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Exposure to various antigens derived from house dust mites (HDM) is considered to be a risk factor for development of certain allergic diseases such as atopic asthma, atopic dermatitis, rhinitis and conjunctivitis. Chitin is an insoluble polysaccharide (β-(1–4)-poly-N-acetyl-d-glucosamine) and a major component in the outer shell of HDMs. Mice exposed to chitin develop asthma-like airway eosinophilia. On the other hand, several lines of evidence show that the effects of chitin on immune responses are highly dependent on the size of chitin particles. In the present study, we show that chitin induced production of IL-33 and TSLP by alveolar and bronchial epithelial cells, respectively, in mice. IL-25, IL-33 and TSLP were reported to be important for group 2 innate lymphoid cell (ILC2)-, but not Th2 cell-, dependent airway eosinophilia in a certain model using chitin beads. Here, we show that—in our murine models—epithelial cell-derived IL-33 and TSLP, but not IL-25, were crucial for activation of resident lung Th2 cells as well as group 2 innate lymphoid cells (ILC2s) to produce IL-5, resulting in development of chitin-induced airway eosinophilia. Our findings provide further insight into the underlying mechanisms of development of HDM-mediated allergic disorders.

Chitin, which is a major component of house dust mites (HDM), fungi, crustaceans, etc., can activate immune cells, suggesting that it contributes to development of allergic disorders such as asthma. Although the pathophysiological sensitization route of asthmatic patients to allergens is considered via the respiratory tract, the roles of intranasally-administered chitin in development of asthma remain unclear. After ovalbumin (OVA) challenge, development of airway inflammation was profoundly exacerbated in mice sensitized with OVA in the presence of chitin. The exacerbation was dependent on IL-33, but not IL-25, thymic stromal lymphopoietin or IL-17A. Chitin enhanced IL-33-dependent IL-1β production by dendritic cells (DCs). Furthermore, chitin- and IL-33-stimulated DC-derived IL-1β promoted OVA-specific Th2 cell activation, resulting in aggravation of OVA-induced airway inflammation. These findings indicate the adjuvant activity of chitin via a new mechanism and provide important clues for development of therapeutics for allergic disorders caused by HDM, fungi and crustaceans.

In this study, we investigated the correlation between the microbiological characteristics of Clostridium difficile clinical isolates and the recurrence of C. difficile -associated disease (CDAD). Twenty C. difficile isolates recovered from 20 single infection cases and 53 isolates from 20 recurrent cases were analyzed by pulsed-field gel electrophoresis (PFGE) and PCR ribotyping, and the cytotoxicity, antimicrobial susceptibility, and sporulation/germination rates of the isolates were examined. Recurrent cases were divided into relapse or reinfection cases by the results of C. difficile DNA typing. Among the 20 recurrent cases, 16 cases (80%) were identified to be relapse cases caused by the initial strain and the remaining 4 cases (20%) were identified to be reinfection cases caused by different strains. All 73 isolates were susceptible to both vancomycin and metronidazole, but resistance against clindamycin, ceftriaxone, erythromycin, and ciprofloxacin was found in 87.7%, 93.2%, 87.7%, and 100% of the isolates, respectively. No correlations between DNA typing group, cytotoxicity, and sporulation rate of isolates and infection status, i.e., single, relapse, or reinfection, were observed. However, the isolates recovered from relapse cases showed a significantly higher germination rate when incubated in medium lacking the germination stimulant sodium taurocholate. These results indicate that the germination ability of C. difficile may be a potential risk factor for the recurrence of CDAD.

Human Mycoplasma pneumoniae (MP) pneumonia is characterized by alveolar infiltration with neutrophils and lymphocytes and lymphocyte/plasma cell infiltrates in the peri-bronchovascular area (PBVA). No mouse model has been able to mimic the pathological features seen in human MP pneumonia, such as plasma cell-rich lymphocytic infiltration in PBVA. To figure out the mechanism for inflammation by MP infection using a novel mouse model that mimics human MP pneumonia, mice were pre-immunized intraperitoneally with Th2 stimulating adjuvant, alum, alone or MP extracts with an alum, followed by intratracheal challenge with MP extracts. The toll-like receptor-2, which is the major receptor for mycoplasma cell wall lipoproteins, was strongly up-regulated in alveolar macrophages in a latter group after the pre-immunization but prior to the intratracheal challenge. Those findings demonstrated that acceleration of innate immunity by antecedent antigenic stimulation can be an important positive-feedback mechanism in lung inflammation during MP pneumonia.

2'-O-(1-Naphthyl)uridine and 2'-O-(2-naphthyl)uridine were synthesized by a microwave-mediated reaction of 2,2'-anhydrouridine with naphthols. Using the 3'-phosphoramidite building blocks, these 2'-O-aryluridine derivatives were incorporated into 2'-O-methylated oligoribonucleotides. Incorporation of five 2'-O-(2-naphthyl)uridines into a 2'-O-methylated RNA sense strand significantly increased the thermostability of the duplex with a 2'-O-methylated RNA antisense strand. Circular dichroism spectroscopy and molecular dynamic simulation of the duplexes formed between the modified RNAs and 2'-O-methyl RNAs suggested that there are π-π interactions between two neighboring naphthyl groups in a sequence of the five consecutively modified nucleosides.

Oligodeoxynucleotides acylated with a 2-(trimethylsilyl)benzoyl (TMSBz) group at the 5′ or 3′ terminus were synthesized according to the general method used for DNA synthesis. The acylated DNA oligomers could be easily purified due to the high lipophilicity of the TMSBz group and showed enhanced hybridization ability and resistance to exonucleases.

We report the synthesis and properties of nucleoside derivatives acylated by 2-(trimethylsilyl)benzoyl (TMSBz) that proved to be extremely stable under basic conditions when introduced into the 5′-hydroxyl group of thymidine, the 4-amino group of deoxycytidine and the 2′-hydroxyl group of uridine. In particular, 2′-O-TMSBz-uridine could be isolated and was more stable in pyridine, while it isomerized in CH2Cl2 in the presence of Et3N to yield a mixture of the 2′-O- and 3′-O-acylated species.

We compared the performance of two commercial toxin detection kits, C. difficile toxin A/B (C. difficile TOX A/B II test; TOX A/B II) and C. difficile toxin A (Uniquick), for (i) detection using highly purified toxin A solution; (ii) cross-reactivity using culture supernatants of toxin A-positive and B-positive C. difficile, toxin A-negative and B-positive C. difficile, and toxin A-negative and B-negative C. difficile strains and other bacteria; and (iii) sensitivity and specificity using clinical specimens. Results indicated that TOX A/B II detected toxin A at concentrations of 0.35 ng/mL and Uniquick at concentrations of 0.7 ng/mL. Uniquick performance was specific for detecting toxin A alone, while TOX A/B II detected toxin A/B specifically. Kit performance was then evaluated using 99 fecal specimens--43 specimens from patients with toxin B-positive C. difficile and 56 from those without. Sensitivity of TOX A/B II vs Uniquick was 95.3% vs 76.7%, specificity 98.2% vs 98.2%, positive predictive 97.6% vs 97.1%, and negative predictive value 96.5% vs 84.6%. Findings thus indicate that TOX A/B II is a more suitable diagnostic aid for CDAD than Uniquick because it correlates well with toxin B-positive C. difficile culture results. Stool culture for C. difficile is also required, however.

In this study, we developed new 3′-terminal deoxyribonucleoside-loading reagents 1 with a new silyl-type linker. These reagents could increase the efficiency of introduction of 3′-terminal deoxyribonucleoside components into polymer supports to a level of 17–29 μmol/g. The efficiency was higher than that of previous T-loading reagents because reagents 1 contain a 4-aminobutyryl residue as a spacer. Moreover, we could synthesize not only unmodified DNA oligomers but also a base-labile modified DNA oligomer using resins 9a–d in the activated phosphite method without base protection.

We propose a new strategy called the 'Protected DNA Probes (PDP) method' in which appropriately protected bases selectively bind to the complementary bases without the removal of their base protecting groups. Previously, we reported that 4-N-acetylcytosine oligonucleotides (ac(4)C) exhibited a higher hybridization affinity for ssDNA than the unmodified oligonucleotides. For the PDP strategy, we created a modified adenine base and synthesized an N-acylated deoxyadenosine mimic having 6-N-acetyl-8-aza-7-deazaadenine (ac(6)az(8)c(7)A). It was found that PDP containing ac(4)C and ac(6)az(8)c(7)A exhibited higher affinity for the complementary ssDNA than the corresponding unmodified DNA probes and showed similar base recognition ability. Moreover, it should be noted that this PDP strategy could guarantee highly efficient synthesis of DNA probes on controlled pore glass (CPG) with high purity and thereby could eliminate the time-consuming procedures for isolating DNA probes. This strategy could also avoid undesired base-mediated elimination of DNA probes from CPG under basic conditions such as concentrated ammonia solution prescribed for removal of base protecting groups in the previous standard approach. Here, several successful applications of this strategy to single nucleotide polymorphism detection are also described in detail using PDPs immobilized on glass plates and those prepared on CPG plates, suggesting its potential usefulness.

5-Carboxy-2′-deoxycytidine ( dC COO - ) was synthesized as an anion-carrier to seek a new possibility of modified oligodeoxynucleotides capable of stabilization of duplexes and triplexes. The base pairing properties of this compound were evaluated by use of ab initio calculations. These calculations suggest that the Hoogsteen-type base pair of dC COO - -G is less stable than that of the canonical C+-G pair and the Watson–Crick-type base pair of dC COO - -G is slightly more stable than the natural G-C base pair. The modified cytosine base showed a basicity similar to that of cytosine (pKa 4.2). It turned out that oligodeoxynucleotides 13mer and 14mer incorporating dC COO - could form duplexes with the complementary DNA oligomer, which were more stable than the unmodified duplex. In contrast, it formed a relatively unstable triplex with the target ds DNA.

New oligonucleotides with a long-chain linker (6,9-dioxa-3,12-diazatetradecane-1,14-diyl) in their backbone were synthesized, and their hybridization properties were studied by measurement of their Tm curves and fluorescence spectra. The Tm analyses revealed that these oligonucleotides could bind to their complementary strands despite the presence of the long-chain linker. We also demonstrated interesting fluorescence properties of oligodeoxynucleotides with an anthracen-9-ylmethyl group on one of the two N-atoms in the long-chain linker. The fluorescence intensity of these oligonucleotides increased upon their hybridization to the complementary strands and was sensitive to the presence of the mismatch base pairs at a specific position.

DNA chips consisting of DNA oligonucleotide probes immobilized on the surface of solid supports are very powerful tools for rapid analysis of multiple samples. In this Letter we describe a new method for the efficient synthesis of DNA probes without their serious elimination by use of a new hydrophobic 16-hyroxydecanoic linker and a new non-aqueous reagent of MeNH2/THF for the deprotection of the base and phosphate protecting groups on CPG resins. The elimination of DNA probes in this new method could be suppressed more than 20-fold compared with the previous method using a hexaethylene glycol linker and concd NH4OH. Moreover, we carried out SNPs detection by use of our DNA–CPG conjugate to show the utility of our new linker and deprotection conditions.

New bi- and tricyclic deoxycytidine derivatives (dChpd, dCmpp, dCtpp, dCppp) were synthesized as analogues of a fluorescent nucleoside, dChpp, previously reported. The carbamoyl group of dChpd and the 5-position of the cytosine ring are bridged via an ethylene linker so that the modified group forms a nonplanar structure with the cytosine ring. The fluorescent study of dChpd indicated that the coplanar structure between the carbamoyl group and the cytosine ring is of importance. N-Methylation of the carbamoyl group (dCmpp) weakened the intensity of the fluorescence of dChpp, and the derivative (dCtpp), which had a thiocarbamoyl group, lost its fluorescent property. Moreover, addition of a pyrrolo-ring (dCppp) to dChpp enhanced the intensity of fluorescence, and an emission light was observed with a marked Stokes shift of 120 nm.

Helicobacter pylori is associated with gastric disorders in humans and some experimental animals, and possesses the luxS/type 2 autoinducer (AI-2) system. The effects of a specific luxS mutation on the characteristics of H. pylori were examined. On 0.3 % agar medium, motility of H. pylori HPKY08 (luxS : : cat) was significantly lower than that of wild-type H. pylori TK1402. The luxS-complemented strain HPKY21 exhibited motility comparable to that of H. pylori TK1402. It was shown that the luxS/AI-2 system plays an important role in H. pylori motility. The luxS mutant exhibited a reduced infection rate relative to the wild-type parent strain TK1402 in a Mongolian gerbil model. At 3 months after oral inoculation, lower numbers of H. pylori were detected by semi-quantitative real-time reverse transcription PCR (qRT-PCR) in luxS(-) mutant-infected gerbils than in TK1402-infected gerbils. Gastric inflammation and increased antibody titre for H. pylori were observed in TK1402-infected gerbils only.

Deoxycytidine derivatives modified with various N-substituted carbamoyl groups at the 4-amino group were synthesized. The detailed 1H NMR studies suggest that the 3′,5′-O-disilylated N-carbamoyldeoxycytidine derivative 11 exists as a species having an intramolecular hydrogen bond between the N3 atom and the carbonyl oxygen atom in MeOD but upon addition of CDCl3, the amount of a homodimer species, having intermolecular hydrogen bonds, gradually increases. Oligodeoxyribonucleotides incorporating various 4-N-carbamoyldeoxycytidine derivatives were also synthesized. These modified oligodeoxynucleotides can hybridize with the complementary strands without disturbing the structure of DNA duplexes, hence changing the orientation of the carbamoyl group in such a manner that a stable Watson–Crick base pair can be formed with the guanine base at the opposite site. It should be noted that the base recognition ability (G against T, C and A) of these modified cytosine bases can be preserved satisfactorily. These results suggest that the 4-N-carbamoyl group is useful as a backbone structure of the linker between various functional residues and oligonucleotides. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)

A set of diastereomeric TpT phosphoramidite building blocks, 1-f and 1-s, containing an alkaline-labile hydroxymethylphosphonate (HMP) linkage were synthesized. Dodecadeoxynucleotides incorporating the HMP bond were also synthesized by use of each stereochemically pure dimer block under mild conditions in the N-unprotected phosphoramidite method. Moreover, it turned out that HMP-oligonucleotides derived from a fast-eluted dimer block exhibit higher affinity for DNA single and double strands than those containing a slow-eluted one.

[reaction: see text] A new route to N-unprotected deoxynucleoside 3'-phosphoramidite building blocks by use of highly selective N-deacylation of commercially available N-acylated deoxynucleoside 3'-phosphoramidites is described. These compounds could be readily converted to other types of N-protected species by facile N-acylations with acylating reagents.

The effect of Helicobacter pylori on DNA synthesis of human cultured cells was examined. Viable H. pylori strains were able to stimulate DNA synthesis by gastric cancer MKN45 cells (eight of eight strains) and laryngeal cancer HEp-2 cells (seven of eight strains) but not by lung cancer A549 cells or fetal intestine Int407 cells. In contrast, neither heat-killed H. pylori nor culture supernatants of H. pylori other than SS-1 strain affected DNA synthesis of MKN45 and HEp-2 cells. In contrast, water extracts of H. pylori strains inhibited DNA synthesis. Soluble outer membrane protein (OMP) of the SS-1 strain stimulated DNA synthesis of MKN45 cells but produced no alteration in cell cycle or apoptosis induction of MKN45 cells. To identify those proteins in the soluble OMP that were capable of stimulating DNA synthesis, the OMP was fractionated by fast performance liquid chromatography with a Mono Q column. The results suggest that two proteins with molecular weights of 18 and 45 kDa, respectively, are associated with stimulation of DNA synthesis by MKN45 cells.

Modified oligodeoxynucleotides incorporating 4- N -( N -arylcarbamoyl)-dC derivatives 1a – c were synthesized. The 1 H NMR spectra of 1a – c suggest that the carbamoyl group forms an intramolecular hydrogen bond with the cytosine ring nitrogen atom so that formation of a Watson–Crick base pair with the complementary guanine base is inhibited. The hybridization properties of oligodeoxynucleotides containing 1a – c were investigated by use of T m analysis. The hybridization properties of 4- N -( N -phenylcarbamoyl)-dC ( 1a ) were similar to those of 4- N -( N -alkylcarbamoyl)-dC derivatives reported previously. In sharp contrast to 1a , it turned out that 4- N -( N -napht-1-yl) and ( N -quionol-5-yl)-dC ( 1b , c ) have a unique property as a universal base.

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for an increasing number of serious nosocomial and community-acquired infections, and accurate methods to detect such strains are needed. We tested the susceptibility of 3 kinds of MRSA isolation medium, MRSA Screen Agar, Oxacillin Resistance Screening Agar and CHROMagar MRSA. Both sensitivity and specificity of CHROMagar MRSA were 100%. The sensitivity and specificity of both MRSA Screen Agar and Oxacillin Resistance Screening Agar were 100% and 91.5%, respectively. It is suggested that CHROMagar MRSA is a useful medium to detect MRSA including mecA positive and oxacillin susceptible strains.

We have attempted to establish a gnotobiotic mouse model monoassociated withMycoplasma pneumoniaefollowing single or repeated infection to examine the mechanism of pathogenesis followingM. pneumoniaeinfection.M. pneumoniaeinoculated into germfree mice colonized equally well at 105CFU/lung in both single infection and repeated infection. In histopathological observation, repeatedly infected mice showed pneumonia with mild infiltration of mononuclear cells and macrophages. Antibody titers againstM. pneumoniaerose in the repeatedly infected mice but not in the singly infected mice. The percentage of CD4-positive, CD8-positive, and CD25-positive lymphocytes infiltrated in the lung was increased in the repeatedly infected mice. In contrast, the lymphocyte subset in the spleen was not significantly different among mock-, singly, and repeatedly infected mice. In the study of cytokine productivity of spleen cells, production of interleukin (IL)-4 and IL-10 was significantly increased and that of gamma interferon was remarkably increased in the mice following repeated infection. These results indicate that a gnotobiotic mouse model monoassociated withM. pneumoniaewas established and that immune mechanisms might be involved in the pathogenesis in pneumonia followingM. pneumoniaeinfection.

A mouse enterohaemorrhagic Escherichia coli (EHEC) infection model was developed with a combination of germ-free (GF) mice and hyper-toxigenic EHEC (HT-EHEC) O157:H7 strain 6. The HT-EHEC strain 6 produced both Shiga-like toxin (SLT)-1 1.0 microg/mI and SLT-2 8.2 microg/ml in vitro. Eight-week-old germ-free mice were inoculated intragastrically with 5.0 x 10(7) cfu of HT-EHEC strain 6. All GF mice challenged with the HT-EHEC developed ruffled fur and convulsion of limbs or hindleg weakness within 3 days after the challenge, culminating in death within 5 days. The HT-EHEC colonised well at a level of 10(8) - 10(9) cfu/g of faeces 5 days after the challenge. Both SLT-1 and SLT-2 were demonstrated in the faeces of the mice for 5 days after challenge. Histological examination of the colons of the infected mice showed congestion of the lamina propria mucosa, mild inflammatory cell infiltration and goblet cell depletion. In proximal tubules of the renal cortex, epithelial swelling with scattered necrotic cells was recognised. Endothelial swelling and mononuclear cell infiltration were also observed in the glomeruli. The brain showed acute neuronal necrosis in the cerebrum and slight loss of Purkinje cells in the cerebellum. Passive immunisation with anti-SLT antisera prolonged the life of the mice without anyneural symptoms. Microscopically, all tissue specimens from the passively immunised mice were not remarkable. These results indicate that the infection of GF mice with HT-EHEC is a useful animal model to study the pathogenicity of SLT-producing E. coliand the toxins.

To determine the pathological significance of the coccoid form of Helicobacter pylori, its adhesion to and induction of secretion of interleukin-8 (IL-8) by gastric epithelial (MKN45) cells were studied. By flow cytometry, the adhesion of the coccoid form to MKN45 cells was significantly lower than that of the helical form. The monoclonal antibody A20 recognising lipopolysaccharide of H. pylori inhibited the adhesion of the coccoid form to MKN45 cells as much as that of the helical form. There was significantly lower induction of IL-8 secretion by MKN45 cells exposed to the coccoid form (two of four strains) as compared with the helical form.

In mycoplasmal pneumonia, the bronchi are histopathologically filled with polymorphonuclear leukocytes. The EGFR pathway is involved in IL-8 production. We investigated the contribution of the EGFR pathway to IL-8 production by bronchial epithelial cells (A549) stimulated with Mp-Ag. The IL-8 production by A549 cells stimulated with Mp-Ag was decreased by the addition of an EGFR kinase inhibitor or transfection with small interfering RNA against EGFR. The levels of epiregulin mRNA in A549 cells were increased by stimulation with Mp-Ag. In conclusion, the EFGR pathway participates in IL-8 production by bronchial epithelial cells stimulated with Mp-Ag.

Ketones were obtained in good yields by titanocene(II)-promoted reaction of thioacetals with alkanenitriles. The regioselective formation of α-substituted ketone was observed when the reaction was carried out in the presence of methyl iodide or benzyl bromide.

Antagonistic interaction between Clostridium butyricum strain MIYAIRI 588 and enterohemorrhagic Esherichia coli (EHEC) strain O157:H7 006 was examined using streptomycin-treated SPF mice and germ free mice. All SPF mice pretreated with streptomycin were colonized with EHEC O157:H7. On the other hand, only 20% of the SPF mice pretreated with streptomycin and C. butyricum were colonized with EHEC O157:H7. In addition, germ free mice died within 4-7 days after infection with EHEC O157:H7. In contrast, all gnotobiotic mice mono-associated with C. butyricum survived after the challenge with EHEC O157:H7. Both the number of EHEC and the amounts of shiga-like cytotoxin (SLT, type 1 and type 2) in fecal contents of gnotobiotic mice treated with C. butyricum were less than those of mice infected with only EHEC O157:H7. In conclusion, the probiotic bacterium, C. butyricum strain MIYAIRI 588, has a preventive effect against EHEC O157:H7 infection.

Co-magaldrox (Maalox) is used world-wide as an antacid and as a cytoprotective agent for gastritis and peptic ulcer diseases. We examined the effects of co-magaldrox on Helicobacter pylori.Adhesion of H. pylori to human gastric epithelial cells (MKN45) was evaluated by flow cytometry. Morphologic changes in H. pylori caused by co-magaldrox were determined by scanning electron microscopy. Induction of interleukin-8 (IL-8) from MKN45 cells was examined with enzyme-linked immunosorbent assay, and the intracellular and extracellular expression of heat-shock protein 60 (HSP60) was analyzed with sodium dodecyl sulphate-polyacrylamide gel electrophoresis and flow cytometry.Adhesion of H. pylori to MKN 45 cells was significantly inhibited by 1.25%-5% comagaldrox. H. pylori aggregated with co-magaldrox according to an electron microscopic examination. IL-8 secretion from MKN45 cells after H. pylori infection was also inhibited by co-magaldrox. Extracellular expression of HSP60 on the surface of H. pylori was decreased after treatment with comagaldrox, whereas the intracellular synthesis of HSP60 was not. HSP60-induced IL-8 secretion was significantly inhibited by co-magaldrox in a dose-dependent manner.These results show that co-magaldrox suppressed the expression of the following virulence factors: adhesion, IL-8 inducibility, and expression of extracellular HSP60. Therefore, co-magaldrox is a potent anti-H. pylori and cytoprotective drug.

The antimicrobial activities of aqueous cacao mass extract against enterohemorrhagic Escherichia coli (EHEC) O157:H7 006 strain were studied. Hot water extract of cacao mass (cocoa extract) was shown to inhibit the growth of EHEC O157:H7 006 strain in PBS or CAYE medium. In addition, the production of verotoxins (types 1 and 2) of EHEC O157:H7 006 strain was significantly inhibited by 8.0% cocoa extract. The cocoa extract did not neutralize the cytotoxity of verotoxins, but had inhibitory effect on adhesion of verotoxins to the target Vero cells. These results demonstrate that cacao mass has antimicrobial effects on EHEC O157:H7.

Monoclonal antibodies (MAbs) that inhibit adhesion of Helicobacter pylori to human gastric cancer (MKN45) cells were established to clarify the mechanism of adhesion of H. pylori. Of 53 hybridoma clones screened by the primary inhibition assay for adhesion, MAb A20 of IgM class was selected on the basis of both its reactivity to whole cells of H. pylori by ELISA and its inhibitory effect on adhesion of H. pylori. The adhesion of H. pylori strain TK1029 to MKN45 cells was inhibited by MAb A20, depending on the concentration of the MAb. The MAb recognised the surface antigen, lipopolysaccharide (LPS) of H. pylori, suggesting that LPS is associated with adhesion of H. pylori to human gastric epithelial cells.

The treatment of carbonyl compounds with the organotitanium species formed from methoxybis(phenylthio)methane and low valent titanium species Cp 2 Ti[P(OEt) 3 ] 2 gave 1-alkenyl ethers in good yields. 1-Alkenyl sulfides were also obtained by the similar reaction using triphenyl trithioorthoformate. These reactions were successfully applied to carboxylic esters and thiolesters to produce 1,2-diheteroatom substituted 1-olefins.

Among the various virulence factors of Helicobacter pylori the role of its heat shock protein 60 (HSP60, HspB) in mucosal inflammation after H. pylori infection was examined. In flow cytometric analysis, the expression of HSP60 on the cell surface was different, depending on the H. pylori strain used. The HSP60 epitope was also detected on the surface of both human gastric cancer cells (MKN45, KATOIII, and MKN28) and human gastric biopsy specimens. The intensity of the expression of HSP60 on the cell surface correlated significantly with the adhesion of H. pylori to MKN45 cells, but not with urease activity and production of vacuolating cytotoxin. A monoclonal antibody to H. pylori HSP60 inhibited the adhesion of H. pylori to MKN45 cells. These results suggest that HSP60 of H. pylori might act as an important virulence factor after H. pylori infection.

Adhesion of Helicobacter pylori to both human gastric carcinoma cell lines (MKN45, MKN28 and KATO III) and prepared primary human gastric epithelial cells were analyzed with flow-cytometry. All strains adhered to human gastric carcinoma cells. Especially, these strains strongly adhered to MKN45 cells. Adhesion of H. pylori strains to prepared primary human gastric epithelial cells was also observed. However, the adherence rates of H. pylori to these cells were different among the cells used. These results suggested that the host factor might be important for adhesion of the bacteria to human gasgric cells. In addition, H20 monoclonal antibody directed to H. pylori HSP60 inhibited the adhesion of H. pylori to both cells. These results indicate that H. pylori HSP60 might be associated with the adhesion of the bacteria to human gastric epithelial cells.

The H20mAb recognizing the 60-kilodalton protein, which existed in the outer membrane and was induced by heat shock at 42 C, was established. The molecule recognized with the mAb was a heat-shock protein 60 (HSP60) of Helicobacter pylori. To understand the role of HSP60 on the cell surface of H. pylori, whether or not H20mAb affects the growth of H. pylori was investigated. When bacteria were cultured with H20mAb, growth was markedly inhibited after 24 hr, although an initial 5 hr-incubation with the mAb induced no significant inhibition of H. pylori growth. The 24- and 48 hr growth of the bacteria after washing to remove the mAb at 5 hr was also inhibited though the inhibitory effect was not strong. In electron microscopical analysis, the spots with high electron density in the cytoplasm of the bacteria treated with H20mAb were increased, depending on the length of incubation time from 5 to 24 hr. After 24 hr treatment with H20mAb, bacterial destruction was also observed, indicating bactericidal activity by H20mAb. These results suggest that the HSP60 on the cell surface of H. pylori might have an essential role in the growth of the bacteria.

Two monoclonal antibodies (MAbs), designated as H9 (IgG2a) and H20 (IgM), directed against heat-shock protein 60 (HSP60) of Helicobacter pylori strain TK1029 were established. Affinity-purified antigens cross-reacted in immunoblots with MAb H9 and MAb H20 respectively. These antigens also reacted with the 3C8 MAb previously established in this laboratory, which recognised Yersinia enterocolitica HSP60. By amino-acid sequence analysis, the N-terminal amino-acid sequence of the protein recognised by both H9 and H20 MAbs was confirmed as the amino-acid sequence of H. pylori HSP60 reported previously. Both MAbs reacted with nine strains of H. pylori in enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. In addition, MAb H9 reacted with extracts of other bacteria including H. mustelae, Pseudomonas aeruginosa, Vibrio cholerae, Serratia marcescens, Proteus mirabilis, Escherichia coli and Shigella sonnei. In contrast, MAb H20 reacted only with strains H. pylori. These results suggest that both the species-specific epitope recognised by MAb H20 and the common epitope recognised by MAb H9 exist on HSP60 of the bacterial cell. Both MAbs also reacted with the 60-kDa protein in the lysate of human gastric carcinoma (MKN45) cells. It was shown by immunohistochemical staining that gastric epithelial cells of four out of six biopsy specimens examined stained positively with MAb H20. These results suggest that there is a common epitope in H. pylori HSP60 and human gastric epithelial cells.

A previous study reported a relationship between the expression of heat-shock protein 60 (HSP60) by Helicobacter pylori and its adhesion to human gastric carcinoma (MKN45) cells. To examine whether the HSP60 homologue of H. pylori is associated with the adhesion of H. pylori to human gastric epithelial cells, an inhibition assay of adhesion of H. pylori to MKN45 cells was performed by flow cytometric analysis with monoclonal antibody (MAb) designated as H20 recognising HSP60 of H. pylori. The rate of adhesion of H. pylori pretreated with MAbH20 to MKN45 cells was lower than that of untreated H. pylori. Primary human gastric epithelial cells from a patient with gastric cancer were also prepared for comparison in the inhibition assay with MAbH20. H. pylori adhered to the primary human gastric epithelial cells, and this adhesion was significantly inhibited by MAbH20. These results suggest that the H. pylori HSP60 homologue recognised by MAbH20 might be associated with the adhesion of H. pylori to primary human gastric epithelial cells as well as to cultured gastric cancer cells.

The adhesion of Helicobacter pylori to gastric carcinoma cells (MKN45, KatoIII and MKN28) and Intestine-407 cells was tested by flow cytometric analysis. The mean adhesion rates of H. pylori strains to MKN45, KatoIII and Intestine-407 cells were 90.5, 42.7 and 15.1%, respectively. There was no statistical correlation between the adhesion rates to MKN45 cells and haemagglutination (HA) activity of H. pylori strains, although H. pylori strains with high HA activity with human type O erythrocytes tended to adhere effectively to MKN45 cells. No correlation between adhesion and production of vacuolating toxin was observed.

The adhesion of Vibrio cholerae O1 strains to human intestinal epithelial cell, Intestine 407, was analyzed by flow cytometer. According to positive percentages of Intestine 407 cells adhered by V. cholerae, two groups of V. cholerae strains were classified as follows: more adhesive (more than 50%), less adhesive (less than 50%) strains. In addition, the fluorescence intensity after attachment of V. cholerae was directly correlated to the number of the microorganisms. It was concluded that flow cytometry is a useful and objective method for analyzing adhesion of V. cholerae to cultured cells.

We examined the effect of sofalcone, a mucosal protective agent that has been reported to inhibit growth of Helicobacter pylori, on adherence, production of vacuolating toxin (VT), and induction of interleukin-8 (IL-8) secretion by H. pylori. Mixing VT with various concentrations (1.5-100 micrograms/ml) of sofalcone resulted in a 50% decrease in the VT titer. When toxigenic H. pylori strains were incubated in the presence of sofalcone (20 and 40 micrograms/ml), although bacterial growth was not inhibited significantly there was significant inhibition of VT production. Notable inhibition of IL-8 secretion by human gastric cancer cells (MKN 45) was detected in the presence of sofalcone (20-100 micrograms/ml) after co-incubation with H. pylori strains. In flow cytometric analysis, adherence of H. pylori strains to MKN 45 cells was significantly inhibited by treatment with sofalcone (20-60 micrograms/ml), indicating at least one reason for the inhibition of IL-8 secretion. These results show that sofalcone is an effective mucosal protective agent that inhibits both production of VT and induction of IL-8 secretion.

The expression of a 60-kDa heat shock protein (HSP60) on the cell surface of Helicobacter pylori was analysed by flow cytometry with polyclonal antibody directed to HSP60. All 13 strains of H. pylori examined expressed HSP60 on the cell surface, although the intensity of expression was different among the strains and depended on culture conditions. There was a correlation between the intensity of HSP60 expressed on the cell surface and the rate of adherence to human gastric carcinoma cells (MKN45) by H. pylori, but not with urease activity and production of vacuolating toxin. By flow cytometric analysis with monoclonal antibody (MAb) 3C8 against HSP60, the reactive epitope in the HSP60 of H. pylori was detected on the surface of MKN45 cells. Furthermore, it was shown that gastric epithelial cells were positively stained with MAb 3C8 in one of two biopsy specimens examined. These results suggest that there is a common epitope showing homology between H. pylori HSP60 and human gastric epithelial cells.

Summary The immunocytochemical localization of the 60-kDa heat shock protein (HSP) in Vibrio cholerae strain 569B was studied by transmission electron microscopy using a combination with the antigen-specific monoclonal antibody (5C3) and immunogold labelling. The labelling with gold particles in V. cholerae detected 2 types; the gold particles were exclusively detected in the cytoplasm for one type and in the periplasmic space for another type, suggesting that the 60-kDa HSP of V. cholerae corresponding immunologically to Escherichia coli GroEL may translocate from the cytoplasm to the periplasmic space in the V. cholerae cell.

To determine amino acid sequences of the epitopes recognized by monoclonal antibodies (mAbs) 3C8 and 5C3 directed against Yersinia enterocolitica heat-shock protein (HSP60), a dot blot analysis was performed using synthesized peptides of Y. enterocolitica HSP60 such as peptides p316-342, p327-359, p340-366, p316-326, p316-321, p319-323, and p321-326 which represent positions of amino acids in Y. enterocolitica HSP60. The dot blot analysis revealed that 5C3 mAb reacted with p316-342, p316-326 and p321-326, and 3C8 mAb p316-342 and p316-326. These results indicate that the epitopes recognized by the mAbs were associated with eleven amino acids, Asp Leu Gly Gln Ala Lys Arg Val Val Ile Asn, of p316-326. The sequence homology between p316-326 of Y. enterocolitica HSP60 and the rest of the HSP60 family suggests that the five amino acids of Lys, Arg, Val, Ile and Asn, which are highly conserved in the HSP60 family, might be related with the epitope recognized by 3C8. In contrast, it was also demonstrated that three amino acids of Leu, Gly and Val, which are not well conserved in the HSP60 family, might be related to the epitope recognized by 5C3.

A comparative study of indirect and direct flow cytometric analysis for adherence of Vibrio cholerae to Intestine 407 cells was performed. The direct flow cytometric analysis employed the lipophilic dye PKH-2. It was concluded that direct flow cytometry using the lipophilic dye PKH-2 is useful and convenient for analyzing bacterium-host cell interactions, since it does not require any specific antibody as the first antibody.

Stereospecific alkylation of (Z)-β-hydroxymethylvinylsilanes proceeded by treatment with copper(I) tert-butoxide and alkyl halides. Tri- and tetra-substituted allylic alcohols were obtained with complete retention of configuration.

In mycoplasmal pneumonia, the bronchi are histopathologically filled with polymorphonuclear leukocytes. The EGFR pathway is involved in IL-8 production. We investigated the contribution of the EGFR pathway to IL-8 production by bronchial epithelial cells (A549) stimulated with Mp-Ag. The IL-8 production by A549 cells stimulated with Mp-Ag was decreased by the addition of an EGFR kinase inhibitor or transfection with small interfering RNA against EGFR. The levels of epiregulin mRNA in A549 cells were increased by stimulation with Mp-Ag. In conclusion, the EFGR pathway participates in IL-8 production by bronchial epithelial cells stimulated with Mp-Ag.

Probiotics involving Lactobacillus, Bifidobacterium, Saccharomyces , Clostridium butyricum, etc. have been reported to have inhibitory effects on Helicobacter pylori infections in in vitro and in vivo studies. In addition, these probiotics have been reported to be effective in clinical studies; the patients treated with triple therapy combined with probiotics had a higher H. pylori eradication rate than those with triple therapy only, and antibiotic-associated gastrointestinal side effects during H. pylori eradication therapy were reduced in the patients treated with a probiotics supplemented regimen. It was also reported that probiotic supplementation reduced side effects and permitted a slight improvement in eradicating H. pylori in secondline eradication therapy.