Zhong Jie Ma

Role of endogenous zinc in the enhancement of bone protein synthesis associated with bone growth of newborn rats

Abstract The role of endogenous zinc in protein synthesis in the bone tissues of newborn rats was investigated in the present study. Femoral–diaphyseal and metaphyseal tissues were obtained at 1, 7, 14, 21, and 28 days after birth. Many protein molecules were found to be present in the diaphyseal and metaphyseal tissues using sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis. Bone protein synthesis activity was enhanced by increasing age, and reached a plateau 21 days after birth. Protein synthesis in the diaphyseal and metaphyseal tissues obtained from 7- or 14-day-old rats was significantly decreased by the addition of dipicolinate (10−3 M), a chelator of zinc ion, into the reaction mixture, while it was significantly enhanced by zinc sulfate (10−4 M). When the diaphyseal and metaphyseal tissues obtained from 7- or 14-day-old rats were cultured for 48 h in a medium containing dipicolinate (10−3 M), bone protein synthesis was significantly reduced. This decrease was blocked completely by culture with the addition of zinc (10−4 M). Culture with zinc (10−5 and 10−4 M) alone had a stimulatory effect on the bone protein synthesis. Zinc (10−4 M)-induced increases in bone protein synthesis were completely blocked by culture with cycloheximide (10−6 M) or actinomycin D (10−7 M). The present study suggests that bone protein synthesis is enhanced with increasing age of newborn rats, and that endogenous zinc in bone tissues has a stimulatory role in the enhancement of protein synthesis with bone growth.

Inhibitory effect of menaquinone-7 (vitamin K2) on osteoclast-like cell formation and osteoclastic bone resorption in rat bone tissues in vitro.

The effect of menaquinone-7 (MK-7; vitamin K2) on oateoclast-like cell formation and osteoclastic bone resorption in rat femoral tissues in vitro was investigated. The bone marrow cells were cultured for 7 days in a α-minimal essential medium (α-MEM) containing a well-known bone resorbing agent [parathyroid hormone (1–34) (PTH) or prostaglandin E2 (PGE2)] with an effective concentration. Osteoclast-like cells were estimated by staining for tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The presence of PTH (10–8 M) or PGE2 (10–6 M) induced a remarkable increase in osteoclast-like multinucleated cells. These increases were significantly inhibited by MK-7 (10–8–10–5 M). MK-7 (10–7 and 10–6 M) significantly inhibited phorbol 12-myristate 13-acetate-induced osteoclast-like cell formation, whereas MK-7 did not inhibit dibutyryl cyclic adenosine monophosphate (DcAMP) (10–5 M)-induced osteoclast-like cell formation. These results suggest that the inhibitory action of MK-7 is partly involved in protein kinase C signaling. The bone cells isolated from rat femoral tissues were cultured for 48 h in an α-MEM containing either vehicle or MK-7 (10–8–10–5 M). The presence of MK-7 (10–6 and 10–5 M) caused a significant decrease in the number of mature osteoclasts. Such a decrease was also seen in the presence of calcitonin (10–10–10–8 M), DcAMP (10–6 and 10–5 M), or calcium chloride (10–3 and 10–3 M). The effect of MK-7 (10–6 M) in decreasing the number of osteoclasts was not further enhanced in the presence of calcitonin (10–8 M), DcAMP (10–5M), or calcium chloride (10–3 M), and was completely abolished by the presence of dibucaine (10–6 M) or staurosporine (10–7 M), which are inhibitors of Ca2+-dependent protein kinases. These results suggested that MK-7 has a suppressive effect on osteoclasts. Moreover, the femoral-metaphyseal tissues obtained from rats were cultured for 48 h in Dulbeccos modified Eagles medium containing either vehicle, PTH (10–7 M), or PGE2 (10–5 M) in the absence or presence of MK-7 (10–7–10–5 M). The presence of PTH or PGE2 induced a significant decrease in bone calcium content. These decreases were significantly blocked by MK-7 (10–7–10–5 M). This study demonstrates that MK-7 has an inhibitory effect on osteoclastic bone resorption in vitro.

Alternation in bone components with increasing age of newborn rats: role of zinc in bone growth

Abstract The effect of zinc on bone growth in newborn rats supplied with lactation by maternal rats was investigated. Newborn rats were killed between 1 and 35 days after birth. Increasing age caused a significant increase in zinc content, calcium content, and alkaline phosphatase activity in the femoral-diaphyseal and metaphyseal tissues, while the bone deoxyribonucleic acid (DNA) content was significantly decreased because of elevation of mineral content. Oral administration of zinc sulfate (2.0 mg/100 g body weight; four times at 24-h intervals) to maternal rats from 1 day after birth induced a significant increase in zinc, alkaline phosphatase activity, DNA, and calcium content in the femoral-diaphyseal and metaphyseal tissues of newborn rats compared with those 7 or 14 days old. The results indicate that the increase in bone components results from lactation with zinc-containing milk of maternal rats. The femoral-metaphyseal tissues of newborn rats obtained at 7 days after birth were cultured for 24 h in a medium containing either vehicle or zinc sulfate (10−6 to 10−4 M) in vitro. Bone alkaline phosphatase activity and calcium and DNA content were significantly increased by zinc addition. These increases were completely prevented by the presence of dipicolinate (10−3 M), a chelator of zinc ion, or cycloheximide (10−6 M), an inhibitor of protein synthesis. The present study suggests that zinc plays a role in the development of bone growth in newborn rats.

Synergistic effect of genistein and zinc on bone components in the femoral-metaphyseal tissues of female rats

Abstract: The effect of genistein and zinc on bone components in rats was investigated. Femoral-metaphyseal tissues obtained from female rats (4 weeks old) were cultured for 24 h in a medium containing either vehicle or genistein (10−7–10−5 M) in the absence or presence of zinc sulfate (10−6–10−4 M) in vitro. The presence of genistein (10−6 and 10−5 M) or zinc (10−6 and 10−5 M) caused a significant increase in alkaline phosphatase activity, deoxyribonucleic acid (DNA), and calcium content in the metaphyseal tissues. These increases were significantly enhanced by the combination of each compound. The synergistic effect on bone components was seen in the combination with genistein (10−6 and 10−5 M) plus zinc (10−5 M). Such an effect was completely blocked by the presence of cycloheximide (10−6 M), an inhibitor of protein synthesis. Moreover, the oral administration of genistein (100 and 300 μg/kg body weight) or zinc sulfate (1 and 5 mg Zn/kg) to rats for 3 days caused a significant elevation of alkaline phosphatase activity, DNA, and calcium content in the femoral-metaphyseal tissues. These increases were significantly enhanced by the combination of each compound. The synergistic effect was seen in the case of genistein (100 and 300 μg/kg) plus zinc (5 mg/kg). These results demonstrate that the anabolic effect of genistein on bone components is synergistically enhanced by zinc in vitro and in vivo. This study further supports the view that the combination of nutritional factors has a potent anabolic effect on bone metabolism.

Potential role of regucalcin as a specific biochemical marker of chronic liver injury with carbon tetrachloride administration in rats

The potential sensitivity of liver specific protein regucalcin as a biochemical marker of chronic liver injury with carbon tetrachloride (CCl4) administration in rats was investigated. CCl4 (10%; 1.0 ml/100 g body wt) was orally given 5 times at 3-day intervals to rats, and the animals were killed by bleeding at 3, 6, 18, and 30 days after the first administration of CCl4. The body weight of rats was significantly lowered 3 and 6 days after CCl4 administration as compared with that of control rats administered with corn oil, and then the weight was restored at 18 and 30 days. Serum glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) activities were significantly increased 3 days after the administration, while a significant increase in serum γ-glutamyltranspeptidase (γ-GTP) activity was seen at 3 and 6 days after the administration. Serum GOT, GPT, and γ-GTP activities were restored to control levels at 18 and 30 days after CCl4 administration. Serum albumin, α-fetoprotein, and ammonium levels were not changed by CCl4 administration. Meanwhile, serum regucalcin concentration was markedly increased 3 and 6 days after CCl4 administration, and a significant increase in serum regucalcin concentration was observed 18 and 30 days after the administration. Liver regucalcin mRNA and liver cytosolic regucalcin levels were significantly decreased 18 and 30 days after CCl4 administration. Liver content of calcium, which intracellular calcium homeostasis is maintained, was significantly increased between 3 and 30 days after CCl4 administration. Hepatic mitochondrial succinate dehydrogenase activity was significantly increased 30 days after the administration. The present study demonstrates that serum regucalcin has a potential sensitivity as a specific biochemical marker of chronic liver injury with CCl4 administration in rats.

Novel protein RGPR-p117: the gene expression in physiologic state and the binding activity to regucalcin gene promoter region in rat liver.

The binding activity of a novel regucalcin gene promoter region‐related protein (RGPR‐p117) to the TTGGC sequence of the rat regucalcin gene promoter region was investigated. The expression of RGPR‐p117 mRNA was seen in the liver tissues of male and female rats. The sexual difference of this expression was not found. Liver RGPR‐p117 mRNA expression was not changed with increasing age (1–50 weeks old), and its expression was not altered by fasting or refeeding. Nuclear factor I‐A1 (NF1‐A1) has been identified to be a transcription factor in stimulating the rat regucalcin gene promoter activity (Misawa and Yamaguchi [2002a] J Cell Biochem 84:795–802]. Recombinant nuclear factor I‐A1 (NF1‐A1) and RGPR‐p117 proteins were used gel mobility shift assay. RGPR‐p117 could not bind to TTGGC motif of the sequence between −525 and −504, which has been defined as a functional promoter element II‐b. NF1‐A1 was specifically bound to the II‐b oligonucleotide. Moreover, RGPR‐p117 was not bound to the II‐b oligonucleotide in the presence of NF1‐A1 or rat liver nuclear protein. The binding of NF1‐A1 to the II‐b oligonucleotide was not altered in the presence of RGPR‐p117. This study demonstrates that RGPR‐p117 mRNA, is expressed stably for physiologic change in rat liver, and that recombinant the protein does not directly bind to the TTGGC motif in rat regucalcin gene promoter. J. Cell. Biochem. 88: 1092–1100, 2003.

Anabolic effect of phosphogenistein and phosphodaidzein on bone components in rat femoral-metaphyseal tissues in vitro.

Abstract. The effect of phosphogenistein and phosphodaidzein, which are phosphorylated for the hydroxyl group (OH) at the 7-position of genistein and daidzein, on bone components was investigated. Femoral-metaphyseal tissues obtained from male rats (4 weeks old) were cultured for 24–72 h in Dulbeccos modified Eagles medium (high glucose, 4.5%) supplemented with antibiotics and bovine serum albumin. The presence of phosphogenistein (10−5 M) caused a significant increase in calcium content, alkaline phosphatase activity, and deoxyribonucleic acid (DNA) content in bone tissues cultured for 24 h. Phosphodaidzein (10−5 M) significantly elevated bone calcium and DNA content. These effects were completely prevented by the presence of cycloheximide (10−6 M), an inhibitor of protein synthesis. When femoral-metaphyseal tissues were cultured for 48 h in the presence of parathyroid hormone (1–34) (PTH; 10−8 M) or prostaglandin E2 (PGE2; 10−6 M), bone calcium content was significantly decreased. This decrease was significantly blocked by the presence of phosphogenistein (10−6 and 10−5 M) or phosphodaidzein (10−6 and 10−5 M). The presence of PTH (10−8 M) or PGE2 (10−6 M) caused a significant increase in glucose consumption and lactic acid production by bone tissues. These increases were significantly inhibited by the presence of phosphogenistein (10−5 M) or phosphodaidzein (10−5 M), indicating their inhibitory effect on bone resorption. The present study has demonstrated that both phosphogenistein and phosphodaidzein have an anabolic effect on bone metabolism in rat femoral-metaphyseal tissues in vitro.