Toll-like Receptors as a Target of Food-derived Anti-inflammatory Compounds

Toll-like receptors (TLRs) play a key role in linking pathogen recognition with the induction of innate immunity. They have been implicated in the pathogenesis of chronic inflammatory diseases, representing potential targets for prevention/treatment. Vegetable-rich diets are associated with the reduced risk of several inflammatory disorders. In the present study, based on an extensive screening of vegetable extracts for TLR-inhibiting activity in HEK293 cells co-expressing TLR with the NF-κB reporter gene, we found cabbage and onion extracts to be the richest sources of a TLR signaling inhibitor. To identify the active substances, we performed activity-guiding separation of the principal inhibitors and identified 3-methylsulfinylpropyl isothiocyanate (iberin) from the cabbage and quercetin and quercetin 4′-O-β-glucoside from the onion, among which iberin showed the most potent inhibitory effect. It was revealed that iberin specifically acted on the dimerization step of TLRs in the TLR signaling pathway. To gain insight into the inhibitory mechanism of TLR dimerization, we developed a novel probe combining an isothiocyanate-reactive group and an alkyne functionality for click chemistry and detected the probe bound to the TLRs in living cells, suggesting that iberin disrupts dimerization of the TLRs via covalent binding. Furthermore, we designed a variety of iberin analogues and found that the inhibition potency was influenced by the oxidation state of the sulfur. Modeling studies of the iberin analogues showed that the oxidation state of sulfur might influence the global shape of the isothiocyanates. These findings establish the TLR dimerization step as a target of food-derived anti-inflammatory compounds.     

Studies on the bacterial spore coat 6 effects of alkali extraction on the spore of Bacillus thiaminolyticus.

Thin sections of the spore of Bacillus thiaminolyticus Matsukawa and Misawa show a characteristic surface structure with five ridges, and a series of three district layers. The outer layer of the spore coat was peeled off by SDS sonic treatment, and than the middle layer was solubilized by alkali extraction of the SDS sonic-treated spore. The spores subjected to these treatments were still refractile, heat resistant, and contained dipicolinic acid, but lost their resistance to mechanical shock.     

Fast backward steps and detachment of single kinesin molecules measured under a wide range of loads

To understand force generation under a wide range of loads, the stepping of single kinesin molecules was measured at loads from −20 to 42 pN by optical tweezers with high temporal resolution. The optical trap has been improved to halve positional noise and increase bandwidth by using 200-nm beads. The step size of the forward and backward steps was 8.2 nm even over a wide range of loads. Histograms of the dwell times of backward steps and detachment fit well to two independent exponential equations with fast (~0.4 ms) and slow (>3 ms) time constants, indicating the existence of a fast step in addition to the conventional slow step. The dwell times of the fast steps were almost independent of the load and ATP concentration, while those of the slow backward steps and detachment depended on those. We constructed the kinetic model to explain the fast and slow steps under a wide range of loads.     

Biodegradation of aniline, anthracene, chlornitrophen, fenitrothion and linear alkylbenzene sulphonate in pond water

Biodegradation of five chemicals (aniline, anthracene, chlornitrophen (CNP), fenitrothion (FNT) and linear alkylbenzene sulphonate (LAS)) by aquatic bacteria in three different types of ponds was determined according to the cultivation method developed by this group. The degradability toward these chemicals was varied among the ponds, except for LAS which was decomposed well in all samples. Higher degradability towards the two agrochemicals, CNT and FNT, was found in the pond surrounded by paddy fields, whereas aniline and anthracene were decomposed more rapidly in the pond located in the industrial area. Water from the pond in the botanical garden, with the least exposure to any chemicals, exhibited the lowest degradation toward all chemicals tested. There was no significant seasonal variation in the biodegradation of chemicals in these ponds. It was deduced that biodegradability toward certain chemicals could be a result of acclimatization of the microbial community by chemical contamination present and past, suggesting the possible use of biodegradation profiles as an indicator for chemical pollution in the aquatic environment.     

Amino acid sequences of the two kinds of regulatory light chains of adductor smooth muscle myosin from Patinopecten yessoensis

Smooth muscle myosin from scallop (Patinopecten yessoensis) adductor muscle contains two kinds of regulatory light chains (regulatory light chains a and b), and myosin having regulatory light chain a is suggested to be suitable for inducing "catch contraction" rather than myosin having regulatory light chain b (Kondo, S. & Morita, F. (1981) J. Biochem. 90, 673-681). The amino acid sequences of these two light chains were determined and compared. Regulatory light chain a consists of 161 amino acid residues, while regulatory light chain b consist of 156 amino acid residues. Amino acid substitutions and insertions were found only in the N-terminal regions of the sequences. The structural difference between the two light chains may contribute to the functional difference between myosins having regulatory light chains a and b.     

Augmentation of LDL receptor activities on lymphocytes by interleukin-2 and anti-CD3 antibody: a flow cytometric analysis

Dormant lymphocytes are known to show little LDL receptor (LDL-R) activities. The present study was designed to determine whether or not LDL-R activities of lymphocytes from normal subjects were high enough to be measured by flow cytometry after the cells had been stimulated with recombinant interleukin-2 (IL-2) and anti-CD3 monoclonal antibody (mAb). IL-2 or anti-CD3 mAb individually provokes proliferation of lymphocytes in a serum-free medium. Proliferation rate was accelerated when the two reagents were used in combination. Stimulated cells cultured for 5 days expressed more than 85% CD3 positive, less than 0.5% CD14 positive, and less than 1.5% CD20 positive. The LDL-R activities of the cells were examined by the uptake of a fluorescence probe, DiI-labeled LDL (DiI-LDL) and analyzed by flow cytometry. Stimulated cells showed increased uptake of DiI-LDL and 84 +/- 9% were positive, whereas only 3.0 +/- 2.5% of the cells without stimulation were positive (P less than 0.001). Under the same conditions stimulated lymphocytes from a homozygous familial hypercholesterolemia (FH) patient showed little LDL-R activities; 14% of the cells were positive. Displacement assays reveal that the uptake of LDL by these cells is occurring by way of its specific pathway. These data imply the lymphocytes stimulated with the reagents used in the study might be used for detecting defects in LDL-R, perhaps defects in other genomic systems as well.     

Zinc deficiency states and carbonic anhydrase isozyme in experimental hemolytic and bleeding anemia of rabbits

Zinc deficiency states were produced in rabbit erythrocytes by experimentally induced bleeding anemia and hemolytic anemia. Parallel decreases in total zinc levels and the contents for major zinc protein, carbonic anhydrase I and II isozymes were observed in the erythrocytes. During the process of the anemias the zinc status in the erythrocytes varied remarkably and the relative increase of zinc ions other than that derived from carbonic anhydrase was observed, suggesting that the former zinc ions play an important role in forming a zinc pool in the erythrocytes under the anemic conditions.     

Bcl-2 expression modulates cell adhesion and migration promoting branching of ureteric bud cells

Bcl-2 is the founding member of a family of proteins that influence apoptosis. During kidney development bcl-2 not only acts as a survival factor, but may also impact cell adhesive mechanisms and by extension branching morphogenesis. The interrelationship between cell adhesion, migration and apoptosis, important during development, is poorly understood. Here we examined the impact lack of bcl-2, an inhibitor of apoptosis, has on ureteric bud (UB) cell adhesion, migration, and branching morphogenesis. Bcl-2 -/- UB cells demonstrated increased cell migration, increased cell invasion and decreased adhesion to vitronectin and fibronectin compared with wild-type cells. Bcl-2 +/+ UB cells readily branched in collagen gel and Matrigel while bcl-2 -/- UB cells did not undergo significant branching in either matrix. Re-expression of bcl-2 in bcl-2 -/- UB cells restored their ability to undergo branching morphogenesis in Matrigel. Consistent with our in vitro data, we show that in the absence of bcl-2, embryonic kidneys undergo decreased UB branching. We observed decreased numbers of UB branch points, UB branch tips and a decreased distance to the first UB branch point in the absence of bcl-2. The alterations in bcl-2 -/- UB cell adhesion and migration was also associated with a significant alteration in expression of a number of extracellular matrix proteins. Bcl-2 -/- UB cells exhibited increased fibronectin expression and decreased thrombospondin-1 and osteopontin expression. Taken together, these data suggest that bcl-2 is required for the proper regulation of cell adhesive and migratory mechanisms, perhaps through modulation of the cellular microenvironment.     

Modulation of control of muscle sympathetic nerve activity during orthostatic stress in humans

We tested the hypothesis that orthostatic stress would modulate the arterial baroreflex (ABR)-mediated beat-by-beat control of muscle sympathetic nerve activity (MSNA) in humans. In 12 healthy subjects, ABR control of MSNA (burst incidence, burst strength, and total activity) was evaluated by analysis of the relation between beat-by-beat spontaneous variations in diastolic blood pressure (DAP) and MSNA during supine rest (CON) and at two levels of lower body negative pressure (LBNP: -15 and -35 mmHg). At -15 mmHg LBNP, the relation between burst incidence (bursts per 100 heartbeats) and DAP showed an upward shift from that observed during CON, but the further shift seen at -35 mmHg LBNP was only marginal. The relation between burst strength and DAP was shifted upward at -15 mmHg LBNP (vs. CON) and further shifted upward at -35 mmHg LBNP. At -15 mmHg LBNP, the relation between total activity and DAP was shifted upward from that obtained during CON and further shifted upward at -35 mmHg LBNP. These results suggest that ABR control of MSNA is modulated during orthostatic stress and that the modulation is different between a mild (nonhypotensive) and a moderate (hypotensive) level of orthostatic stress.