Beneficial Effect of Heat-Killed Lactic Acid Bacterium Lactobacillus johnsonii No. 1088 on Temporal Gastroesophageal Reflux-Related Symptoms in Healthy Volunteers: A Randomized, Placebo-Controlled, Double-Blind, Parallel-Group Study.

A randomized, placebo-controlled, double-blind, parallel-group clinical study was conducted to examine the effects of ingesting a heat-killed lactic acid bacterium, Lactobacillus johnsonii No. 1088 (LJ88) on temporal gastroesophageal reflux-related symptoms in healthy volunteers. A total of 120 healthy Japanese volunteers of both sexes, aged between 21 and 63 years, whose Frequency Scale for the Symptoms of Gastroesophageal Reflux Disease (FSSG) total score was 8 or greater, but who were not diagnosed with functional dyspepsia according to the Rome IV classification, were enrolled. They were randomly assigned to either the LJ88 or placebo group and instructed to ingest the test food (1 billion heat-killed LJ88 or placebo) once a day for six weeks. Gastroesophageal reflux-related symptoms were evaluated using FSSG scores as a primary endpoint. The Gastrointestinal Symptoms Rating Scale (GSRS), stomach state questionnaire, and serum gastrin concentration were used as secondary endpoints. In the FSSG evaluation, the heartburn score was significantly improved at 6 weeks in the LJ88 group compared to the placebo group. No severe adverse events related to the test food were observed. In conclusion, daily ingestion of heat-killed LJ88 improved temporal heartburn symptoms in non-diseased individuals.

Improvement of Aspects of Subjective Sleep Quality of Healthy Volunteers by Ingestion of Porcine Placental Extract: A Randomized Cross-Over Pilot Study.

Objectives: This study assessed the effects of oral porcine placental extract (PPE) on sleep quality of healthy volunteers not satisfied with their sleep. Design: This study used a randomized, placebo-controlled, double-blind, cross-over clinical pilot study. Setting: This study was conducted under an outpatient multicenter setting in Japan. Interventions: A total of 20 healthy Japanese volunteers aged between 28 and 73, whose Pittsburgh Sleep Quality Index global scores were between 6 and 10, successfully completed the study. At first, PPE at 300 mg/kg or placebo was ingested for 2 weeks. Then, after a 2-week washout period, each group ingested under a cross-over setting the opposite sample (placebo or PPE) for another 2 weeks. Main Outcome Measures: Objective measurement of the sleep made with an activity tracker and subjective measurements of sleep quality by use of St. Mary's Hospital Sleep Questionnaire were done just before and after the administration time slots. Results: No effect of PPE on the sleep length was observed. Several measures in the subjective St. Mary's Hospital Sleep Questionnaire, i.e., changes in Q5 (sleep depth) and Q9 (sleep wellness) between pre- and post-ingestions, were significantly different between groups in the direction of improvement of subjective sleep quality in the PPE group. Conclusions: Although oral PPE at 300 mg/day for 2 weeks did not affect the length of sleep itself, it significantly improved several measures of subjective sleep quality. These results suggest that PPE might be a way to improve sleep quality without hypnotic drugs. Clinical Trial Registration: www.umin.ac.jp/ctr/, identifier: UMIN000026468.

Synergistic effect of anti-Helicobacter pylori urease immunoglobulin Y from egg yolk of immunized hens and Lactobacillus johnsonii No.1088 to inhibit the growth of Helicobacter pylori in vitro and in vivo.

Helicobacter pylori is a pathogenic bacterium that infects the stomach, causing chronic gastritis; and it is also considered to be related to the occurrence of gastric cancers. Although some eradication regimens including multiple antibiotics have been developed, the emergence of resistance to antibiotics becomes problematic. Therefore, other approaches to compensate or augment the effects of standard regimens are needed. In this study, we examined the possible synergistic effects of anti-H. pylori urease IgY and Lactobacillus johnsonii No.1088 (LJ88) both in vitro and in vivo. Anti-H. pylori urease IgY was purified from egg yolks laid by the hens immunized with urease purified from H. pylori. LJ88 is a unique strain of lactic acid bacterium isolated from human gastric juice, and it has been reported to inhibit H. pylori both in vitro and in vivo. The in vitro mixed culture study showed that anti-H. pylori urease IgY augmented the anti-H. pylori activity of LJ88 against both clarithromycin-sensitive and -resistant H. pylori strains. In a germ-free mice infection model, combined administration of daily anti-H. pylori urease IgY and weekly living LJ88 significantly reduced H. pylori infections, whereas either monotherapy did not. In an in vivo human gut microbiota-associated mice model, not only daily administration of living LJ88 but also heat-killed one significantly reduced an H. pylori infection in the stomach when combined with anti-H. pylori urease IgY. The extent of reduction of the stomach H. pylori by such a combination therapy was larger than that reported for LJ88 monotherapy. These results taken together revealed a synergistic effect of anti-H. pylori urease IgY and living or heat-killed LJ88, thus suggesting that such a combination might be a promising therapy to possibly compensate and/or augment standard anti-H. pylori regimens.

A porcine placental extract prevents steatohepatitis by suppressing activation of macrophages and stellate cells in mice.

Nonalcoholic fatty liver disease (NAFLD) is caused by ectopic fat accumulation in the liver. NAFLD is associated with hepatic inflammation and oxidative stress, resulting in nonalcoholic steatohepatitis (NASH) with advanced fibrosis. Placental extracts have been used to treat various chronic diseases due to their antioxidative effect. However, the effects of the extracts on the development of NASH have yet to be elucidated. Here, we demonstrated that supplementation with an oral porcine placental extract (PPE) attenuated lipid accumulation and peroxidation, insulin resistance, inflammatory and stress signaling, and fibrogenesis in the liver of NASH model mice fed a high-cholesterol and high-fat diet. The PPE reduced the number of M1-like liver macrophages, but increased the number of anti-inflammatory M2-like macrophages, resulting in a predominance of M2 over M1 macrophage populations in the liver of NASH mice. Accordingly, the PPE suppressed lipopolysaccharide-induced M1 polarization in isolated murine peritoneal macrophages, whereas it facilitated interleukin 4-induced M2 polarization. Furthermore, the PPE reduced the hepatic stellate cell (HSC) activation associated with the attenuated transforming growth factor-ß/Smad3 signaling, both in the liver of NASH mice and in RI-T cells, a HSC line. The PPE may be a potential approach to prevent NASH by limiting lipid peroxidation, promoting M2 macrophage polarization, and attenuating HSC activation.

Anti-Helicobacter pylori activity of non-living, heat-killed form of lactobacilli including Lactobacillus johnsonii No.1088.

Some strains of lactic acid bacteria are reported to inhibit the growth of Helicobacter pylori and proposed to be useful to support so-called triple therapy for H. pylori. Although most strains must be alive to exert their anti-H. pylori activity, some lactobacilli strains are effective even when dead. One possible underlying mechanism of such an activity of non-living lactobacilli is reportedly co-aggregation with H. pylori. In this study, we found that a non-living heat-killed form of Lactobacillus johnsonii No.1088 (HK-LJ88) and also that of some other lactobacilli inhibited the growth of H. pylori in vitro. Furthermore, the number of H. pylori in the infected stomach of germ-free mice was significantly decreased by the repeated oral administration of HK-LJ88. Observation by scanning electron microscopy revealed that no co-aggregation had occurred between H. pylori and HK-LJ88; instead, deformations of H. pylori (e.g. disappearance of spiral, bending of cell body, coccoid formation, degradations, etc.) appeared after incubation for 24 h with HK-LJ88. These results suggest that HK-LJ88 inhibited H. pylori activity probably not by co-aggregation but by some unknown mechanism involving HK-LJ88's cell surface molecules and that even non-living lactobacilli are possibly useful to support H. pylori eradication therapy.

A highly acid-resistant novel strain of Lactobacillus johnsonii No. 1088 has antibacterial activity, including that against Helicobacter pylori, and inhibits gastrin-mediated acid production in mice.

A novel strain of Lactobacillus johnsonii No. 1088 was isolated from the gastric juice of a healthy Japanese male volunteer, and characterized for its effectiveness in the stomach environment. Lactobacillus johnsonii No. 1088 was found to have the strongest acid resistance among several lactobacilli examined (>10% of cells survived at pH 1.0 after 2 h), and such a high acid resistance property was a specific characteristic of this strain of L. johnsonii. When cultured with various virulent bacteria, L. johnsonii No. 1088 inhibited the growth of Helicobacter pylori, Escherichia coli O-157, Salmonella Typhimurium, and Clostridium difficile, in which case its effectiveness was more potent than that of a type strain of L. johnsonii, JCM2012. In addition to its effect in vitro, L. johnsonii No. 1088 inhibited the growth of H. pylori in human intestinal microbiota-associated mice in both its live and lyophilized forms. Moreover, L. johnsonii No. 1088 suppressed gastric acid secretion in mice via decreasing the number of gastrin-positive cells in the stomach. These results taken together suggest that L. johnsonii No. 1088 is a unique lactobacillus having properties beneficial for supporting H. pylori eradication by triple therapy including the use of a proton pump inhibitor (PPI) and also for prophylaxis of gastroesophageal reflux disease possibly caused after H. pylori eradication as a side effect of PPI.

Novel humanized anti-CD20 antibody BM-ca binds to a unique epitope and exerts stronger cellular activity than others.

Cellular activity of BM-ca, a novel humanized anti-CD20 antibody, was quantitatively compared with that of two other anti-CD20 antibodies used for clinical practice, rituximab and ofatumumab. The results of a complement-dependent cytotoxicity (CDC) assay revealed that the strongest antibody was ofatumumab, followed by BM-ca, with rituximab being the weakest. Ofatumumab and BM-ca were effective not only against rituximab-sensitive SU-DHL-4 cells but also against rituximab-resistant RC-K8 cells. In an antibody-dependent cell-mediated cytotoxicity (ADCC) assay, although the effective concentrations against SU-DHL-4 cells were almost the same among these three antibodies, the maximum cytotoxic level was the highest for BM-ca. In an anti-cell proliferation assay using SU-DHL-4 cells, BM-ca was the most effective and ofatumumab, the weakest. Against RC-K8 cells, only BM-ca was effective. When combined with each of four cancer chemotherapeutics (prednisolone, vincristine, hydroxydaunorubicin, and cisplatin), BM-ca exerted the most effective combinatorial anti-cell proliferation activity. To assess the in vivo effect of BM-ca, we intravenously administered BM-ca into cynomolgus monkeys and found that the peripheral B-cell levels did not decrease in half of the animals. Sequencing of cDNA encoding CD20 of cynomolgus monkeys revealed that the responders and nonresponders had Leu/Pro (hetero) and Leu/Leu (homo) at amino acid (a.a.) position 160, respectively, suggesting that the epitope recognized by BM-ca was around this a.a. By analyzing reactivity to synthetic peptides, the epitope recognized by BM-ca was estimated to be a.a.'s 156-166, not shared with rituximab and ofatumumab. These results suggest BM-ca to be a promising anti-CD20 antibody having superior properties and recognizing a unique epitope.

Proteomic biomarkers for acute interstitial lung disease in gefitinib-treated Japanese lung cancer patients.

Interstitial lung disease (ILD) events have been reported in Japanese non-small-cell lung cancer (NSCLC) patients receiving EGFR tyrosine kinase inhibitors. We investigated proteomic biomarkers for mechanistic insights and improved prediction of ILD. Blood plasma was collected from 43 gefitinib-treated NSCLC patients developing acute ILD (confirmed by blinded diagnostic review) and 123 randomly selected controls in a nested case-control study within a pharmacoepidemiological cohort study in Japan. We generated ∼7 million tandem mass spectrometry (MS/MS) measurements with extensive quality control and validation, producing one of the largest proteomic lung cancer datasets to date, incorporating rigorous study design, phenotype definition, and evaluation of sample processing. After alignment, scaling, and measurement batch adjustment, we identified 41 peptide peaks representing 29 proteins best predicting ILD. Multivariate peptide, protein, and pathway modeling achieved ILD prediction comparable to previously identified clinical variables; combining the two provided some improvement. The acute phase response pathway was strongly represented (17 of 29 proteins, p = 1.0×10(-25)), suggesting a key role with potential utility as a marker for increased risk of acute ILD events. Validation by Western blotting showed correlation for identified proteins, confirming that robust results can be generated from an MS/MS platform implementing strict quality control.

i-RUBY: a novel software for quantitative analysis of highly accurate shotgun-proteomics liquid chromatography/tandem mass spectrometry data obtained without stable-isotope labeling of proteins.

We developed a novel software named i-RUBY (identification-Related qUantification-Based strategY algorithm for liquid chromatography/tandem mass spectrometry (LC/MS/MS) data) that enables us to perform fully automatic ion current-based spectral feature analysis of highly accurate data obtained by LC/MS/MS. At the 1st step, this software utilizes accurate peptide/protein identification information for peak detection and peak matching among measurements. Then, at the 2nd step, it picks yet unidentified peaks and matches them to the peaks identified at the 1st step by a linear interpolation algorithm. The analysis of human plasma externally spiked with a known amount of yeast alcohol dehydrogenase 1 showed a good linear relationship between the amount of protein spiked and the quantitative values obtained by i-RUBY analysis. Experiment using human plasma digests spiked with a mixture of known amounts of synthetic peptides derived from two yeast proteins, alcohol dehydrogenase 1 and glucose-6-phospate isomerase, showed the expansion by the 2nd step of i-RUBY of the lower quantification limits to 1/10 to 1/1000 of those reached only by identified peaks at the 1st step. Good correlations between the i-RUBY results and the amount of proteins were confirmed by the analysis of real samples, i.e., sera of normal subjects and cancer patients, by comparing quantitative values of acute-phase proteins obtained by i-RUBY analysis of LC/MS/MS data with those obtained by an immunological method using Bio-Plex. These results taken together show that i-RUBY is a useful tool for obtaining dependable quantitative information from highly accurate shotgun-proteomics LC/MS/MS data.

Selected reaction monitoring by linear ion-trap mass spectrometry can effectively be applicable to simultaneous quantification of multiple peptides.

Selected reaction monitoring (SRM) mass spectrometry (MS) is becoming a popular approach for targeted quantitative proteomics. Triple-quadrupole mass spectrometers have been historically considered as the instrument of choice for this type of quantitative analysis. Recently, however, it has been reported that the SRM MS with a linear ion-trap (LIT) mass spectrometer is rather more appropriate for quantitative analysis of large peptides than the triple-quadrupole ones. In this study, we demonstrate that the SRM MS performed with a LIT mass spectrometer can simultaneously analyze multiple peptides and can quantify specific peptides in biological specimens without the use of stable isotope (SI)-labeled standard peptides. Firstly, a mixture of 10 synthesized peptides derived from yeast proteins and bovine serum albumin (BSA) was simultaneously analyzed by the LIT SRM. The ion peak areas of the 10 peptides were linearly correlated with the input amounts between 1 fmol and 10 pmol. Furthermore, the same peptide mixture spiked into human plasma was analyzed, and a linear response was found. Next, the amount of a BSA tryptic peptide was quantified by using an SI-labeled or a non SI-labeled peptide as an external reference standard. The difference in the quantified amounts of the BSA tryptic peptide was less than 10% between the 2 methods, suggesting that the "externally pulsed" non SI-labeled standard peptides derived from another species are useful. These results indicate that the SRM MS conducted with a LIT mass spectrometer is applicable to targeted quantitative proteomics of peptides at least up to 10 in number.

Unique profiles of changes in cell membrane fluidity during ethanol-induced yeast-to-pseudohyphal transition in Candida tropicalis.

A dimorphic transition from the yeast form to filamentous one in Candida tropicalis pK233 is triggered by the addition of ethanol into the glucose semi-defined liquid medium and the process of filamentation accompanies temporal depolarization of yeast cells. The transition is completely prevented by further supplementation of myo-inositol at the start of cultivation. The addition of ethanol caused an increase in membrane fluidity during the process of depolarization, and then fluidity was gradually lowered to the level equivalent with that of the stationary-phase yeast cells in accordance with filamentation. The increase in membrane fluidity of ethanol-induced cells appeared parallel with reduction in the content of membrane phosphatidylinositol, which was rich in saturated palmitic acid. Introduction of exogenous myo-inositol or 1 M sorbitol into the ethanol-supplemented culture at the start of cultivation restored yeast growth and the reduction of membrane fluidity occurred, coupled with the recovery of the phosphatidylinositol content.

Protein lysine methyltransferase G9a acts on non-histone targets.

By methylation of peptide arrays, we determined the specificity profile of the protein methyltransferase G9a. We show that it mostly recognizes an Arg-Lys sequence and that its activity is inhibited by methylation of the arginine residue. Using the specificity profile, we identified new non-histone protein targets of G9a, including CDYL1, WIZ, ACINUS and G9a (automethylation), as well as peptides derived from CSB. We demonstrate potential downstream signaling pathways for methylation of non-histone proteins.

Global analysis of gel mobility of proteins and its use in target identification.

SDS-PAGE is a basic method that has long been used for separation of proteins according to their molecular sizes. Despite its simplicity, it provides information on characteristics of proteins beyond their molecular masses because gel mobility of proteins often reflects their physicochemical properties and post-translational modifications. Here we report on a global analysis of gel mobility of the proteome, which we term the "mobilitome," covering 93.4% of the fission yeast proteome. To our surprise, more than 40% of proteins did not migrate to their calculated positions. Statistical analyses revealed that the discrepancy was largely dependent on the hydrophobicity of proteins. This experimental data set, with a high coverage rate of real mobility, made it feasible to identify proteins detected on the gel without using any specialized techniques. This approach enabled us to detect previously unknown post-translational modifications of a protein; for example, we revealed that eIF5A is novel substrate of a Sir2-related deacetylase Hst2. Furthermore, we concomitantly identified twelve acetylated and eight methylated proteins using specific anti-acetylated and anti-methylated lysine antibodies, most of which had not been known to be subject to the modifications. Thus, we propose the general usefulness of the mobilitome and electrophoresis-based methodology for the identification and characterization of proteins detected on the gel.

Genome-wide expression analysis of yeast response during exposure to 4 degrees C.

Adaptation to temperature fluctuation is essential for the survival of all living organisms. Although extensive research has been done on heat and cold shock responses, there have been no reports on global responses to cold shock below 10 degrees C or near-freezing. We examined the genome-wide expression in Saccharomyces cerevisiae, following exposure to 4 degrees C. Hierarchical cluster analysis showed that the gene expression profile following 4 degrees C exposure from 6 to 48 h was different from that at continuous 4 degrees C culture. Under 4 degrees C exposure, the genes involved in trehalose and glycogen synthesis were induced, suggesting that biosynthesis and accumulation of those reserve carbohydrates might be necessary for cold tolerance and energy preservation. The observed increased expression of phospholipids, mannoproteins, and cold shock proteins (e.g., TIP1) is consistent with membrane maintenance and increased permeability of the cell wall at 4 degrees C. The induction of heat shock proteins and glutathione at 4 degrees C may be required for revitalization of enzyme activity, and for detoxification of active oxygen species, respectively. The genes with these functions may provide the ability of cold tolerance and adaptation to yeast cells.

Proteomic analysis of organ-specific post-translational lysine-acetylation and -methylation in mice by use of anti-acetyllysine and -methyllysine mouse monoclonal antibodies.

Post-translational lysine-acetylation and -methylation are two major PTMs of lysine residues in proteins. Recently, we established pan-reactive anti-acetyllysine mouse mAbs, which can bind to Nepsilon-acetylated lysine residues in various contexts of amino acid sequences. In the present study, we established pan-reactive anti-methyllysine mouse mAbs comparable to the anti-acetyllysine ones. By using these anti-acetyllysine and -methyllysine antibodies, we found that the pattern of lysine-acetylated and -methylated proteins in mouse organs showed extreme variation from organ to organ. We selected brain and skeletal muscle as model cases to be further analyzed by 2-DE followed by Western blotting. In brain, alpha-tubulin at its basal level was found to be extremely acetylated; and alpha-enolase was shown to be a newly recognized possibly acetylated protein. NF-L protein, Hsc70, alpha-tubulin fragments, beta-actin, and brain-type creatine kinase were identified as putative lysine-methylated proteins in mouse brain. In skeletal muscle, lysine-methylation of alpha-actin and both lysine-acetylation and -methylation of muscle-type creatine kinase were found as novel putative lysine-modified proteins. The approach presented here might be useful to find novel disease markers and/or drug target molecules that would not be noticed by use of the traditional proteomic approach only.

HIV-1 Tat targets Tip60 to impair the apoptotic cell response to genotoxic stresses.

HIV-1 transactivator Tat uses cellular acetylation signalling by targeting several cellular histone acetyltransferases (HAT) to optimize its various functions. Although Tip60 was the first HAT identified to interact with Tat, the biological significance of this interaction has remained obscure. We had previously shown that Tat represses Tip60 HAT activity. Here, a new mechanism of Tip60 neutralization by Tat is described, where Tip60 is identified as a substrate for the newly reported p300/CBP-associated E4-type ubiquitin-ligase activity, and Tat uses this mechanism to induce the polyubiquitination and degradation of Tip60. Tip60 targeting by Tat results in a dramatic impairment of the Tip60-dependent apoptotic cell response to DNA damage. These data reveal yet unknown strategies developed by HIV-1 to increase cell resistance to genotoxic stresses and show a role of Tat as a modulator of cellular protein ubiquitination.

Analysis of eluted peptides from type 1 diabetes-susceptible HLA class II molecules identified novel islet protein, heparin/heparan sulfate-interacting protein.

Identification of peptides derived from pancreatic islet and presented by type 1 diabetes-susceptible MHC class II molecules has great significance to elucidate the pathogenesis of type 1 diabetes. A bulk culture of Epstein-Barr virus-transformed B-cells, which were established from a 22-year-old type 1 diabetic woman with HLA-DR4 and -DQw8, was pulsed with the homogenate of a human embryonic pancreas-derived cell line 1B2C6, and another culture was not pulsed with antigen. Peptide fractions were obtained by treatment of affinity-purified HLA-DR and -DQ molecules with 0.1% trifluoroacetic acid, and were subjected to reverse-phase high performance liquid chromatography (RP-HPLC). The RP-HPLC profiles of peptides derived from DR molecules revealed three peaks that specifically appeared after pulsing, but no such peaks were obtained from DQ molecules. From one of these three peaks, a peptide that consisted of 14 amino acids (AKSXNHTXXNQXRK, where X represents the undetermined amino acids) was identified. This peptide was derived from heparin/heparan sulfate-interacting protein (HIP). Immunostaining of pancreatic sections using antiserum for HIP peptide revealed exclusive staining of the islets. Thus, HIP was identified as an islet protein naturally processed and presented by HLA-DR4 molecules.

Chlamydocin-hydroxamic acid analogues as histone deacetylase inhibitors.

Chlamydocin-hydroxamic acid analogues were designed and synthesized as histone deacetylase (HDAC) inhibitors based on the structure and HDAC inhibitory activity of chlamydocin and trichostatin A. Chlamydocin is a cyclic tetrapeptide containing an epoxyketone moiety in the side chain that makes it an irreversible inhibitor of HDAC. We replaced the epoxyketone moiety of chlamydocin with hydroxamic acid to design potent and reversible inhibitors of HDAC. In addition, a number of amino-cycloalkanecarboxylic acids (Acc) are introduced instead of the simple amino-isobutric acid (Aib) for a variety of the series of chlamydocin analogues. The compounds synthesized were tested for HDAC inhibitory activity and the results showed that many of them are potent inhibitors of HDAC. The replacement of Aib residue of chlamydocin with an aromatic amino acid enhances the in vivo and in vitro inhibitory activity. We have carried out circular dichroism and molecular modeling studies on chlamydocin-hydroxamic acid analogue and compared it with the solution structure of chlamydocin.

Synthesis and histone deacetylase inhibitory activity of cyclic tetrapeptides containing a retrohydroxamate as zinc ligand.

Cyclic tetrapeptide retrohydroxamic acids were prepared as histone deacetylase (HDAC) inhibitors and evaluated the inhibitory activity and found that they have potential as anticancer drugs.

Screening of genes that respond to cryopreservation stress using yeast DNA microarray.

We studied the response of yeast cells after cryopreservation treatment using DNA microarray technology. Genes that contribute to "Cell rescue, defense and virulence," "energy," and "metabolism," were significantly induced. These genes were classified as encoding heat shock proteins, oxidative stress scavenger, and enzymes involved in glucose metabolism. The expression profile of mRNA after cryopreservation treatment was calculated to be closer to that following treatment with detergent or plant oils rather than by other stress factors such as heavy metals and agricultural chemicals. These results suggest that the cryopreservation treatment caused damage to the structure of the cell wall and cellular organelles. This was supported by the localization of the products of the induced genes at the cell wall and within cellular organelles.