Hiroaki Yajima

Roles of Disulfide Linkage and Calcium Ion-Mediated Interactions in Assembly and Disassembly of Virus-Like Particles Composed of Simian Virus 40 VP1 Capsid Protein

ABSTRACT The simian virus 40 capsid is composed of 72 pentamers of VP1 protein. Although the capsid is known to dissociate to pentamers in vitro following simultaneous treatment with reducing and chelating agents, the functional roles of disulfide linkage and calcium ion-mediated interactions are not clear. To elucidate the roles of these interactions, we introduced amino acid substitutions in VP1 at cysteine residues and at residues involved in calcium binding. We expressed the mutant proteins in a baculovirus system and analyzed both their assembly into virus-like particles (VLPs) in insect cells and the disassembly of those VLPs in vitro. We found that disulfide linkages at both Cys-9 and Cys-104 conferred resistance to proteinase K digestion on VLPs, although neither linkage was essential for the formation of VLPs in insect cells. In particular, reduction of the disulfide linkage at Cys-9 was found to be critical for VLP dissociation to VP1 pentamers in the absence of calcium ions, indicating that disulfide linkage at Cys-9 prevents VLP dissociation, probably by increasing the stability of calcium ion binding. We found that amino acid substitutions at carboxy-terminal calcium ion binding sites (Glu-329, Glu-330, and Asp-345) resulted in the frequent formation of unusual tubular particles as well as VLPs in insect cells, indicating that these residues affect the accuracy of capsid assembly. In addition, unexpectedly, amino acid substitutions at any of the calcium ion binding sites tested, especially at Glu-157, resulted in increased stability of VLPs in the absence of calcium ions in vitro. These results suggest that appropriate affinities of calcium ion binding are responsible for both assembly and disassembly of the capsid.

Quercetin-3-O-glucuronide induces ABCA1 expression by LXRα activation in murine macrophages

Reverse cholesterol transport (RCT) removes excess cholesterol from macrophages to prevent atherosclerosis. ATP-binding cassette, subfamily A, member 1 (ABCA1) is a crucial cholesterol transporter involved in RCT to produce high density lipoprotein-cholesterol (HDLC), and is transcriptionally regulated by liver X receptor alpha (LXRα), a nuclear receptor. Quercetin is a widely distributed flavonoid in edible plants which prevented atherosclerosis in an animal model. We found that quercetin-3-O-glucuronide (Q3GA), a major quercetin metabolite after absorption from the digestive tract, enhanced ABCA1 expression, in vitro, via LXRα in macrophages. In addition, leaf extracts of a traditional Asian edible plant, Nelumbo nucifera (NNE), which contained abundant amounts of quercetin glycosides, significantly elevated plasma HDLC in mice. We are the first to present experimental evidence that Q3GA induced ABCA1 in macrophages, and to provide an alternative explanation to previous studies on arteriosclerosis prevention by quercetin.

TRANSFER OF SV40 TEMPERATURE-SENSITIVE EARLY GENE INTO HUMAN EPIDERMAL KERATINOCYTES BY THE RECOMBINANT ADENOVIRUS VECTOR

SummaryWe constructed a recombinant adenovirus vector that contained the origin-defective SV40 early gene, coding temperature-sensitive T antigen. This vector transferred the SV40 early gene into human epidermal keratinocytes with high efficiency. T antigen conferred the ability of keratinocytes to grow with limited differentiation in the presence of serum and high calcium concentration at the permissive temperature (34°C), although normal keratinocytes were induced to differentiate and stop growing under the same conditions. The serum/Ca++-resistant cells did not proliferate at the nonpermissive temperature (40°C), indicating that they depended on T antigen for their proliferation. The temperaturesensitive T antigen dissociated from the tumor suppressor gene products, p53, at 40°C. The serum/Ca++-resistant cells still had the ability to proceed to terminal differentiation when injected into SCID mice as cultured keratinocytes. However, they did not form an apparent basal layer. This indicated that the tissue remodeling process in the serum/Ca++-resistant keratinocytes was abnormal. All of these epidermoid cysts disappeared within 8 wk and no tumor developed for 6 mo. We consider that ΔE1/SVtsT is a useful tool to examine multistep carcinogenesis of human epithelial cells in vitro.

Roasted Barley Extract (Mugi-cha) Containing Cyclo(d-Phe-l-Pro) Prevents Lowering of the Cutaneous Blood Flow and Skin Temperature under Air Conditioning: A Randomized, Double-Blind, Placebo-Controlled, Crossover Study

Roasted barley extract (RBE), also known as mugi-cha, is a well-known healthy non-caffeinated beverage, and its health functionality has been widely reported. Our previous clinical study showed that RBE affects the cutaneous blood flow and skin temperature after cold-water immersion and that cyclo(d-Phe-l-Pro) is responsible for its effect. In this study, we investigated whether cyclo(d-Phe-l-Pro)-containing RBE prevents the decrease in the cutaneous blood flow and skin temperature. Subjects remained in the air-conditioned room while ingesting RBE or a placebo. We measured the cutaneous blood flow and skin temperature. We evaluated the effect of RBE administration by two-way repeated measures analysis of variance. A total of 15 subjects were enrolled. The change in cutaneous blood flow in the RBE and placebo groups was -0.79 ± 0.38 and -2.03 ± 0.35 mL min-1 100 g-1, respectively ( p value of 0.041). The change in the skin temperature in the RBE and placebo groups was -1.85 ± 0.35 and -3.02 ± 0.30 °C, respectively ( p value of <0.001). We also did subclass analysis with cold-feeling subjects. For the seven subjects who had cold sensation, the change in the cutaneous blood flow in the RBE and placebo groups was -0.48 ± 0.58 and -2.56 ± 0.48 mL min-1 100 g-1, respectively ( p value of 0.008). The change in the skin temperature in the RBE and placebo groups was -1.46 ± 0.74 and -2.89 ± 0.39 °C, respectively ( p value of 0.009). Thus, RBE containing cyclo(d-Phe-l-Pro) prevents the decrease in the cutaneous blood flow and skin temperature under air conditioning.