Young Kil Choi

Non-association of estrogen receptor genotypes with bone mineral density and bone turnover in Korean pre-, peri-, and postmenopausal women

Abstract: Estrogen is known to play a critical role in both skeletal maturity and the rate of bone loss. This suggests the possibility that the estrogen receptor (ER) gene is one of the candidate genes that determines peak bone density and/or bone turnover rate. We investigated two established restriction fragment length polymorphisms (RFLPs) in intron 1 at the ER gene, represented as PvuII and XbaI. In 598 healthy Korean women aged 20–74 years, we examined the association of these ER genotypes with bone mineral density (BMD) and bone turnover status. The distribution of the PvuII and XbaI RFLPs was as follows: pp 205 (34.3%), Pp 308 (51.5%), PP 85 (14.2%) and xx 384 (64.2%), Xx 180 (30.1%), XX 34 (5.7%), respectively (where capital letters signify the absence of, and lower-case letters signify the presence of, the restriction site of each RFLP). No significant genotypic differences were found in BMD and bone markers. We grouped the subjects into three categories according to their menstrual status: 104 premenopausal women with regular menstruation, 182 perimenopausal women who had amenorrhea of not less than 3 months and not more than 12 months’ duration, and 312 postmenopausal women whose last menstruation was at least 12 months previously. No significant genotypic difference in either BMD or bone markers was found in any of these three groups. Furthermore we categorized women in peri- and postmenopause into a high loser group and a normal loser group according to the level of bone resorption markers. There was no difference in genotypic proportions between the high and normal loser groups. Our data suggest that these ER polymorphisms are not associated with BMD or bone turnover in Korean women.

Oncogenic Osteomalacia Caused by a Phosphaturic Mesenchymal Tumor of the Oral Cavity: A Case Report

We report a case of oncogenic osteomalacia associated with a phosphaturic mesenchymal tumor in a 31-year-old woman. She was presented with severe generalized bone and muscle pain and was restricted to bed. She lost 20cm in height over the 8 years since she had first noticed a pain in her thigh. A walnut-sized, hard, soft tissue tumor was found very easily beside her lower molar teeth. Radiologic examination revealed a remarkable decrease in bone density and multiple pathologic fractures of spine, femur and phalangeal bones. Severe hypophosphatemia, hyperphosphaturia, low plasma 1,25-dihydroxyvitamin D3 level and high plasma PTH level were disclosed at presentation. Histomorphometric examination revealed an extensive area of unmineralized osteoid and little mineralizing activity. A pharmacologic dose of 1α-hydroxyvitamin D3 or 1,25-dihydroxyvitamin D3 slightly increased the serum phosphate level and renal tubular reabsorption of phosphate, and slightly decreased plasma PTH level without any symptomatic improvement. Histologic examination of the tumor revealed a mixed connective tissue tumor that consisted of central woveh bones and surrounding primitive spindle cells with prominent vascularities. After removal of the tumor, all biochemical, hormonal and radiologic abnormalities disappeared with remarkable symptomatic improvement.

Effect of Cilostazol on the Neuropathies of Streptozotocin-Induced Diabetic Rats

Objectives This study examined the effect of cilostazol, a potent phosphodiesterase inhibitor, on the progression of neuropathies associated with streptozotocininduced diabetes mellitus in Sprague-Dawley rats. Methods Eight weeks after streptozotocin treatment, a pelleted diet containing 0.03% cilostazol (15mg/kg body weight) was given for four weeks. Body weight, blood glucose level, motor nerve conduction velocity(MNCV), myelinated fiber density and size distribution of sciatic nerves were compared between age-matched normal rats (Group 1), control diabetic rats (Group 2) and cilostazol-treated diabetic rats (Group 3). Results Body weight was significantly reduced and blood glucose level was significantly increased in diabetic rats (Group 2 and 3) compared to normal rats. MNCV and cAMP content of sciatic nerves were significantly reduced in diabetic rats 12 weeks after streptozotocin treatment. Myelinated fiber size and density were also significantly reduced, and thickening of the capillary walls and duplication of the basement membranes of the endoneural vessels were observed in the diabetic rats. Whereas both body weight and blood glucose level of Group 3 did not differ significantly from those of Group 2, cilostazol treatment significantly increased MNCV and cAMP content of sciatic nerves in Group 3 but not to the levels observed in Group 1. MNCV positively correlated with cAMP content of sciatic nerves (r=0.86; p < 0.001). Cilostazol treatment not only restored myelinated fiber density and size distribution but reversed some of the vascular abnormalities. Conclusion These findings suggest that a reduced cAMP content in motor nerves may be involved in the development of diabetic neuropathy, and that cilostazol may prevent the progression of diabetic neuropathy by restoring functional impairment and morphological changes of peripheral nerves.

Chrysanthemum zawadskii extract protects osteoblastic cells from highly reducing sugar-induced oxidative damage

In this study, Chrysanthemum zawadskii extract (CZE) was investigated to determine its effects on 2-deoxy-D-ribose (dRib)-induced oxidative damage and cellular dysfunction in the MC3T3-E1 mouse osteoblastic cell line. Osteoblastic cells were treated with the highly reducing sugar, dRib, in the presence or absence of CZE. Cell viability, apoptosis and reactive oxygen species (ROS) production were subsequently examined. It was observed that dRib reduced cell survival, while it markedly increased the intracellular levels of ROS and apoptosis. However, pre-treatment of the cells with CZE attenuated all the dRib-induced effects. The antioxidant, N-acetyl-L-cysteine (NAC), also prevented dRib-induced oxidative cell damage. In addition, treatment with CZE resulted in a significant increase in alkaline phosphatase (ALP) activity and collagen content, as well as in the expression of genes associated with osteoblast differentiation [ALP, collagen, osteopontin (OPN), osteoprotegerin (OPG), bone sialoprotein (BSP), osteocalcin (OC) and bone morphogenetic protein (BMP)2, BMP4 and BMP7]. In mechanistic studies of the antioxidative potential of CZE, we found that CZE reversed the dRib-induced decrease in the expression of phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT)1 and AKT2 genes, which are master regulators of survival-related signaling pathways. CZE also upregulated the gene expression of the antioxidant enzymes, superoxide dismutase (SOD)2, SOD3 and glutathione peroxidase 4 (GPx4), which was inhibited by dRib. Taken together, these results suggest that CZE attenuates dRib-induced cell damage in osteoblastic cells and may be useful for the treatment of diabetes-associated bone disease.

Responses of Osteoblastic Cell Line MC3T3-E1 Cell to the Calcium Channel Blocker Diltiazem and Verapamil

The effects of the calcium channel blockers, diltiazem and verapamil, on osteoblastic functions were investigated in cultured osteoblastic cells MC3T3-E1. DNA synthesis was evaluated by the incorporation of [3H]thymidine, and collagen synthesis by measuring the incorporation of [3H]proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP). Diltiazem inhibited the DNA synthesis of osteoblastic cells by up to 57.6 and 54.6% at concentrations of 25 and 50 microM. Verapamil also significantly inhibited DNA synthesis by up to 61.6 and 40.9% at concentrations of 25 and 50 microM. The percent control of CDP formation were decreased by up to 76.7% in 5 microM and 44.3% in 50 microM of diltiazem. Verapamil also decreased CDP synthesis to 49.7% at 10 microM and 32.6% at 50 microM. NCP synthesis was decreased by the calcium channel blocker but inhibition of the CDP formation was greater than that of NCP. The calculated percent collagen synthesis was decreased at a calcium channel blocker concentration of 10 microM. The inhibitory effects of diltiazem and verapamil on percent collagen synthesis were not reversed by increasing the calcium concentration of culture media by either 1 or 5 mM. From this study, we conclude that calcium channel blockers have a direct inhibitory effect on osteoblastic function. Long-term administration of diltiazem or verapamil produces adverse effects on normal bone metabolism.

Ipriflavone for the treatment of osteoporosis

Osteoporosis is a major public health problem occurring primarily among the postmenopausal population. Postmenopausal osteoporosis is characterized not only by a reduction in bone mass but also by bone microarchitecture alterations, which result in greater bone frailty and in an increased fracture risk [1]. However, osteoporosis is a preventable disease. Primary prevention of osteoporosis rests on regular exercising and adequate intake of dietary calcium [2,3]. For secondary prevention in women undergoing menopause, replacement estrogen therapy given for at least 10 years is associated with substantial reductions in fractures of the radius, hip and spine [4,5]. Many drugs have been studied to determine whether they prevent bone loss or reduce the incidence of additional fractures in patients with established osteoporosis. These drugs capable of arresting postmenopausal bone loss include parenteral, nasal or rectal calcitonin and bisphosphonates. Drug therapy for osteoporosis can be divided operationally into two main categories: those that inhibit bone resorption, and thus reduce bone tumover, and those that stimulate bone formation, exerting an anabolic effect. Therapeutic agents that inhibit bone remodelling would appear to be best suited to those patients with high tumover osteoporosis (about 30%). Included in this category are calcium, vitamin D and its metabolites, gonadal steroids, calcitonin, ipriflavone and bisphosphonates. Although estrogen replacement therapy has been proven to be effective in older women, calcitonin appears to be the treatment of choice for this population since it stabilizes or increases bone mass and also has reported analgesic properties [6]. Drugs that stimulate bone remodelling or bone formation would be best suited to patients with low tumover osteoporosis (about 70%) [7]. The agent in this class that is widely used is sodium fluoride. In this study, we evaluated the new molecule ipriflavone (7-isopropoxy-3-phenyl-4H-l-benzopyran-4-one) as another choice for osteoporosis prevention. Ipriflavone is an original molecule. Its introduction took a complicated

Allelic divergence in the human insulin gene provides evidence for intragenic recombination events in the non-coding regions: evidence for existence of new alleles

Intragenic polymorphism of the human insulin gene (INS) was investigated in Korean subjects. The 1.9 kb INS sequence, including the 5′ to 3′ flanking regions, was amplified using the polymerase chain reaction (PCR), and analyzed by direct sequencing. All nucleotide sequences in the coding regions were the same as INS sequences previously reported, and four nucleotides, at positions +216, +1045, +1367, and +1380 in the non-coding regions, were found to be polymorphic. In addition to the previously identified polymorphic alleles αl (A-C-C-C) and β1 (T-G-T-A), new nucleotide arrangements were also identified and designated α4 (A-C-C-A), α5 (A-G-C-C), α6 (A-C-T-C), and β2 (T-C-C-C). It was concluded that the new alleles may originate by intragenic recombination within INS during chromosomal crossing-over between the α1 and β1 alleles. The allele α1 was the predominant form in our sample; the new variant alleles, as well as allele β1, appeared to be much less frequent in INSs genes of the Korean subjects studied. Furthermore, the new alleles were detected only in heterozygous form. These results suggest that intragenic recombination can account for allelic divergence in INS.

Polymorphism of glucokinase gene in non-insulin dependent diabetes mellitus.

Several lines of evidence suggest a strong genetic component to NIDDM. To clarify the role of glucokinase gene in the development of NIDDM, restriction fragment length polymorphism (RFLP) of glucokinase gene and 3′ microsatellite polymorphism analyses by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) were performed in NIDDM and control subjects. Compared to NIDDM with 1.3 kb allele/Pvu I digestion of glucokinase, 10% of NIDDM did not demonstrate 1.3 kb allele and these patients were charcterized by increased insulin secretion. In 3′ microsatellite polymorphism analysis, autoradiography of PCR products revealed three different alleles, including Z, Z+2 and Z+4. Z was the most common allele in both NIDDM and nondiabetic controls. There was no significant allele associated with NIDDM. Frequency of the homozygote Z/Z genotype was significantly lower in NIDDM subjects (16.7%) compared to normal control (46.7%) (p<0.05). There was no difference in clinical findings according to 3′ microsatellite genotypes in NIDDM. These data suggest that there does not appear to be a significant glucokinase allele associated with NIDDM but Z/Z genotype may play a suppressive role in the pathogenesis of a certain type of NIDDM in Korea. Further studies may be required to identify the molecular basis of this association.

Possible Correlation Between Non-Insulin Dependant Diabetes Mellitus NIDDM and Restriction Fragment Length Polymorphism RFLP of the 5`-Flanking Region of Insulin Gene in Korean Diabetic Population

Restriction fragment length polymorphism (RFLP) is a new and precise method for the detection of the disease on the molecular level. The genetic background of Non-insulin dependant diabetes mellitus (NIDDM) has been strongly suggested, but its occurrences are obvious for other expession factors. We first investigated insulin gene polymorphism in twenty-nine Korea NIDDM patients and 11 non-diabetic controls. We found 91 percent and 86 percent of the genotype 1/1 in diabetics and controls respectively. Also genotype 1/3 was found 9 percent and 14 percent each in both groups. Genotype 3/3 revealed 0 percent in two groups. Four acromegalics who had secondary DM showed only genotype 1/1. These findings showed that class 3 allele(2.4 kb) was a weak genetic marker of NIDDM, and had a faint correlation.

Effect of β-endorphin and Cortisol on the PHA Stimulated Lymphoblastogenesis

The mechanism of immune suppression in a severely stressful condition is not known. Since the demonstration of β-endorphin receptor on the surface of the circulating lymphocyte, it was reported that β-endorphin could suppress PHA stimulated lymphoblastogenesis. Because the concentration of β-endorphin was supraphysiologically high, it is doubtful that β-endorphin can suppress the lymphoblastogenesis directly in vivo. We investigated the suppression of PHA stimulated lymphoblastogenesis by β-endorphin in vitro and the effect of β-endorphin in some conditions where β-endorphin increases in vivo. PHA induced lymphoblastogenesis of T lymphocyte was maximal at the concentration of 5 μg/ml. β-endorphin could not suppress the blastogenesis even at the highest concentration. In the five healthy men who received metyrapone the previous night, PHA stimulated blastogeneses were not significantly suppressed. In a patient with Nelson’s syndrome, the lymphoblastogenesis was suppressed at all concentrations of PHA. Cortisol significant suppressed the blastogenesis even at the concentration of 10 μg/dl and its suppressive effect was shown in dose dependant manner. Our results suggested that β-endorphin could not suppress the lymphoblastogenesis directly in vivo.The mechanism of immune suppression in a severely stressful condition is not known. Since the demonstration of β-endorphin receptor on the surface of the circulating lymphocyte, it was reported that β-endorphin could suppress PHA stimulated lymphoblastogenesis. Because the concentration of β-endorphin was supraphysiologically high, it is doubtful that β-endorphin can suppress the lymphoblastogenesis directly in vivo. We investigated the suppression of PHA stimulated lymphoblastogenesis by β-endorphin in vitro and the effect of β-endorphin in some conditions where β-endorphin increases in vivo. PHA induced lymphoblastogenesis of T lymphocyte was maximal at the concentration of 5 μg/ml. β-endorphin could not suppress the blastogenesis even at the highest concentration. In the five healthy men who received metyrapone the previous night, PHA stimulated blastogeneses were not significantly suppressed. In a patient with Nelson’s syndrome, the lymphoblastogenesis was suppressed at all concentrations of PHA. Cortisol significant suppressed the blastogenesis even at the concentration of 10 μg/dl and its suppressive effect was shown in dose dependant manner. Our results suggested that β-endorphin could not suppress the lymphoblastogenesis directly in vivo.