Katsuko Kajiya

Effects of External Factors on the Interaction of Tea Catechins with Lipid Bilayers

Green tea contains a high concentration of such catechins as (−)-epicatechin (EC), (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECg), and (−)-epigallocatechin gallate (EGCg). Their biological activities have been evaluated by in vitro experiments using cultured cells or bacteria, but the order of activity of the various catechins differed with the study. We have been studying the interaction of tea catechins with lipid bilayers, and clarified that the number of hydroxyl groups on the B-ring, the presence of the galloyl moiety, and the stereochemical structure of each catechin govern their affinity for lipid bilayers. We investigated in this present study the effects of various external factors on the affinity of tea catechins for lipid bilayers by using liposomes as model membranes. The amount of tea catechins incorporated into the lipid bilayers increased with increasing salt concentration in an aqueous medium and decreased with increasing negative electric charge of the lipid bilayers. Furthermore, the amount of EGCg or ECg incorporated into the lipid bilayers increased with increasing EC concentration. These results reveal that the salt concentration in an aqueous medium, the electric charge of the membrane, and the presence of other catechins governed the affinity of tea catechins for the lipid bilayers.

Direct evidence of interaction of a green tea polyphenol, epigallocatechin gallate, with lipid bilayers by solid-state Nuclear Magnetic Resonance.

The interaction of a tea catechin, epigallocatechin gallate (EGCg), with the model membrane of dimyristoylphosphatidylcholine (DMPC) was studied by solid-state 31P and 2H NMR. The 31P chemical shift anisotropy of the DMPC phosphate group decreased on addition of EGCg. The 2H NMR spectrum of [4-2H]EGCg, which is deuterated at the 4-position, in the DMPC liposomes gave deuterium nuclei with much smaller quadrupole splittings than those in the solid phase. These 31P and 2H NMR observations provide direct experimental evidence that the EGCg molecule interacts with the lipid bilayers.

Involvement of Fyn tyrosine kinase in actin stress fiber formation in fibroblasts

Lysophosphatidic acid (LPA) and sphingosylphosphorylcholine (SPC) activated Fyn tyrosine kinase and induced stress fiber formation, which was blocked by pharmacological inhibition of Fyn, gene silencing of Fyn, or dominant negative Fyn. Overexpressed constitutively active Fyn localized at both ends of F‐actin bundles and triggered stress fiber formation, only the latter of which was abolished by Rho‐kinase (ROCK) inhibition. SPC, but not LPA, induced filopodia‐like protrusion formation, which was not mediated by Fyn and ROCK. Thus, Fyn appears to act downstream of LPA and SPC to specifically stimulate stress fiber formation mediated by ROCK in fibroblasts.

Chapter 4: Interaction of Plant Polyphenols with Liposomes

Abstract The biological activities of plant polyphenols have been examined by various methods in vitro and in vivo for prediction of their ability to prevent human diseases. Their activities found in in vitro experiments with cultured mammalian cells should reflect the amount incorporated into the cells during incubation. Since no transporter specific to plant polyphenols has been found in mammalian cells, it is supposed that most polyphenols are incorporated into the cells by passive transport and the amount incorporated is related to the affinity of the polyphenol for the cell membranes. In general, measurements of the amount of a polyphenol incorporated into the cell membranes or the cells are difficult and inaccurate, because the amount is very low and some polyphenols are unstable and metabolized immediately after incorporation. We developed a method to measure the amount of polyphenols incorporated into the lipid bilayers of liposomes with a dense internal aqueous phase. The higher relative density of the liposomes than that of water enabled to separate the medium and the liposomes by ultracentrifugation for a short time after incubation. Consequently, the amount of a polyphenol incorporated into the separated liposomes was selectively measured. With this method, the affinities of various kinds of plant polyphenols, including phenyl propanoids, gallic acid esters, curcumin, flavonoids and isoflavones have been investigated. In particular, interaction of tea catechins with lipid bilayers has been investigated in detail. Epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECg) and epigallocatechin gallate (EGCg) are the major components of polyphenols in green tea infusions. EGCg is the most abundant among these compounds. The reported biological activities of EC and EGC were often lower than those of their corresponding gallic acid esters, i.e., ECg and EGCg. We clarified that EC and EGC had lower affinities for the lipid bilayers of liposomes than ECg and EGCg. This indicates that the interaction of tea catechins with the lipid bilayer partly governs their activities. In addition to the presence of the gallic acid ester, the number of the hydroxyl groups on the B-ring and the steric character of the C-ring affected the affinity of the catechins for the lipid bilayers. Furthermore, the external factors such as salt concentration in an aqueous medium of the liposome suspension, the electric charges of the lipid bilayers and the presence of EC governed the affinity of EGCg for the lipid bilayers. The interaction of EGCg with a model membrane was also examined by solid state 31 P and 2 H NMR. These NMR observations provide direct experimental evidence that EGCg molecule interacts with the lipid bilayers. Using liposomes with a dense internal aqueous phase, we also clarified that ECg and EGCg were located on the surface of the lipid bilayers and perturbed the membrane structure.

Sphingosylphosphorylcholine induces stress fiber formation via activation of Fyn-RhoA-ROCK signaling pathway in fibroblasts.

Sphingosylphosphorylcholine (SPC), a bioactive sphingolipid, has recently been reported to modulate actin cytoskeleton rearrangement. We have previously demonstrated Fyn tyrosine kinase is involved in SPC-induced actin stress fiber formation in fibroblasts. However, Fyn-dependent signaling pathway remains to be elucidated. The present study demonstrates that RhoA-ROCK signaling downstream of Fyn controls stress fiber formation in SPC-treated fibroblasts. Here, we found that SPC-induced stress fiber formation was inhibited by C3 transferase, dominant negative RhoA or ROCK. SPC activated RhoA, which was blocked by pharmacological inhibition of Fyn activity or dominant negative Fyn. Constitutively active Fyn (ca-Fyn) stimulated stress fiber formation and localized with F-actin at the both ends of stress fibers, both of which were prevented by Fyn translocation inhibitor eicosapentaenoic acid (EPA). In contrast, inhibition of ROCK abolished only the formation of stress fibers, without affecting the localization of ca-Fyn. These results allow the identification of the molecular events downstream SPC in stress fiber formation for a better understanding of stress fiber formation involving Fyn.

Effects of the DASH-JUMP dietary intervention in Japanese participants with high-normal blood pressure and stage 1 hypertension: an open-label single-arm trial

The Dietary Approaches to Stop Hypertension (DASH) diet is recommended by the American Heart Association to lower blood pressure (BP); however, its effects in Japanese participants have not been rigorously studied. We assessed the effects of the DASH-Japan Ube Modified diet Program (DASH-JUMP), a modified DASH diet, on cardiometabolic and inflammatory biomarkers in Japanese participants with untreated high-normal BP or stage 1 hypertension. Fifty-eight participants (30 men and 28 women; mean age 54.1±8.1 years) with untreated high-normal BP or stage 1 hypertension followed the DASH-JUMP (salt 8.0 g per day) for 2 months. After the intervention period, they resumed their usual diets for 4 months. The DASH-JUMP significantly decreased the participants’ body mass index values (24.6±3.5 kg m−2 at baseline23.2±3.3 kg m−2 at 2 months, P=0.000), BP (153±14/91±11 mm Hg at baseline130±16/80±9 mm Hg at 2 months, P=0.000 and 139±16/85±10 mm Hg at 6 months, P=0.000), fasting serum glucose level (100±26 mg dl−194±15 mg dl−1 at 2 months, P=0.003) and fasting insulin level (6.9±5.9 μIU ml−14.4±2.7 μIU ml−1 at 2 months, P=0.000). The mean compliance of the participants for the DASH-JUMP diet was 88.5%. The DASH-JUMP diet reduced cardiovascular risk factors and may be an effective nutritional strategy for preventing cardiovascular events.