and failed to induce tolerance in these mice to the lethality of P. gingivalis LPS. These results indicate that P. gingivalis LPS plays the central role in the activation of non-LPS-responsive C3H/HeJ mice.

A substance that inhibits the activity of bacterial endotoxin (LPS) was found in cinnamon bark. The inhibitor, extracted from dry cinnamon bark with 67% ethanol/water, was purified by using Limulus gelation activity as an indicator of endotoxin activity. The inhibitor suppressed the activity of the LPS when it was mixed with the inhibitor prior to the assay. The reduction of the LPS activity depended on the concentration of both the inhibitor and LPS when mixed, and also on the incubation time. The inhibitor suppressed the activity of all LPS and lipid A preparations tested regardless of the origin of the bacteria. The inhibitor alone did not affect the Limulus system. These results indicate that the inhibition was caused by direct interaction of the inhibitor with the LPS molecule. Furthermore the inhibitor abrogated the pyrogenicity of the LPS. Although it is uncertain whether the inhibitor actually plays a role in the defense mechanism in cinnamon bark, this is the first report that an inhibitor of bacterial endotoxin exists in a plant.

The chemical structure of lipid A of lipopolysaccharide isolated from Comamonas testosteroni was determined by quantitative analysis, methylation analysis, mass spectrometry and NMR spectroscopy. The lipid A backbone was found to consist of 6-O-(2-deoxy-2-amino-beta-D-glucopyranosyl)-2-deoxy-2-amino-alpha-D-g luc ose which was phosphorylated in positions 1 and 4'. Hydroxyl groups at positions 4 and 6' were unsubstituted, and position 6' of the non-reducing terminal residue was identified as the attachment site of the polysaccharide part. Liquid secondary-ion/mass spectrometry revealed a pseudomolecular ion at m/z 1572 [M-H]- as a major diphosphoryl lipid component carrying six acyl groups. Fatty acid distribution analysis and electrospray ionization/mass spectrometry of the lipid A showed that positions 2,2',3, and 3' of the sugar backbone were N-acylated or O-acylated by (R)-3-hydroxydecanoic acid, and that the hydroxyl groups of the amide-linked residues attached to positions 2 and 2' were further O-acylated by tetradecanoic and dodecanoic acids, respectively.

The synthetic disaccharide precursor of lipid A (406: identical to lipid IVA) was found to reduce its endotoxic activity in mice by an order of 105 or more, by replacing the hydroxyl groups with succinyl or acetyl residues. Both the succinylated and acetylated 406 were also found to antagonize the endotoxic mitogenicity on murine splenocytes. Previous studies demonstrated that the succinylated or acetylated synthetic complete lipid A preparations retained the whole endotoxic activity [1994, Infect. Immunol. 62, 1705]. The drastic contrast in all of these results suggests the importance of the substituents on the hydroxyl groups of 3-hydroxy fatty acids of non-reducing glucosamine of lipid A for the activity and for transformation to the antagonistic structure.

Publisher Summary This chapter discusses the induction of prostaglandin release from macrophages by bacterial endotoxin. Endotoxin is chemically a lipopolysaccharide (LPS), consisting of a hydrophilic polysaccharide part and a hydrophobic lipid part, named lipid A, which has been proven to be the active center of almost all the biological activities of endotoxin. The concept of involvement of prostaglandins in endotoxic action is supported partly by the observation that certain endotoxic activities such as the induction of fever, abortion, and the early phase of endotoxic shock are suppressed by an inhibitor of cyclooxygenase. It has also been suggested that endotoxin shock is closely related to increased concentrations of several groups of prostanoids in blood and lymph. The chapter describes the preparation of bacterial endotoxin, cell (macrophage) preparation, and measurement of prostaglandins. Prostaglandins and their metabolites are generally measured by such techniques as radioimmunoassay (RIA), gas chromatography (GC), GC–mass spectrometry, and high-performance liquid chromatography (HPLC).

SUMMARY: Chemically modified lipopolysaccharides of Salmonella abortus-equi were tested for mitogenicity on mouse spleen cells as well as antagonism of the mitogenicity of intact lipopolysaccharide (LPS). All the lipopolysaccharide preparations deacylated by different alkaline treatments suffered a drastic loss of mitogenicity. The mitogenic activity of lipid A was also lost when succinic residues were introduced on hydroxyl groups. Partially deacylated alkaline-treated preparations (but not completely deacylated preparations) inhibited the activation of splenic B-cells by LPS. They were found to be toxic to spleen cells, however, and to suppress not only the mitogenicity of LPS but that of concanavalin A as well. This inhibitory action was not exhibited when all of the fatty acid was eliminated. Succinylated lipid A, on the other hand, was not toxic to the cells and inhibited the B-cell mitogenicity of lipopolysaccharide (but not the T-cell mitogenicity of concanavalin A). Chemical analysis revealed that about 4.6 mol of succinic acid had been introduced into lipid A by succinylation, and that the fatty acid and phosphate composition was unchanged by this treatment. Macrophages do not seem to participate in this inhibition. Inhibition was observed when succinylated lipid A was added either at the same time or after lipid A mitogen, but optimal inhibition was expressed when it was added to the culture 3 h before LPS. Inhibition was not affected by washing the cells before adding LPS. Inhibition increased as the ratio of suppressor to mitogen increased, suggesting that the succinylated lipid A competes with intact LPS.

To confirm the revised lipid A structure of Escherichia coli and to establish the structure responsible for its functions, biological activities of the synthetic compounds based on the presented structure of E. coli lipid A were investigated. Compound 506, 2-deoxy-6-O-(2-deoxy-2-[(R)-3-dodecanoyloxytetradecanoylamino]-3-O [(R)3-tetradecanoyloxytetradecanoyl]-beta-D-glucopyranosyl]-3-O-[(R) -3-hydroxytetradecanoyl]-2-[(R)-3-hydroxytetradecanoylamino]-alpha -D-glucopyranose 1,4'-bis(phosphate), exhibited activities identical to those of natural E. coli lipid A in eliciting Shwartzman reaction and tests on lethality, pyrogenicity, interferon- and tumor necrosis factor-inducing activities as well as in B-cell activating activity and Limulus amebocyte lysate gelating activity. With the exception of the Shwartzman reaction the monophosphorylated synthetic compounds at either the 1 or 4' position showed slightly lower activities than the compound with the bisphosphorylated compound (Compound 506). The compound without the phosphate group showed no or only very weak activities. The structural requirements for each activity (i.e. binding position and composition of fatty acids and presence of phosphate groups) are discussed taking into account the results of previous investigations.

Synthetic lipid A part structures corresponding structurally to a biosynthetic lipid A disaccharide precursor have been analyzed for endotoxic activity in several systems in vivo and in vitro. It was found that a synthetic beta-1,6-linked D-glucosamine disaccharide, which carries four molar equivalents of (R)-3-hydroxytetradecanoyl residues in positions 2, 3, 2' and 3' and phosphoryl groups in positions 1 and 4' (preparation 406), exhibited lethal toxicity, B lymphocyte mitogenicity, the capacity to engender prostaglandin formation in macrophages and to induce endotoxic tolerance, as well as serological lipid A antigenicity. On a weight basis, preparation 406 was of comparable activity to lipid A precursor and bacterial free lipid A. Preparation 406, like lipid A precursor, lacked, however, the ability to induce the local Shwartzman phenomenon and both preparations were of moderate pyrogenicity. Two further synthetic analogues which contained only one phosphoryl group (preparation 404 at C-4', preparation 405 at C-1) showed comparable or diminished activity depending on the test system employed, except in the capacity to inactivate complement where they exhibited, in contrast to preparation 406, significant activity. The results show that the endotoxic principle of lipopolysaccharides, as postulated previously is embedded in the lipid A component. Our results also suggest initial conclusions on the structural requirements for the expression of endotoxin activities.

Resistance against ascites tumor development and interferon-inducing activity were demonstrated in lipopolysaccharide derived from the protein-lipopolysaccharide complex obtained from an autolysate of Pseudomonas aeruginosa. Lipid A obtained from the lipopolysaccharide was sufficient to induce interferon in vitro but no antitumor activity was found if lipid A or the polysaccharide derived from lipopolysaccharide was injected into the animal. Chemical modification of the polysaccharide portion or deacylation of the lipopolysaccharide also diminished antitumor activity. In contrast, interferon was induced by these incomplete lipopolysaccharides. These results indicate that both the lipid A portion and covalently linked polysaccharide are necessary for the inhibition of ascites tumor development, whereas incomplete lipid A with amide-linked fatty acids is sufficient to induce interferon in vitro.