Apple latent spherical virus (ALSV) vectors were evaluated for virus-induced gene silencing (VIGS) of endogenous genes among a broad range of plant species. ALSV vectors carrying partial sequences of a subunit of magnesium chelatase (SU) and phytoene desaturase (PDS) genes induced highly uniform knockout phenotypes typical of SU and PDS inhibition on model plants such as tobacco and Arabidopsis thaliana, and economically important crops such as tomato, legume, and cucurbit species. The silencing phenotypes persisted throughout plant growth in these plants. In addition, ALSV vectors could be successfully used to silence a meristem gene, proliferating cell nuclear antigen and disease resistant N gene in tobacco and RCY1 gene in A. thaliana. As ALSV infects most host plants symptomlessly and effectively induces stable VIGS for long periods, the ALSV vector is a valuable tool to determine the functions of interested genes among a broad range of plant species.

The effect of dietary vitamin K2 (menaquinone-7) on bone loss in ovariectomized (OVX) rats was investigated. OVX rats were freely given experimental diets containing menaquinone-4 (MK-4; 12mg/100g diet) or menaquinone-7 (MK-7; 18.1mg/100g diet) for 24 days; MK-4 and MK-7 were equal in molar concentrations. This feeding caused a remarkable increase of MK-4 and MK-7 concentrations in the serum and femur of OVX rats. OVX-induced decrease in the femoral dry weight and femoral calcium content was prevented by the feeding of dietary MK-4 or NK-7. In separate experiments, OVX rats were freely given experimental diets containing the fermented soybean (natto; including 9.4 microg MK-7/100g diet) without or with added MK-7 (37.6 microg/100g diet) for 77 days. Feeding produced a significant elevation of MK-4 and MK-7 concentrations in the serum of OVX rats. In this case, a significant increase in the femoral MK-4 content was observed but MK-7 was not detected in the femoral tissues. OVX-induced decreases in the femoral dry weight and femoral calcium content were significantly prevented by the feeding of diets containing natto with MK-7 added (37.6 microg/100g diets). This study demonstrates that the intake of dietary MK-7 has a preventive effect on bone loss caused by OVX. This effect may be partly caused by MK-4, which is formed by degradation of MK-7.

The effects of prolonged intake of juice prepared from Satsuma mandarin (Citrus unshiu MARC.) containing β-cryptoxanthin on circulating biochemical markers of bone metabolism in subjects, including menopausal woman, were investigated. Ninety volunteers, aged 27-65 years (19 men and 71 women), were enrolled in this study. The 71 females included 35 premenopausal women (ages, 27-50 years) and 36 postmenopausal women (ages, 46-65 years). Volunteers were divided into four groups; placebo juice without β-cyptoxanthin (5 men and 19 women), juice containing β-cyptoxanthin at 1.5 mg/200 ml of juice/day (4 men and 17 women), 3.0 mg/day (5 men and 17 women), and 6.0 mg/day (5 men and 18 women). Placebo or juice (200 ml) was ingested once a day for 28 or 56 days. Serum β-cryptoxanthin concentrations were significantly increased after the intake of juice containing β-cryptoxanthin (1.5, 3.0, or 6.0 mg/day) for 28 or 56 days, and the increases were dose-dependent. Bone-specific alkaline phosphatase and γ-carboxylated osteocalcin are serum bone markers of bone formation, and bone tartrate-resistant acid phosphatase (TRACP) and N-telopeptides of type I collagen are markers of bone resorption. Bone-specific alkaline phosphatase activity was significantly increased after the intake of juice containing β-cryptoxanthin (3.0 or 6.0 mg/ day) for 56 days as compared with the value obtained before intake. γ-Carboxylated osteocalcin concentration was significantly increased after the intake of juice containing β-cryptoxanthin (3.0 or 6.0 mg/day) for 28 or 56 days as compared with the value obtained before intake or after the intake of placebo juice. Serum TRACP activity and type I collagen N-telopeptide concentration were significantly decreased after the intake of juice containing β-cryptoxanthin (3.0 or 6.0 mg/day) for 28 or 56 days as compared with the value obtained before intake or after intake of placebo juice, and significant decreases were also seen after the intake of 1.5 mg/day β-cryptoxanthin as compared with the value obtained before intake. In menopausal women, bone-specific alkaline phosphatase activity and γ-carboxylated osteocalcin concentration were significantly increased after the intake of juice containing β-cryptoxanthin (3.0 or 6.0 mg/day) for 56 days as compared with the value obtained after placebo intake. Also, this intake caused a significant decrease in bone TRACP activity. Meanwhile, serum calcium, inorganic phosphorous, and parathyroid hormone (intact) were not changed after the intake of β-cryptoxanthin-containing juice for 28 or 56 days. This study demonstrates that the prolonged intake of juice fortified with β-cryptoxanthin has stimulatory effects on bone formation and inhibitory effects on bone resorption in humans, and that the intake has an effect in menopausal women.

The characterization of 66 kDa protein molecule, a major protein component which is produced from femoral-diaphyseal tissues with fracture healing (Igarashi and Yamaguchi [2002] Int. J. Mol. Med. 9:503-508), was investigated. Weaning rats were killed at 7 and 14 days after femoral fracture. When the femoral-diaphyseal tissues with fracture healing were cultured for 48 h in a serum-free medium, many proteins in the bone tissues were released into the medium. Analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that a protein molecule of approximately 66 kDa was markedly increased in culture medium from bone tissues with fracture healing. N-terminal sequencing of 66 kDa protein indicated that its N-terminus was identical to that of rat albumin. Western blot analysis of medium 66 kDa protein showed expression of albumin. This expression was significantly enhanced by fracture healing. The expression of albumin was seen in the diaphyseal (cortical bone) and metaphyseal (trabecular bone) tissues of rat femur. When the femoral-diaphyseal tissues obtained at 7 days after femoral fracture were cultured in a serum-free medium containing either vehicle, parathyroid hormone (1-34) (10(-7) M), insulin-like growth factor-I (10(-8) M) or zinc acexamate (10(-4) M), medium albumin was significantly increased in the presence of those bone-stimulating factors. The addition of albumin (0.5 or 1.0 mg/ml of medium) caused a significant increase in calcium and deoxyribonucleic acid contents in the femoral-diaphyseal and -metaphyseal tissues obtained from normal rats in vitro. The present study demonstrates that fracture healing induces a remarkable production of albumin which is a major protein component produced from femoral-diaphyseal tissues of rats, and that albumin has an anabolic effect on bone components.

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