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We evaluated gamma-irradiated Listeria monocytogenes as a killed bacterial vaccine, testing the hypothesis that irradiation preserves antigenic and adjuvant structures destroyed by traditional heat or chemical inactivation. Irradiated Listeria monocytogenes (LM), unlike heat-killed LM, efficiently activated dendritic cells via Toll-like receptors and induced protective T cell responses in mice. Like live LM, irradiated LM induced Toll-like-receptor-independent T cell priming. Cross-presentation of irradiated listerial antigens to CD8(+) T cells involved TAP- and proteasome-dependent cytosolic antigen processing. These results establish that killed LM can induce protective T cell responses, previously thought to require live infection. gamma-irradiation may be potentially applied to numerous bacterial vaccine candidates, and irradiated bacteria could serve as a vaccine platform for recombinant antigens derived from other pathogens, allergens, or tumors.

This paper compares the fiscal disciplines of local governments in various countries, and discusses the characteristics of local government bond systems in the United States, France, Canada, and Sweden studied through local survey. Some points are clarified from the international comparison. In many countries except Japan, local governments face discipline through market mechanism in the market of local government bond. Various actors such as investors and banks in the market support the fiscal discipline. Furthermore, some fiscal rules also play an important role for reduction of local government debt.

The WRNIP1 protein interacts with WRN, the product of the causative gene for Werner syndrome. Mutation of the Saccharomyces cerevisiae gene MGS1, the yeast counterpart of WRNIP1, confers synthetic lethality with mutation of RAD18. To examine the functional relationship between WRNIP1 and Rad18 in higher eukaryotic cells, we generated WRNIP1-/-/-/RAD18-/- lines from chicken DT40 cells and compared them with single mutant cell lines. Unlike the corresponding yeast mutant, WRNIP1-/-/-/RAD18-/- cells are viable but grow more slowly than single mutants and wild type cells, and they show an additive or synergistic elevation in the frequency of sister chromatid exchanges. As reported, WRNIP1-/-/- cells and RAD18-/- cells are moderately and severely sensitive to camptothecin (CPT), respectively. Unexpectedly, the severe CPT sensitivity of RAD18-/- cells is slightly suppressed by disruption of the WRNIP1 gene.

The diethyl ether extracts from aqueous 4-methylphenol solutions in the presence of bromide ion after treatment with chlorine were mutagenic to the Ames salmonella test strain TA100 in the absence of rat liver homogenate. Gas chromatography/mass spectrometry (GC/MS) showed the occurrence of halogenated products in the extracts: bromo-4-methylphenols, bromo-4-methylquinones and brominated 4-methylphenol dimers. The diethyl ether extracts were fractionated into several fractions by polyamide thin-layer chromatography (TLC) . The fractionated components were examined for mutagenicity by means of Ames assays, and were identified by GC/MS. TLC fraction of the extracts revealed that two components present in the extracts are mutagenic. GC/MS analysis indicated the presence of 2, 6-dibromo-4-hydroxy-4-methylcyclohexa-2, 5-lien-1-one (2, 6-dibromotolquinol) as the major mutagen and brominated 4-methylphenol dimers as minor mutagenic compounds in the chlorinated 4-methylphenol solutions containing bromide ion. Production of 2, 6-dibromotolquinol in water was found to be dependent on the equivalents of chlorine per mole of compound and the reaction pH.

The interactions between genetic and environmental factors play a major role in the development of childhood asthma. We hypothesized that a pre-existing Th2/asthmatic response can promote Th2 responses to newly encountered Ags (i.e., phenotype spread). To test this hypothesis, we developed a mouse model in which the requirements for the induction and inhibition of phenotype spread to a clinically relevant neo-allergen (i.e., ragweed) were investigated. Our results indicate that 1) phenotype spread to the neo-allergen can be induced only within the first 8 h after a bronchial challenge with the first Ag (OVA); 2) Th2 differentiation of naive CD4+ T cells occurs in bronchial lymph nodes; 3) trafficking of naive CD4+ T cells to local lymph nodes and IL-4 produced by OVA-activated Th2 cells play essential roles in the differentiation of naive CD4+ T cells to Th2 cells; and 4) suppression of the production of chemokines involved in the homing of naive CD4+ T and Th2 cells to bronchial lymph nodes by a TLR9 agonist inhibited phenotype spread and abrogated the consequent development of experimental asthma. These findings provide a mechanistic insight into Th2 phenotype spread and offer an animal model for testing relevant immunomodulatory interventions.

By conducting a survey of Swedish families and analyzing the data, this paper identifies factors that support the coexistence of high female labor participation and total fertility rate: child-care leave for more than one year, working part-time after leave, child-care service at lower cost and better child benefits. Due to leave, 25 percent of the female labor force age 25-34 are actually absent from the workplace. The survey also finds that more than 90 percent of Swedish legally married couples have experienced co-habitation for several years before their marriage.

Epidemiological evidence points to the inverse relationship between microbial exposure and the prevalence of allergic asthma and autoimmune diseases in Westernized countries. The molecular basis for this observation has not yet been completely delineated. Here we report that the administration of certain toll-like receptor (TLR) ligands, via the activation of innate immunity, induces high levels of indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme of tryptophan catabolism in various organs. TLR9 ligand-induced pulmonary IDO activity inhibits Th2-driven experimental asthma. IDO activity expressed by resident lung cells rather than by pulmonary DCs suppressed lung inflammation and airway hyperreactivity. Our results provide a mechanistic insight into the various formulations of the hygiene hypothesis and underscore the notion that activation of innate immunity can inhibit adaptive Th cell responses.

Background Environmental exposures to toll-like receptor (TLR) ligands have been suggested to provide immunologic protection against allergic diseases. However, some TLRs use unique intracellular signaling pathways, suggesting that ambient TLR ligand exposures might induce a range of host responses. Objective These investigations compared peptidoglycan (PGN; TLR2)–induced and immunostimulatory sequence DNA oligodeoxynucleotide (ISS-ODN; TLR9)–induced innate responses and determined how airway exposures to these TLR ligands affect adaptive immunity and the asthmatic phenotype. Methods In in vitro and in vivo studies innate responses to PGN and ISS-ODN were compared. Alternatively, mice were intranasally immunized with ovalbumin (OVA) alone or OVA plus PGN or ISS-ODN, and adaptive immune profiles and responses to airway OVA challenge were assessed. Results PGN and ISS-ODN induced divergent innate responses predictive of their having T H 2- and T H 1-biasing adjuvant potential, respectively. Consistent with these findings, mice intranasally immunized with OVA alone had relatively weak adaptive responses, whereas intranasal OVA/PGN– and OVA/ISS-ODN–coimmunized mice had much stronger humoral and cellular responses that were T H 2 and T H 1 biased, respectively. Finally, on airway allergen challenge, mice intranasally immunized with OVA alone had modest T H 2-biased airway hypersensitivity responses, whereas airway responses were greatly exaggerated for intranasal OVA/PGN–immunized mice. In contrast, intranasal OVA/ISS-ODN–immunized mice had little evidence of airway hypersensitivity after airway allergen challenge. Conclusions Considered in a larger context, these results suggest that inspired air is likely to contain TLR ligands capable of both preventing and precipitating the asthmatic phenotype.

An HPLC method for the analysis of orange color in food using β‐cryptoxanthin as an indicator was established. Orange color was extracted from food samples with ether, and after evaporation of the extract, the residue was dissolved in methanol, to which 5% sodium hydroxide–methanol was then added. The mixture was occasionally stirred, then allowed to stand for 24 hr at room temperature, in a tightly sealed container shielded from light. Subsequently, distilled water was added and the pH of the mixture was adjusted to be below 4.5 using hydrochloric acid. It was then purified with a C18 cartridge before being subjected to HPLC analysis. The HPLC conditions were as follows: column, Tosoh TSK gel ODS‐80Ts (5 µm, 4.6 × 150 mm); column temperature, 45°C; mobile phase, acetone–water (6:1); flow rate, 1.2 mL/min; detection wavelength, 460 nm. The average recoveries of the orange color were over 73.9% from juice and jelly fortified at the concentrations of 2–100 µg/g. The coefficients of variation were 1...

An HPLC method for the analysis of marigold color in food, using lutein as an indicator, was established. Marigold color was extracted from food samples with ether and, after evaporation of the extract, the residue was dissolved in methanol to which 5% sodium hydroxide–methanol was then added. The mixture was occasionally stirred, then allowed to stand for 24 hours at room temperature in a tightly sealed container shielded from light. Subsequently, distilled water was added and the pH of the mixture was adjusted to below 4.5 using hydrochloric acid. It was then purified with a C18 cartridge before being subjected to HPLC analysis. The HPLC conditions were as follows: column, Tosoh TSK gel ODS‐80Ts (5 µm, 4.6 × 150 mm); column temperature, 40°C; mobile phase, acetonitrile–water (9:1); flow rate, 1.0 mL/min; detection wavelength, 460 nm. The average recoveries of the marigold color were over 78.6% from noodles and juice fortified at the concentrations of 25–500 µg/g. The coefficients of variation w...

Chitin carbamate derivatives including 4-substituted and 3,5-disubstituted phenylcarbamates, 1-phenylethylcarbamates, and cycloalkylcarbamates were synthesized and coated on macroporous silica gel to evaluate their chiral recognition abilities as chiral stationary phases (CSPs) for high-performance liquid chromatography (HPLC). Among the derivatives, the 3,5-dimethylphenyl, 4-chlorophenyl, and 4-trifluoromethylphenylcarbamates showed relatively high-chiral recognition abilities when a hexane–2-propanol mixture was used as the eluent. The CSPs based on the chitin 3,5-dimethylphenyl and 3,5-dichlorophenylcarbamates could be stably used in the presence of chloroform and ethyl acetate as a component of the eluents, and a few racemates were more sufficiently resolved by the addition of a small amount of chloroform in the mobile phase. Some racemates were more efficiently resolved under the reversed phase condition.

The selective extraction of heavy rare-earth metal ions is achieved by using a novel calix[4]arenediphosphonic acid 4, in which two phosphono groups are introduced directly at the lower rim of the ...

Chronic lymphocytic leukemia (CLL), the most common type of leukemia in the Western world, has a clinically variable course, with differences in the pace of malignant B-lymphocyte accumulation and in the time until cytoreductive treatment is needed. In various solid tumors, the expression of stem cell associated markers, such as aldehyde dehydrogenase (ALDH), has been associated with aggressiveness and a poor prognosis. However, the level of ALDH has not been assessed in CLL B cells. Despite their mature appearance, the B cells from patients with CLL possess immature characteristics both functionally and biochemically. CLL B cells often display biochemical markers of cells early in the blood lineage, including ROR1 [1], Wntl6 [2] and LEF1. In addition, CLL B cells have higher levels of reactive oxygen species (ROS) than normal B cells [3] The ALDH enzyme family is thought to protect cells from accumulated aldehydes formed during lipid peroxidation and poorly regulated oxidative metabolism in a low oxygen environment [4]. Cells with a high level of ALDH activity are considered to have improved self-renewal capacity [5]. The goal of this study was to ascertain whether increased ALDH activity is associated with more aggressive disease in CLL. To this end, we compared ALDH activity with two CLL biomarkers, immunoglobulin heavy chain variable gene (IgHV) mutation status and ZAP-70 expression. These biomarkers are regularly used to classify patients with CLL as high-risk: IgHV unmutated (≥98% homology to IgHVgermline gene) or ZAP-70 positive (≥ 20% of CD19 CLL B cells expressing ZAP-70) and low-risk: IgHV mutated and ZAP-70 negative [6]. Blood samples had been previously collected and viably stored from 40 consenting subjects diagnosed with CLL and followed at the University of California San Diego (UCSD) Moores Cancer Center, with the approval of the UCSD Institutional Review Board. Twenty cases were selected with unmutated IgHV or that were ZAP-70 positive (high risk), and an additional 18 cases were selected which had both mutated IgHV and were ZAP-70 negative (low risk) [6]. Two samples were excluded because of insufficient clinical data. All blood samples were collected from patients prior to treatment. In addition, peripheral blood mononuclear cells (PBMCs) were prepared from samples collected from healthy volunteers registered at the San Diego Blood Bank, as previously described [6]. Cells (2.5 × 106) were stained for ALDH activity with an Aldefluor assay kit (StemCell Technologies, Vancouver, Canada) according to the manufacturer’s instructions. The cells were then stained for the B cell surface marker CD19, and analyzed using a FACSCalibur instrument (BD Biosciences, San Diego, CA). A total of 50,000 events were acquired. Data analysis was performed using Flowjo software (Ashland, OR) and Prism (GraphPad Software, La Jolla, CA). The cells were gated for viable CD19+ cells and mean fluorescent activity was determined. Receiver operating characteristic (ROC) curves were computed in R v2.13.0 [7] using the package ROCR, with standard errors and corresponding 95% confidence intervals (CIs) computed based on the standard errors of Somers’ Dxy rank correlation using the function rcorr.cens from the Hmisc package [8]. Significance was attained at the 5% level if the CI for the AUC was entirely above 0.50, demonstrating greater predictive ability than by chance alone. We assayed mean ALDH levels in CD19− B cells in 18 previously characterized low-risk (ZAP-70 negative and IgHV mutated) and 20 high-risk (IgHV unmutated or ZAP-70 positive) patients with CLL, as well as PBMCs [6] from six healthy individuals. The characteristics of the patients are summarized in Table I. Table I Characteristics of patients with CLL. The mean ALDH activity was elevated in the high-risk CLL samples, compared to the low-risk CLL group (p = 0.002, one-sided Wilcoxon test) [Figure 1(A); combined high-risk groups, n = 20, vs. low risk group, n = 18]. Normal CD19− B cells exhibited low levels of ALDH activity. The area under the curve (AUC) for classifying high-risk versus low-risk samples using the ALDH level was 0.77 (95% CI: 0.62, 0.92) [Figure 1(B)], indicating that ALDH activity had a moderate but statistically significant ability to discriminate between these two groups. Considering each biomarker separately, ALDH had a similar ability to classify IgHV mutational status alone [AUC of 0.77; 95% CI: 0.62, 0.92; Figure 1(C)] and ZAP-70 status alone [AUC of 0.76; 95% CI: 0.59, 0.93; Figure 1(D)]. Moreover, for the 16 samples with unmutated IgHV and an ALDH value above 400, ZAP-70 expression correlated with ALDH activity (r = 0.90 with p 400 ALDH]. These data collectively show: (i) that ALDH activity is increased in CLL B cells, (ii) that ALDH has a similar moderate ability to classify patients with CLL according to IgHV mutational status and ZAP-70 expression status alone, and jointly, and (iii) that increased ALDH activity is associated with high-risk CLL. Figure 1 ALDH activity in chronic lymphocytic leukemia. (A) Mean ALDH activities were determined in the gated CD19+ population. Mean ALDH activities of different groups of CLL and CD19+ B cells from healthy volunteers are shown. Bars indicate mean values. (B–D) ... The observation that ALDH activity is increased in CLL samples obtained from patients with high-risk characteristics has implications related to disease pathogenesis and treatment. Recently, elevated ALDH activity in CD34+ CD38− stem-like cells from acute myelogenous leukemia has been correlated with subsequent clinical relapse [9]. Increased ALDH activity in CLL cells might similarly reflect an immature, aggressive phenotype. ALDH enzymes enable cells to remove toxic products formed in a hypoxic environment as byproducts of oxidative metabolism. Together with other factors, the elevated ALDH in high-risk CLL may promote cell survival in stressful tissue compartments. ALDH may also aid in the detoxification of oxidation products resulting from overexpression of c-Myc and activated phosphatidylinositol-3 kinase (PI3K), as commonly observed in CLL [10,11]. In summary, ALDH activity, which can be measured relatively easily, warrants further investigation for use as a screening biomarker to classify patients with CLL according to a high-risk versus low-risk disease course. Future studies are needed to determine the precise role of ALDH in the pathogenesis of CLL.

In the present study, a thin-layer chromatography (TLC) method for the analysis of tomato, orange, and marigold colors in foods was developed. The colors were extracted from food samples with ethyl ether, and after the extract was evaporated, the residue was dissolved in methanol. For the analysis of the tomato color, after the addition of water to the methanol solution, it was then purified through a C18 cartridge before being subjected to the TLC analysis. With respect to the analyses of the orange and marigold colors, after adding 2 mL of a 5% sodium hydroxide–methanol solution to the methanol solution, the mixture was occasionally stirred, kept away from light, and then allowed to stand for 24 hours at room temperature. Subsequently, the pH of the mixture was adjusted to 4.5 or less using 1 mol/L hydrochloric acid. The mixture was then purified through a C18 cartridge before being subjected to the TLC analysis. The TLC conditions were as follows: plate, RP-18F254S (Art. 15389, E. Merck); solv...

Ubc9 is an enzyme involved in the conjugation of SUMO-1 (small ubiquitin related modifier 1) to target proteins. The SUMO-1 conjugation system is well conserved from yeasts to higher eukaryotes, but many SUMO-1 target proteins reported recently in higher eukaryotic cells, including IκBα, MDM2, p53, and PML, are not present in yeasts. To determine the physiological roles of SUMO-1 conjugation in higher eukaryotic cells, we constructed a conditional UBC9 mutant of chicken DT40 cells containing the UBC9 transgene under control of a tetracycline-repressible promoter and characterized their loss of function phenotypes. Ubc9 disappeared 3 days after the addition of tetracycline and the increase in viable cell number stopped 4 days after the addition of drug. In contrast to the cases of ubc9 mutants of budding and fission yeasts, which show defects in progression of G2 or early M phase and in chromosome segregation, respectively, we did not observe accumulation of cells in G2/M phase or a considerable increase in the frequency of chromosome missegregation upon depletion of Ubc9 but we did observe an increase in the number of cells containing multiple nuclei, indicating defects in cytokinesis. A considerable portion of the Ubc9-depleted cell population was committed to apoptosis without accumulating in a specific phase of the cell cycle, suggesting that chromosome damages are accumulated in Ubc9-depleted cells, and apoptosis is triggered without activating checkpoint mechanisms under conditions of SUMO-1 conjugation system impairment.

Synthetic oligodeoxynucleotides (ODNs) containing immunostimulatory sequences (ISS-ODN, also known as CpG-ODNs) have been shown to display in experimental models potent Th1-biassed immunoadjuvant activity upon parenteral or mucosal co-administration with a variety of antigens. In an attempt to potentiate adjuvant activity, and to reduce dose and number of administrations, ISS-ODN was entrapped (up to 90% efficiency) in large (1.5 microm) multilamellar liposomes using a simple and fast (5 min) procedure. Mice were vaccinated once or twice intramuscularly (i.m.) or intranasally (i.n.) with subunit influenza vaccines (consisting of the viral hemagglutinin and neuraminidase, HN) or with hepatitis B surface antigen particles (HBsAg), either non-encapsulated or liposome-encapsulated, together with free or liposomal ISS-ODN (5-25 microg per dose). At 3-12 weeks post-vaccination, the humoral (systemic, mucosal) and cellular responses and protective immunity were assessed. Vaccine formulations containing liposomal ISS-ODN co-administered with either soluble antigen or liposomal antigen (in the same vesicles or in separate vesicles) were up to 30 times more effective than formulations containing un-encapsulated ISS-ODN in inducing: (a) antigen-specific serum and mucosal IgG2a and IgA antibodies; (b) splenocyte proliferative response, cytotoxic activity and IFNgamma production; (c) a DTH response; and (d) protection against virus challenge. The response was Th1-dominant in the influenza model and a mixed Th1+Th2 response in the hepatitis B model. No adverse reactions were noted. Thus, liposomal encapsulation of ISS-ODN further enhances its inherent adjuvant activity.

In the immunocompetent host, Mycobacterium avium is responsible for chronic localized pulmonary disease, which is characterized by the presence of increased numbers of activated T cells and macrophages in the lungs. M. avium organisms as well as sonic extracts of M. avium were found to act as chemoattractants for THP-1 cells as well as monocytes, monocyte-derived macrophages and alveolar macrophages obtained from normal human donors in an in vitro chemotaxis assay, where a significantly higher number of cells were found in wells containing M. avium compared to control wells. Proteolytic treatment of M. avium sonicate resulted in significant loss (50%) of chemotactic activity. Monoclonal antibodies against recombinant human monocyte chemoattractant protein-1 (MCP-1) were found to cross-react with a 34-kDa protein of M. avium sonicate on Western blot and inhibit M. avium sonicate-mediated chemotaxis of THP-1 cells (47%). These data suggest the presence of an 'MCP-1 like' molecule on M. avium. Recruitment of host immune regulatory cells to the site of infection by pathogens may be involved in generating a local immune response or may be a bacterial strategy for survival within the host by recruiting the cells that they infect, i.e. macrophages.

A technique for the analysis of red cabbage color using reversed-phase TLC and scanning densitometry is described. The technique involves the following three steps: 1) clean up of the color with a C18 cartridge, 2) separation of the colors on the reversed-phase C18-TLC using acetonitrile-0.2 mol/L trifluoroacetic acid (1 : 2) as the solvent system, and 3) measurement of visible absorption spectra of the color using scanning densitometry without isolation of the color. In order to investigate the capability of the present method, 45 commercial foods were analyzed, and their chromatographic behaviors and spectra were observed. The obtained separation and the spectra were not affected by coexisting substances, including grape skin color, elderberry color, perilla color, and cochineal color in the foods, and the spots always gave the same Rf values and spectra as the standards with good reproducibility. The present method is considered to be useful for the rapid analysis of red cabbage color in foods.

Immunostimulatory sequence (ISS) DNA containing unmethylated CpG dinucleotides stimulate NK and APC to secrete proinflammatory cytokines, including IFN-αβ and -γ, TNF-α, and IL-6 and -12, and to express costimulatory surface molecules such as CD40, B7-1, and B7-2. Although ISS DNA has little direct effect on T cells by these criteria, immunization of wild-type mice with ISS DNA and OVA results in Ag-specific CTL and Th1-type T helper activity. This investigation examines the mechanisms by which ISS DNA primes CD8+ and CD4+ lymphocyte activities. In this report we demonstrate that ISS DNA regulates the expression of costimulatory molecules and TAP via a novel autocrine or paracrine IFN-αβ pathway. Coordinated regulation of B7 costimulation and TAP-dependent cross-presentation results in priming of Ag-specific CD8+ CTL, whereas CD40, B7, and IL-12 costimulation is required for priming of CD4+ Th cells by ISS-based vaccines.

High mobility group box protein 1 (HMGB1) acts as an endogenous danger molecule that is released from necrotic cells and activated macrophages. We have previously shown that peptide Hp91, whose sequence corresponds to an area within the B-Box domain of HMGB1, activates dendritic cells (DCs) and acts as an adjuvant in vivo. Here we investigated the underlying mechanisms of Hp91-mediated DC activation. Hp91-induced secretion of IL-6 was dependent on clathrin-and dynamin-driven endocytosis of Hp91 and mediated through a MyD88- and TLR4-dependent pathway involving p38 MAPK and NF kappa B. Endosomal TLR4 has been shown to activate the MyD88-independent interferon pathway. Hp91-induced activation of pIRF3 and IL-6 secretion was reduced in IFN alpha beta R knockout DCs, suggesting an amplification loop via the IFN alpha beta R. These findings elucidate the mechanisms by which Hp91 acts as immunostimulatory peptide and may serve as a guide for the future development of synthetic Th1-type peptide adjuvants for vaccines. Funding Agencies|National Institutes of Health/NCI [5U54 CA119335]; U.S. Army Medical Research and Materiel Command [W81XWH-07-1-0412]; Swedish Research Council [AI52731]; Swedish Physicians against AIDS Research Foundation; Swedish International Development Cooperation Agency; SIDA SARC; VINNMER for Vinnova; Linkoping University Hospital Research Fund; CALF; Swedish Society of Medicine

The aim of this study was to investigate risk factors affecting 5- and 10-year survival in autotransplantation of third molars with complete root formation at dental clinics. Participating dentists were requested to provide information on transplantations performed between 1 January 1990 and 31 December 2009. After data screening and elimination, 183 teeth in 171 men aged 20-72 years (mean, 44.8 years) and 205 teeth in 189 women aged 20-74 years (mean, 42.0 years) were included in the study. A single-factor analysis using the log-rank test revealed that the following factors had a significant influence (p

In the present study, a TLC method for the analysis of gardenia yellow in foods using crocetin as an indicator was developed. Gardenia yellow was extracted from food samples with methanol or hydrous methanol, and after the extract was evaporated, the residue was dissolved in water and the pH was adjusted to 11 or above with 1 mol/L NaOH. The resultant mixture was occasionally stirred, then allowed to stand in a water bath at 50°C for 30 minutes. Subsequently, the pH of the mixture was slightly acidified using hydrochloric acid. It was then purified through a C18 cartridge before being subjected to the TLC analysis. The TLC conditions were as follows: plate, RP-18F254S (Art. 15389, E. Merck); solvent system, acetonitrile-tetrahydrofuran-0.1 mol/L oxalic acid (7:8:7). The visible absorption spectrum of the color was measured using scanning densitometry without isolation of the color. In order to investigate the capability of the present method, 37 commercial foods were analyzed, and their chromatographic be...

In the present study, an HPLC method for the analysis of paprika color in food using capasanthin as an indicator was developed. Paprika color was extracted from food samples with ether, and after the extract was evaporated, the residue was dissolved in methanol, to which 5% sodium hydroxide–methanol solution was then added. The resultant mixture was occasionally stirred, then allowed to stand for 24 hours at room temperature in a tightly sealed container kept away from light. Subsequently, distilled water was added and the pH of the mixture was adjusted to be 4.5 or less using hydrochloric acid. It was then purified with a C18 cartridge before being subjected to the HPLC analysis. The HPLC conditions were as follows: column, TOSOH TSK gel ODS-80Ts (5 μm, 4.6 × 150 mm); column temperature, 40°C; mobile phase, acetonitrile–water (3:1); flow rate, 0.8mL/min; detection wavelength, 460nm. According to the present method, the average recoveries of the paprika color when the fortified concentrations were 0.25, 0...

献立作成能力を身に付けるためには,食品や料理についての知識,料理の技術等様々な要素が必要とされる。その中でも「料理の知識」や「自分の作ることのできる料理」は献立作成の上で重要な要素となっている。そこで,現在の大学生がレパートリーにしたいと考える料理の傾向とその選択理由について検討した。対象は平成10,12年度に調理学実習Iを履修した本短期大学部食物栄養学科1年生である。方法は実習で行った料理の中から自分の「レパートリーにしたい料理」を各自3品選び,実際に家で作り,写真を撮り,その選択理由についてもあげるレポートを提出してもらい,これを料理とその選択理由の面から分析した。その結果,学生が「レパートリーにしたい料理」の上位にあげた料理は2年ともほぼ同じ料理であり,これらはいくつかの報告にある家庭で極めて高い率で手作りする料理,日頃よく食べる料理と一致していた。また,西洋風料理を好む傾向があり,調理方法では焼く,煮るの料理を好む傾向があった。選択理由の面からみると,調理操作を第1に考えており,それはより積極的に技術を身に付けたいというものと手軽であるというものの2つに分かれていた。次は食べる人(自分)の好みであり,続いて料理のもつイメージ,使う食材の順で理由の割合が高かった。また,料理によってその選択理由に特徴がみられた。今回は学生がレパートリーにしたいと考える料理を調べ,実際に料理をするときにどんな料理に興味を示すのか,またその理由もあわせて検討したが,今後はこれらをふまえて学生の料理の知識や技術の向上を考え,献立作成能力の向上につなげていきたい。

The Aspergillus nidulans amyR gene and its cDNA were cloned and sequenced. The genomic gene comprised 2,092 bp, interrupted by two short introns, and encoded a cys-6 zinc transcriptional activator (AMYR) of 662 amino acid residues with a calculated molecular mass of 72,862 Da. Disruption of the amyR gene caused defects in the utilization of maltose and starch and abolished expression of the taaG2 gene encoding A. oryzae Taka-amylase A, which is inducibly and abundantly expressed in the wild-type A. nidulans. Expression of the amyR gene was under the control of the carbon catabolite repressor, CREA. The growth defect of the malA1 mutant on maltose was complemented by the amyR gene; and the amyR gene derived from the mutant possessed a single mutation, from A to T, at position 1,483, resulting in a substitution of His478 to Leu. These results indicate that the amyR gene is identical to the genetically defined malA gene. AMYR possessed five domains (Zn and MH1-MH4) homologous to Mal63p, a transcriptional activator for the genes involved in maltose utilization in Saccharomyces cerevisiae. The His478 to Leu substitution lay within the MH3 domain, corresponding to the negative regulatory domain of Mal63p which relieves the inhibitory effect on the activation function in response to maltose.

Two microbial oxidases of acidic d -amino acids have been purified to homogeneity. One is a d -aspartate oxidase of the yeast Cryptococcus humicolus UJ1 that was induced markedly with d -aspartate and is far more active toward d -aspartate than d -glutamate. The other is a d -glutamate oxidase of Candida boidinii 2201 that preferred d -glutamate to d -aspartate as a substrate in terms of k cat / K m , but was not induced very effectively by d -glutamate. The most potent competitive inhibitor of the C. humicolus d -aspartate oxidase was malonate, and that of the C. boidinii d -glutamate oxidase was d -malate. The former enzyme was a homotetramer of 160 kDa consisting of subunits of 40 kDa, each of which contained 1 mol of FAD, while the latter was a monomer of 45 kDa. The N-terminal sequences of both enzymes were similar to those of other FAD enzymes and contained a consensus sequence common to most enzymes binding ADP-containing nucleotides. Peroxisomal localization of the C. humicolus d -aspartate oxidase was shown by subcellular fractionation and morphological analysis via electron microscopy of C. humicolus cells, where induction of the enzyme was accompanied by induction of catalase and development of peroxisomes. The apo-form of C. humicolus d -aspartate oxidase, prepared by removal of FAD was a monomeric protein of 40 kDa, and its binding with FAD proceeded in two stages. The K d for the apoprotein-FAD complex was very low (8.2×10 −12 M) consistent with the observed tight binding. The C. humicolus d -aspartate oxidase was essentially similar to other flavoprotein oxidases of acidic and neutral d -amino acids with respect to its spectral properties and sensitivity to specific modifying reagents for arginyl and histidyl residues.

食品中のウコン色素, クチナシ黄色素及びアナトー色素 (水溶性アナトーを含む) の逆相TLC/スキャニングデンシトメトリーによる分析法を検討した. TLCプレートは逆相C18を用い, 展開溶媒としてアセトニトリル-テトラヒドロフラン-0.1mol/Lシュウ酸 (7 : 8 : 7) を使用することにより, ウコン色素, クチナシ黄色素及びアナトー色素を良好に分離することができた. スキャニングデンシトメータを用いて, TLC上のスポットの可視部吸収スペクトルを測定したところ, 良好なスペクトルが得られた. 本法を市販89食品中のウコン色素, クチナシ黄色素及び水溶性アナトーを含むアナトー色素の分析に応用し, その有用性を確認した.

Mycobacterium avium is a common opportunistic pathogen in immunocompromised patients such as those infected with human immunodeficiency virus. Although M. avium is an intracellular organism replicating predominantly in macrophages, disseminated M. avium infection is seen in AIDS patients with CD4 + cell counts of M. avium . To determine whether CD40-CD40 ligand (CD40L) interactions play a role in M. avium infection, we studied the ability of CD40L to restrict M. avium replication in human monocyte-derived macrophages (MDM) in vitro. MDM were infected with M. avium and cocultured with CD40L-transfected 293 cells for 7 days. Intracellular growth of M. avium in these MDM was assessed by colony counting. CD40L-expressing cells inhibited growth of M. avium in MDM by 86.5% ± 4.2% compared to MDM cultured with control cells. These findings were verified by assays using purified, soluble recombinant human CD40L (CD40LT). CD40LT (5 μg/ml) inhibited intracellular growth of M. avium by 76.9% ± 18.0% compared to cells treated with medium alone. Inhibition by CD40LT was reduced by monoclonal antibodies (MAbs) against CD40 and CD40L. The inhibitory effect of CD40LT was not accompanied by enhancement of interleukin-12 (IL-12) production by M. avium -infected MDM, while CD40L-expressing cells stimulated IL-12 production by these cells. Treatment of M. avium -infected mice with MAb against murine CD40L resulted in recovery of larger numbers of organisms (0.8 to 1.0 log) from the spleens, livers, and lungs of these animals compared to infected mice which received normal immunoglobulin G. These results indicate that CD40-CD40L signaling may be an important step in host immune response against M. avium infection.

It is known that rodlike crystals of aspartame, in which several needle crystals appear to be bundled together (bundlelike crystals), obtained from nonstirred crystallization have remarkably improved physicochemical properties compared with crystals (needlelike crystals) obtained from conventional stirred crystallization. For example, bundlelike crystals have much better thermal stabilities than the needlelike crystals. The difference of thermal stabilities between these crystals could not be explained only by their different crystal sizes, because it still remained even when these crystals were adjusted to have the same crystal size. Characterization of these crystals was carried out using powder X-ray diffraction. It is found that bundlelike crystals have different crystal texture compared with needlelike crystals. It is indicated that differences of crystal texture between these crystals are caused by crystallizing conditions, that is, stirred or nonstirred. It was concluded that differences of physico...

Purpose: We evaluated the availability of archival histopathological prepara-tions for genetic diagnosis of Leber hereditary optic neuropathy (LHON). Methods: Preparations of various tissues of an autopsied case of LHON, andof the optochiasmal arachnoidea of nine cases of bilateral optic neuropathy(BON) were studied to determine the presence of a point mutation of the mito-chondrial DNA nucleotide (nt) 11778 using PCR method. Results: An nt11778 point mutation was detected in all preparations of theautopsied case. Five preparations out of six BON cases who were diagnosed asLHON based on positive family history, revealed this point mutation. Thismutation was also detected in two of three BON patients with no family historyof the disease. Conclusion: The archival preparations were found to be available as materialsof genetic diagnosis for LHON, which indicated that it would be capable to re-evaluate retrospectively the pedigree of LHON and BON cases. Key words: Leber hereditary optic neuropathy – bilateral optic neuropathy – genetic diagnosis –mtDNA nt11778 point mutation – stained histopathological preparations.Acta Ophthalmol. Scand. 1998: 76: 14–19

Mycobacterium avium is an intracellular organism which multiplies predominantly within human macrophages. This organism has previously been shown to induce apoptosis in human macrophages. With a view to identifying M. avium components that induce cell death in infected host cells, sonicated extracts of M. avium as well as individual components isolated from the M. avium sonicate were tested in various assays with a human monocytic cell line (THP-1). THP-1 cells incubated with M. avium sonicate showed significantly reduced viability after a 2-day exposure compared to control cells incubated with media alone. This effect was dose dependent, with only 6.6% +/- 5.2% and 48.8% +/- 10.3% of the cells being viable by trypan blue exclusion at 600 and 300 microg/ml, respectively. Control cells, on the other hand, exhibited a viability of 98.8% +/- 1.0%. In addition, an 80% ammonium sulfate fraction of the M. avium sonicate and the previously characterized 68-kDa protein were found to have similar effects on THP-1 cells. In both cases, the reduction in viability was due to apoptosis characterized by chromatin condensation, DNA fragmentation by agarose gel electrophoresis, or terminal deoxynucleotidyl transferase-mediated d-UTP nick end labeling (TUNEL) and release of nuclear matrix protein (NMP) into the culture medium. M. avium sonicate-induced apoptosis of THP-1 cells was completely inhibited by the commonly used antioxidants pyrrolidinedithiocarbamate (PDTC) and butylated hydroxyanisole (BHA), indicating that the generation of free oxygen radicals may be responsible for inducing cell death. M. avium sonicate was found to induce apoptosis of monocyte-derived macrophages (MDMs) as well. This effect was not reversed in the presence of PDTC and was not accompanied with DNA fragmentation when determined by agarose gel electrophoresis, as seen in the case of THP-1 cells. However, these MDMs were found to contain fragmented DNA by TUNEL. These findings suggest that the mechanism of cell death in MDMs may be different from that observed with THP-1 cells. Furthermore, these results provide new insight into the effect of M. avium components on host cell responses during M. avium infection.

Helicobacter pylori lipid A, characterised by a glucosamine beta (1-6) disaccharide 1-(2-aminoethyl)phosphate acylated by (R)-3-hydroxyoctadecanoic acid and (R)-3-(octadecanoyloxy)octadecanoic acid at the 2- and 2'-positions, respectively, exhibited no or very low endotoxic activities, i.e. lethal toxicity in galactosamine-loaded mice, pyrogenicity for rabbits and the activity of the Limulus test compared with Escherichia coli-type synthetic lipid A (compound 506), which possesses beta-(1-6)-linked glucosamine disaccharide 1,4'-bisphosphate, with two acyloxyacyl groups at the 2'- and 3'-positions and two 3-hydroxytetradecanoyl groups at the 2- and 3-positions. The endotoxic properties of H. pylori lipid A were also a little weaker than those of the low endotoxic lipid A of P. gingivalis, which has 1-phospho beta-(1-6)-linked glucosamine disaccharide with 3-hydroxy-15-methylhexadecanoyl and 3-hexadecanoyloxy-15-methylhexadecanoyl groups at the 2- and 2'-positions, respectively. Further, the mitogenic activity of H. pylori lipid A in murine splenic mononuclear cells was also less than those of P. gingivalis lipid A and compound 506. However, H. pylori lipid A induced comparable production of interleukin-6 (IL-6) by human peripheral blood mononuclear cells (PBMC) compared with P. gingivalis lipid A and compound 506. H. pylori lipid A also increased human natural killer cell activity, and strongly agglutinated rabbit erythrocytes. However, the lipid As of H. pylori and P. gingivalis showed lower activities in inducing tumour necrosis factor alpha (TNF-alpha) production by human PBMC and IL-8 production by human gingival fibroblasts than that of compound 506. The structural feature of H. pylori lipid A may be associated with low endotoxic properties and potent immunobiological activities.

The chemical structure of a novel lipid A, which was obtained as a major component from lipopolysaccharide of Helicobacter pylori strain 206-1, was determined to be a glucosamine beta(1-6) disaccharide 1-(2-aminoethyl)phosphate acylated by (R)-3-hydroxyoctadecanoic acid and (R)-3-(octadecanoyloxy)octadecanoic acid at the 2- and 2'-position, respectively. The absence of a phosphoryl group at the 4'-position and fatty acyl groups at the 3- and 3'-position, and the stoichiometric presence of 2-aminoethyl phosphate at the 1-position are unique features, distinguishing it from the lipid A of enterobacteria.

Attachment to and uptake by host cells are important early events in the pathogenesis of intracellular organisms such as Mycobacterium avium. Monocyte-derived macrophages (MDM) are known to express multiple surface receptors that play a role in binding to and uptake of M. avium. These include complement receptor type 3 (CR3), fibronectin receptor, mannose receptor, and transferrin receptor. In addition to these, we have previously reported that the integrin receptor alpha(v)beta3 also plays a role in binding to M. avium in a nonopsonic environment. Further, we have shown that a 68-kDa surface protein of M. avium binds to human monocytes and plays a role in attachment of M. avium to MDM. The present study provides direct evidence that this protein mediates attachment of M. avium to MDM by binding to alpha(v)beta3. Using the technique of cell surface enzyme-linked immunosorbent assay, we have shown that the M. avium 68-kDa protein inhibits the binding of monoclonal antibodies (MAb) against alpha(v)beta3 to MDM compared to control proteins such as ovalbumin and laminin (P < 0.05). Dual-labeling studies were performed to demonstrate that after phagocytosis, alpha(v)beta3 is present along with M. avium in phagosomes of M. avium-infected MDM. In addition, we have demonstrated that this interaction between alpha(v)beta3 and the M. avium 68-kDa protein resulted in enhancement of CR3 expression, which is known to play a role in complement-mediated uptake of M. avium. Attachment of MDM to wells coated with the M. avium 68-kDa protein resulted in a twofold increase in CR3 expression compared to attachment of MDM to wells coated with ovalbumin. This enhancement was completely inhibited by pretreatment of MDM with MAb against alpha(v)beta3. In summary, M. avium binds to MDM via alpha(v)beta3 with the help of the M. avium 68-kDa protein, and this ligation enhanced the expression of CR3 on MDM. Since CR3 has been known to play a role in M. avium uptake, enhanced expression of this receptor mediated by M. avium-alpha(v)beta3 interaction indicates a complex mechanism of communication among different receptors that participate in M. avium attachment and uptake. These findings add to current understanding of the roles played by multiple receptor-ligand systems in uptake and pathogenesis of intracellular pathogens such as M. avium.

栄養学各論実習を履修した学生の献立作成における料理選択・料理構成の現状,および授業を受講した後の変化を検討し以下の結果を得た。1)主食・主菜・副菜に加え,汁物などの液体料理を加えた基本的献立構成能力は,殆どの学生が有しており,学習前・後で変化はなかった。2)献立を構成するための皿数は,実習前・後で差がなかったが,実習後では分散が小さくなり,朝食で4皿,夕食で4-5皿に集中した。3)朝食の献立における料理選択および料理構成はほぼ画一化され,使用される食材料・料理は限定されており,学習前・後で大きな変化はなかった。4)学習前は,夕食の主菜料理の50%がハンバーグと焼き魚で占められていたが,学習後はその割合が20%に低下し,主菜料理の料理種類および調理方法が広く分散した。5)朝食・夕食ともに生食する野菜料理の頻度が低下し,その分,煮物,和え物,温野菜サラダなどの加熱野菜料理の頻度が高くなった。6)学習後,献立作成の際の料理イメージがより明確にされたことによって,献立を構成する料理の食材料が明記される頻度が高くなった。今回は個人の献立作成能力の検討ではなく,集団としての学習による変化を検討したものであるが,今後は献立作成能力に関与する要因等を検討することで,個人の能力を向上させるための方法を探っていきたいと考えている。

Pulmonary administration of Toll-like receptor (TLR) ligands protects hosts from inhaled pathogens. However, systemic side effects induced by TLR stimulation limit clinical development. Here, a small-molecule TLR7 ligand conjugated with phospholipid, 1V270 (also designated TMX201), was tested for innate immune activation and its ability to prevent pulmonary infection in mice. We hypothesized that phospholipid conjugation would increase internalization by immune cells and localize the compound in the lungs, thus avoiding side effects due to systemic cytokine release. Pulmonary 1V270 administration increased innate cytokines and chemokines in bronchial alveolar lavage fluids, but neither caused systemic induction of cytokines nor B cell proliferation in distant lymphoid organs. 1V270 activated pulmonary CD11c+ dendritic cells, which migrated to local lymph nodes. However, there was minimal cell infiltration into the pulmonary parenchyma. Prophylactic administration of 1V270 significantly protected mice from lethal infection with Bacillus anthracis, Venezuelan equine encephalitis virus and H1N1 influenza virus. The maximum tolerated dose of 1V270 by pulmonary administration was 75 times the effective therapeutic dose. Therefore, pulmonary 1V270 treatment can protect the host from different infectious agents by stimulating local innate immune responses while exhibiting an excellent safety profile.

A cell-based high-throughput screen to identify small molecular weight stimulators of the innate immune system revealed substituted pyrimido[5,4-b]indoles as potent NFκB activators. The most potent hit compound selectively stimulated Toll-like receptor 4 (TLR4) in human and mouse cells. Synthetic modifications of the pyrimido[5,4-b]indole scaffold at the carboxamide, N-3, and N-5 positions revealed differential TLR4 dependent production of NFκB and type I interferon associated cytokines, IL-6 and interferon γ-induced protein 10 (IP-10) respectively. Specifically, a subset of compounds bearing phenyl and substituted phenyl carboxamides induced lower IL-6 release while maintaining higher IP-10 production, skewing toward the type I interferon pathway. Substitution at N-5 with short alkyl substituents reduced the cytotoxicity of the leading hit compound. Computational studies supported that active compounds appeared to bind primarily to MD-2 in the TLR4/MD-2 complex. These small molecules, which stimulate innate immune cells with minimal toxicity, could potentially be used as adjuvants or immune modulators.

The main reasons for loss of autotransplanted teeth are different from those involved in natural teeth loss. The aim of this study was to investigate which procedures were employed to treat spaces vacated when autotransplanted teeth were lost. Participating dentists were requested to provide information on transplantations they had undertaken. A total of 614 teeth in 552 patients (37 dentists) ranging in age from 17 to 79 years (mean age: 44.1 years) were examined. A total of 102 transplanted teeth were lost during the observation period. Procedures for treatment of spaces vacated were not influenced by main reason for transplanted tooth loss. The procedure used to treat depended on the original prosthodontic treatment of the transplanted teeth. For single crowns, the spaces were left empty (33.9%) or replaced by bridge work (30.5%), implants (20.3%), or dentures (10.2%). For single crowns in the upper and lower second molar regions, the spaces were usually left empty (upper 100%, lower 71.4%), while for those in the upper and lower first molar regions, the spaces were often replaced by bridge work (upper 41.7%, lower 50.0%). For bridge abutments, spaces were replaced by dentures (42.9%), implants (33.3%), or left empty (14.3%), and in the lower second molar region, they were mostly replaced by implants (5 cases, 41.7%). For most denture abutment cases, the spaces were replaced by dentures (88.9%). During the survival period of the transplanted teeth, the masticatory burden on the other teeth is reduced and the adjacent teeth are supported by the transplanted tooth. Even if transplanted teeth are eventually lost, traditional procedures can be performed to fill the vacated space.

Sparing sensitive healthy tissue from chemotherapy exposure is a critical challenge in the treatment of cancer. The work described here demonstrates the localized in vivo photoactivation of a new chemotherapy prodrug of doxorubicin (DOX). The DOX prodrug (DOX-PCB) was 200 times less toxic than DOX and was designed to release pure DOX when exposed to 365 nm light. This wavelength was chosen because it had good tissue penetration through a 1 cm diameter tumor, but had very low skin penetration, due to melanin absorption, preventing uncontrolled activation from outside sources. The light was delivered specifically to the tumor tissue using a specialized fiber-optic LED system. Pharmacokinetic studies showed that DOX-PCB had an α circulation half-life of 10 min which was comparable to that of DOX at 20 min. DOX-PCB demonstrated resistance to metabolic cleavage ensuring that exposure to 365 nm light was the main mode of in vivo activation. Tissue extractions from tumors exposed to 365 nm light in vivo showed the presence of DOX-PCB as well as activated DOX. The exposed tumors had six times more DOX concentration than nearby unexposed control tumors. This in vivo proof of concept demonstrates the first preferential activation of a photocleavable prodrug in deep tumor tissue.

High-speed counter-current chromatography (HSCCC) has been successfully applied to the separation of the ivermectin components. A 25-mg quantity of the sample was separated using a two-phase solvent system of n -hexane-ethyl acetate-methanol-water (19:1:10:10, v/v). The fractions were analyzed by high-performance liquid chromatography, nuclear magnetic resonance, and fast-atom bombardment mass spectrometry. The separation yielded 18.7 mg of 99.9% pure ivermectin B1a, 1.0 mg of 96.0% pure ivermectin B1b, and 0.3 mg of 98.0% pure avermectin Bla (precursor of ivermectin).

Leber's hereditary optic neuropathy (LHON) is a disorder considered to be due to a mutation in the mitochondrial DNA (mtDNA). The authors analyzed the mtDNA genotype of peripheral blood leukocytes from two family members with LHON, and further analyzed mtDNA in various tissues originating from the separate germ layers (skin, hair, leukocyte, muscle, arachnoid, CSF cells, and buccal mucosa) in two LHON patients. A G-to-A transition of mtDNA np 11778 (Wallace mutation) was detected by the elimination of a SfaN I site and the acquisition of a Maelll site in all individuals and tissues examined.