小林 優多郎

The pathogenesis of Alzheimer’s disease (AD) remains unclear, but an imbalance between the production and clearance of amyloid-β (Aβ) peptides is known to play a critical role in AD progression. A promising preventative approach is to enhance the normal Aβ clearance activity of brain phagocytes such as microglia. In mice, the intraperitoneal injection of Toll-like receptor 4 agonist was shown to enhance Aβ clearance and exhibit a preventative effect on AD-related pathology. Our previous clinical study demonstrated that orally administered Pantoea agglomerans-derived lipopolysaccharide (LPSp) exhibited an LDL (low-density lipoprotein)-lowering effect in human volunteers with hyperlipidemia, a known risk factor for AD. In vitro studies have shown that LPSp treatment increases Aβ phagocytosis by microglial cells however it is still unclear whether orally administered LPSp exhibits a preventive effect on AD progression. We show here that in senescence-accelerated prone 8 (SAMP8) mice fed a high-fat diet, oral administration of LPSp at 0.3 or 1 mg/kg body weightday for 18 weeks significantly improved glucose metabolism and lipid profiles. The LPSp treatment also reduced pro-inflammatory cytokine expression and oxidative-burst activity in the peripheral blood. Moreover, LPSp significantly reduced brain Aβ burden and memory impairment as seen in the water maze test, although we could not confirm a significant enhancement of Aβ phagocytosis in microglia isolated from the brains after treatment. Taken together, our results show that LPSp holds promise as a preventative therapy for AD or AD-related diseases induced by impairment of metabolic functions.

Pantoea agglomerans (P. agglomerans) is a Gram-negative bacterium that grows symbiotically with various edible plants, and the oral or sublingual administration of lipopolysaccharide derived from P. agglomerans (LPSp) have been suggested to contribute to prevention of immune-related diseases. Our previous study indicated that orally administered LPSp was shown to exhibit an LDL-lowering effect in hyperlipidemic volunteers however, a preventive effect of LPSp on atherosclerosis is unclear. The present study attempted to evaluate the anti-atherosclerotic effect by LPSp in a mouse model of high-fat diet (HFD)-induced atherosclerosis. For 16 weeks, apoE-deficient mice were fed an HFD and received drinking water containing LPSp (0.3 or 1 mg/kg body weight/day). The results showed that the orally administered LPSp decreased body weight. A significant reduction in atherosclerotic plaque deposition was observed even with the lower dose of LPSp. The biochemical analyses showed that LPSp markedly improved glucose tolerance and reduced plasma LDL and oxidized LDL levels. In addition, LPSp significantly reduced the production of pro-inflammatory mediators including MCP-1 (in the plasma), TNF-aα and IL-6 (in the colon), and decreased the oxidative burst activities in the peripheral blood sample. Taken together, these results suggest the possibility that oral administration of LPSp can effectively ameliorate HFDinduced hyperlipidemia and inflammatory/oxidative responses to prevent atherosclerosis and related metabolic.

Denaturation of actin and myosin in myofibrils induced by heating at 50°C was investigated to reveal the mechanism of irreversible liberation of actin from myofibrils on heating at lower temperatures than conventional cooking. Denaturation of these proteins was determined by Mg2+-ATPase (adenosine triphosphatase) and Ca2+-ATPase activities. When minced meat was heated for 20 min, actin was liberated accompanying denaturation of 80% of actin and 50% of myosin. Heating of the myofibrillar fraction (MFF) isolated from meat homogenate induced much slower denaturation of actin than myosin. When MFF was heated with sarcoplasmic fractions, denaturation of actin was facilitated, suggesting that sarcoplasmic fractions contain factors to facilitate actin denaturation. Inosine-5′-monophosphate, a component of sarcoplasmic fractions, was shown to have no effect on actin and myosin denaturation. These results suggest that heating meat at 50°C dissociates binding (‘Bond A’) between actin and myosin participating in ATPase activities, resulting in denaturation of both proteins under influence of sarcoplasmic components. Although denaturation of actin and myosin disrupted Bond A, actin was not liberated simultaneously, suggesting the presence of another bond (‘Bond B’, more heat-stable than Bond A) between both proteins and necessity of disruption of Bond B for actin release from myofibrils.

Background/Aim: Phagocytes recognize pathogens that enter the body as well as other abnormal and foreign materials that may exist within an organism (such as dead cells, oxidized lipids, and denatured proteins), and phagocytose and eliminate them to maintain a healthy state. In a previous study a simple prototype device was used, under development by Hamamatsu Photonics (Prototype), that detects fluorescence to determine the phagocytic activity of the murine macrophage cell line J774.1. The present study aimed to determine whether it was possible to detect phagocytic activity in a slight amount of human peripheral blood without using hemolysis. Materials and Methods: Three microliters of human peripheral blood was drawn from the fingertip and mixed with 30 mu g of pH-sensitive fluorescent particles. The fluorescence intensity of the human peripheral blood sample was then measured using the Prototype in development, cultured for 2 h at 37 degrees C, and then re-measured. The phagocytes were observed under fluorescence microscopy and the phagocytosis rate of CD11b-positive cells was verified with a flow cytometer. Result: The phagocytic activity of non-hemolyzed human peripheral blood was measured using the Prototype under development; fluorescence after phagocytosis was detected. Furthermore, this was confirmed by both fluorescence microscopy and flow cytometry. The precision of the measurements of human peripheral blood phagocytic activity was verified with the Prototype using samples from three healthy individuals. The relationship between blood sugar levels and phagocytic activity before and after meal times was determined. Concerning exercise, phagocytic activity tended to decrease, although salivary amylase level increased in the healthy individual examined after exercise. Conclusion: The simple Prototype can measure phagocytic activity in a small amount of peripheral blood without hemolysis. The device allows for rapid and minimally-invasive detection of changes in phagocytic activity, which has conventionally been difficult. These findings provide promising evidence that assessment of individual phagocytic capacity can be made easier using this novel device.

Background/Aim: Phagocytic activity is affected by a number of different stress and age-dependent factors. An easy measurement of phagocytic activity is thought to allow an indicator of an individual's health. In this study, we investigated conditions of measurement to easily evaluate the activity of phagocytosis of phagocytic cells (macrophages and neutrophils) using an easy-to-use prototype, which was improved from the device by Hamamatsu Photonics K.K., to detect neutrophil activity using subtle fluorescence. Materials and Methods: pH-sensitive fluorescent particles (pHrodo-Green E. coli Bio particles, GE particles) were added to mouse-derived macrophage cell lines (J774.1) and then incubated for 2 h at 37 degrees C. For negative control, the phagocytosis inhibitor cytochalasin D (CyD), was added prior to culture. Next, fluorescence intensity was measured by the Prototype to evaluate the phagocytic activity of macrophages and neutrophils. Phagocytosis was also confirmed by flow cytometry. Results: The Prototype detected a steady fluorescence increase in 5 sec in J774.1 after phagocytosis, using GE particles as a negative control in the presence of CyD. Furthermore, detection was possible at 10(4) cells/test, a concentration where the flow cytometer had difficulty for detection. Conclusion: The Prototype enables measurement of the phagocytic activity within a short period of time, even with a small sample amount, thus establishing the basic conditions of measurements of phagocytosis.

Background/Aim: Bacterial lipopolysaccharide (LPS) is involved in the activation of the innate immune responses on monocytes/macrophages in vitro, and by intravenous injection. Although small quantities of LPS are usually found in traditional Chinese medicines, vegetables and fruits, the mode of action of orally administered LPS is still unclear. Materials and Methods: LPS derived from Pantoea agglomerans (LPSp) was orally administered to C3H/HeN or C3H/HeJ mice ad libitum. Results: The LPSp treatment enhanced phagocytosis by resident peritoneal macrophages of C3H/HeN mice but not of C3H/HeJ mice. This activation can be defined as primed activation because no augmentation of inflammatory cytokines production was detected. LPSp in peritoneal fluid was detected and successfully quantified. Moreover, the LPSp reduced the expression of avian reticuloendotheliosis viral oncogene-related B (RelB) in the macrophages without degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cell inhibitor, alpha (I kappa B alpha). Conclusion: Orally administered LPSp can reach the peritoneum, and enhance phagocytosis via Toll-like receptor 4 signaling pathway in resident peritoneal macrophages.

The aim of this study was to establish, through a standard addition method, a convenient quantification assay for dipeptides (GY, YG, SY, YS, and IY) in soybean hydrolysate using 2,4,6-trinitrobenzene sulfonate (TNBS) derivatization-aided LC-TOF-MS. Soybean hydrolysate samples (25.0 mg mL(-1)) spiked with target standards were subjected to TNBS derivatization. Under the optimal LC-MS conditions, five target dipeptides derivatized with TNBS were successfully detected. Examination of the standard addition curves, with a correlation coefficient of r(2) >0.979, provided a reliable quantification of the target dipeptides, GY, YG, SY, YS, and IY, in soybean hydrolysate to be 424 +/- 20, 184 +/- 9, 2188 +/- 199, 327 +/- 16, and 2211 +/- 133 mu g g(-1) of hydrolysate, respectively. The proposed LC-MS assay is a reliable and convenient assay method, with no interference from matrix effects in hydrolysate, and with no requirement for the use of an isotope labeled internal standard. (C) 2015 Elsevier Ltd. All rights reserved.

The use of hesperidin in the pharmaceutical field is limited by its aqueous insolubility. The effects of natural compounds in tea on the solubility of hesperidin were evaluated and the underlying mechanism was investigated by nuclear-magnetic resonance (NMR) and quantum mechanical calculations. The solubility of hesperidin was measured by liquid chromatography time-of-flight mass spectrometry; the structure of the hesperidin/theasinensin A complex was characterized by H-1-NMR, diffusion-ordered NMR spectroscopy, and rotating frame NOE spectroscopy, as well as theoretically by quantum mechanical calculations. Among the natural compounds in tea, theasinensin A was the most effective in improving hesperidin solubility. The complexation of hesperidin with theasinensin A led to changes in the chemical shift of protons in hesperidin (Delta delta: 0.01-0.27 ppm) and diffusion coefficient (Delta D: 0.66-1.32 x 10(-10) m(2)/s) of hesperidin. ROE correlation signals between hesperidin and theasinensin A and quantum mechanical calculations revealed that two hesperidin molecules formed a stable complex with theasinensin A (2:1 complex) with a Delta G energy of -23.5 kJ/mol. This is the first study that provides insight into the enhanced solubility of hesperidin through interactions with theasinensin A via a 2:1 complex formation between hesperidin and theasinensin A.

Trp-His, the anti-atherosclerotic dipeptide, exerted an antiproliferative effect on vascular smooth muscle cells by L-type Ca2+ channel blocker-like effect. The beneficial potential by the blockade of Ca2+ channels on chronic intestinal inflammation, including inflammatory bowel disease (IBD), is unclear. Trp-His (100 or 250 mg/kg body weight/day) was administered for 14 days to BALB/c mice, and 5% dextran sodium sulfate (DSS) was administered to induce colitis in the last 7 days. Trp-His reduced DSS-induced typical colitis symptoms and cytokine expression in the colon. Trp-His inhibited interleukin (IL)-8 secretion in tumor necrosis factor (TNF)-alpha-stimulated HT-29 cells. The inhibitory effect of Trp-His, as well as that of Ca2+ channel blockers, was impaired by the presence of Ca2+ channel agonist Bay K 8644. The TNF-alpha-induced activation of mitogen-activated protein kinases (MAPKs) and I kappa B alpha were decreased by Trp-His. These results indicated that the anti-inflammatory effect of Tip-His may be involved in the blockade of L-type Ca2+ channels.

Ovotransferrin (OVT), one of the major hen egg white proteins, was shown to possess antimicrobial and antioxidant activities in vitro. However, there is no information regarding the in vivo preventative effect in chronic inflammatory diseases such as inflammatory bowel disease (IBD). The aim of the present study is to evaluate the anti-inflammatory effects of OVT in a mouse model of dextran sodium sulfate (DSS)-induced colitis. OVT (50 or 250 mg/kg BW) was given orally for 14 days to female BALB/c mice, and 5% DSS (MW 36-50 kDa) was used to induce acute colitis (days 7-14) via drinking water. The current in vivo study demonstrated that OVT significantly reduced clinical signs, weight loss, shortening of the colon, and inflammatory cytokine markers of disease. The histopathological analysis of the colon revealed that OVT reduced histological scores. These results indicate that the use of OVT may be a potential promising candidate for the prevention of IBD.

Background: Tryptophan-histidine (Trp-His) was found to suppress the activity of the Ca2+/calmodulin (CaM)-dependent protein kinases II (CaMKII), which requires the Ca2+-CaM complex for an initial activation. In this study, we attempted to clarify whether Trp-His inhibits Ca2+-CaM complex formation, a CaMKII activator. Methods: The ability of Trp-His and other peptides to inhibit Ca2+-CaM complex formation was investigated by a Ca2+-encapsulation fluorescence assay. The peptide-CaM interactions were illustrated by molecular dynamic simulation. Results: We showed that Trp-His inhibited Ca2+-CaM complex formation with a 1:1 binding stoichiometry of the peptide to CaM, considering that Trp-His reduced Hill coefficient of Ca2+-CaM binding from 2.81 to 1.92. His-Trp also showed inhibitory activity, whereas Trp + His, 3-methyl His-Tip, and Phe-His did not show significant inhibitory activity, suggesting that the inhibitory activity was due to a peptide skeleton (irrespective of the sequence), a basic amino acid, a His residue, the N hydrogen atom of its imidazole ring, and Trp residue. In silica studies suggested the possibility that Trp-His and His-Trp interacted with the Ca2+-binding site of CaM by forming hydrogen bonds with key Ca2+-binding residues of CaM, with a binding free energy of -49.1 and -68.0 kJ/mol, respectively. Conclusions: This is the first study demonstrating that the vasoactive dipeptide Trp-His possesses inhibitory activity against Ca2+-CaM complex formation, which may elucidate how Tip-His inhibited CaMKII in a previous study. General significance: The results provide a basic idea that could lead to the development of small peptides binding with high affinity to CaM and inhibiting Ca2+-CaM complex formation in the future. (C) 2014 Elsevier B.V. All rights reserved.

Adenine had a concentration-dependent relaxation action on the phenylephrine-contracted aorta ring, with an EC50 value of 0.40 +/- 0.12 mM. This effect was also observed in the endothelium-denuded aorta. Among the adenine analogues, N-methyladenine and benzimidazole still evoked an apparent relaxation effect, while 1-, 3- or 7-methyladenine and imidazole were no longer vasodilators. These findings demonstrate that the imino group from the uncharged imidazolium moiety in adenine played a key role in the relaxation of the contracted aorta.

Trp-His is the only vasoactive di-peptide known to regulate intracellular Ca2+ ([Ca2+](i)) and prevent the onset of atherosclerosis in mice. In this study, we showed that Trp-His reduced the [Ca2+] i elevation in phospholipase C-activated vascular smooth muscle cells (VSMCs), while a mixture of the corresponding constituent amino acids did not show significant reduction. Furthermore, Trp-His suppressed calmodulin-dependent kinase II (CaMK II) activity in angiotensin II-stimulated VSMCs, resulting in the inhibition of phosphorylation of voltage-dependent L-type Ca2+ channels (VDCC). Therefore, Trp-His potentially regulates the VDCC phosphorylation cascade through Ca2+-CaM/CaMK II. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Our previous findings regarding the biological activities of small peptides revealed that a di-peptide Trp-His (WH) could play a role in the prevention of vascular lesions including cell proliferation and atherosclerosis Its vasoprotective effects could be associated with suppression of the vasocontraction signaling cascade but the underlying mechanism(s) remains obscure In this study we attempted to elucidate the vasoprotective mechanism of WH in opposing the proliferation of rat vascular smooth muscle cells (VSMCs) In VSMCs from 8 week-old male Wistar rat thoracic aortae WH evoked a significant dose-dependent anti-proliferation effect without cytotoxicity In mitogen-stimulated cell experiments 300 mu M WH inhibited cytosolic Ca2+ elevation in VSMCs induced by 10 mu M angiotensin II (Ang II) Furthermore WH suppressed extracellular Ca2+ entry Into CaCl2-stimulated VSMCs The biological capacity of WH as an intracellular Ca2+ ([Ca2+](1)) suppressor was also proven when 50 mu M Bay K8644 was used to enhance Ca2+ entry via a voltage-dependent L-type Ca2+ channel (VDCC) and 300 mu M WH elicited a 23% reduction in [Ca2+](1) The absence of a reduction of the [Ca2+](1) by the mixture of tryptophan and histidine revealed the importance of the peptide backbone in the [Ca2+](1) reduction effect Furthermore the WH-induced [Ca2+](1) reduction was abolished by verapamil but not by nifedipine indicating that WH likely binds to an extracellular site of the VDCC at a site similar to that of the dihydropyridine type-Ca2+ channel blockers (C) 2010 Elsevier Inc All rights reserved