Prenylflavonoids isolated from Macaranga tanarius stimulate odontoblast differentiation of human dental pulp stem cells and tooth root formation via the mitogen-activated protein kinase and protein kinase B pathways.

AIM: To identify odontogenesis-promoting compounds and examine the molecular mechanism underlying enhanced odontoblast differentiation and tooth formation. METHODOLOGY: Five different nymphaeols, nymphaeol B (NB), isonymphaeol B (INB), nymphaeol A (NA), 3'-geranyl-naringenin (GN) and nymphaeol C (NC) were isolated from the fruit of Macaranga tanarius. The cytotoxic effect of nymphaeols on human DPSCs was observed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effect of nymphaeols on odontoblast differentiation was analysed with Alizarin Red S staining and odontoblast marker expression was assessed using real-time polymerase chain reaction and Western blot analysis. The molecular mechanism was investigated with Western blot analysis. In order to examine the effect of INB on dentine formation in the developing tooth germ, INB-soaked beads were placed under the tooth bud explants in the collagen gel; thereafter, the tooth bud explant-bead complexes were implanted into the sub-renal capsules for 3 weeks. Tooth root formation was analysed using micro-computed tomography and histological analysis. Data are presented as mean ± standard error (SEM) values of three independent experiments, and results are compared using a two-tailed Student's t-test. The data were considered to have statistical significance when the P-value was less than 0.05. RESULTS: Three of the compounds, NB, INB, and GN, did not exert a cytotoxic effect on human DPSCs. However, INB was most effective in promoting the deposition of calcium minerals in vitro (P < 0.001) and induced the expression of odontogenic marker genes (P < 0.05). Moreover, this compound strongly induced the phosphorylation of mitogen-activated protein (MAP) kinases and protein kinase B (AKT) (P < 0.05). The inhibition of p38 MAP, c-Jun N-terminal kinase (JNK), and AKT substantially suppressed the INB-induced odontoblast differentiation (P < 0.001). In addition, isonymphaeol B significantly induced the formation of dentine and elongation of the tooth root in vivo (P < 0.05). CONCLUSIONS: Prenylflavonoids, including INB, exerted stimulatory effects on odontoblast differentiation and tooth root and dentine formation via the MAP kinase and AKT signalling pathways. These results suggest that nymphaeols could stimulate the repair processes for dentine defects or injuries.

Gestational diabetes is associated with high energy and saturated fat intakes and with low plasma visfatin and adiponectin levels independent of prepregnancy BMI.

BACKGROUND/OBJECTIVES: Gestational diabetes mellitus (GDM) risk factors are well established for Caucasians, but not for Asians. We hypothesized that nutrient intakes, plasma adipokines and/or gestational hormones might be linked to GDM development among pregnant Korean women. This study sought to identify new risk factors for GDM and adverse pregnancy outcomes according to body weight at prepregnancy. SUBJECTS/METHODS: All subjects were pregnant women visiting the Cheil General Hospital and Women's Healthcare Center between June 2006 and March 2009. Non-GDM (n=531) and GDM (n=215) participants were divided into normal-weight and overweight groups according to prepregnancy body mass index (BMI) above or below 23 kg/m(2) at 24-28th week of gestation. At that time, glucose tolerance, insulin resistance as homeostatic model assessment for insulin resistance, insulin secretory capacity as homeostatic model assessment for ß-cell function, anthropometric measurement, nutrient intakes, and plasma levels of adipokines and gestational hormones were determined. RESULTS: GDM women gained more weight in early pregnancy than non-GDM among normal-weight women. GDM was mainly associated with increased insulin resistance in overweight women and decreased insulin secretory capacity in normal-weight women. Plasma visfatin and adiponectin were lower and progesterone levels higher in GDM than non-GDM independent of BMI while plasma resistin levels were higher in non-GDM, but not GDM, overweight women. Energy and saturated fat intakes were higher in GDM independent of body weight, whereas taurine intakes were lower in GDM than non-GDM only in normal-weight women. CONCLUSIONS: Low visfatin and adiponectin and high progesterone levels in the circulation and high energy and saturated fat intakes were common risk factors for GDM and pregnancy outcome such as large for gestational age. Daily reference intakes for energy and fat during pregnancy need to be re-evaluated according to prepregnancy BMI.

Efficacy and safety of glimepiride/metformin sustained release once daily vs. glimepiride/metformin twice daily in patients with type 2 diabetes.

AIMS: The study investigated the clinical equivalence in reducing haemoglobin A1c (A1C) between glimepiride/metformin sustained release (GM-SR) 2/500 mg, a fixed-dose combination, once daily and glimepiride/metformin (GM) 1/250 mg, a fixed-dose combination, twice daily in patients with type 2 diabetes (T2D). METHODS: A multicentre, randomised, double-blind, double-dummy study was conducted in 14 hospitals in Korea. Inclusion criteria were age 30-75 years, T2D diagnosis no longer than 10 years previously, A1C between 7% and 10%, and body mass index <40 kg/m(2) . A total of 207 subjects were randomised into the GM-SR group (n=101) or the GM group (n=106). Participants were assessed at baseline, 8 weeks and 16 weeks after treatment. RESULTS: After 16 weeks treatment, no difference in baseline-adjusted changes of A1C (primary efficacy variable) was observed between the two groups (-0.59% for GM-SR group vs. -0.61% for GM group, 95% CI: -0.17 to 0.21; p=0.84). In addition, there were no significant differences in secondary efficacy parameters between the two groups, including changes in A1C up to week 8, changes in fasting plasma glucose (FPG) and 2-h-postprandial plasma glucose up to week 8 and week 16, response rate, drug compliance and hypoglycaemic events. However, there was a difference in baseline-adjusted changes of FPG between the two groups (-1.01 mmol/l for GM-SR group vs. -1.52 mmol/l for GM group, p=0.01 in the intention to treat set). CONCLUSIONS: GM-SR 2/500 mg once daily was as effective as GM 1/250 mg twice daily in lowering A1C. In addition, no difference was noted in hypoglycaemic events between the two groups.

Vitamin D and diabetes in Koreans: analyses based on the Fourth Korea National Health and Nutrition Examination Survey (KNHANES), 2008-2009.

AIMS: A causal relationship between vitamin D deficiency and the incidence of diabetes mellitus has been suggested, but little research has been conducted on the Korean population. METHODS: We analysed the glucose tolerance status and serum 25-hydroxyvitamin D concentrations in 12263 subjects >19 years old who were registered for the Korea National Health and Nutrition Examination Survey, 2008-2009. RESULTS: Various demographic variables such as gender, age, season, resident area, physical activity, smoking, alcohol, marital status, education and occupation were associated with serum 25-hydroxyvitamin D concentrations. After adjusting for these variables as confounders, 25-hydroxyvitamin D concentrations in subjects with diabetes were significantly lower than those in subjects with normal glucose tolerance and those with impaired fasting glucose (P=0.005). Compared with the ≥ 75 nmol/l subgroup of serum 25-hydroxyvitamin D concentration, the odds ratios and 95% confidence intervals for diabetes mellitus were 1.206 (95%CI 0.948-1.534) in the 50- to 74-nmol/l subgroup, 1.339 (1.051-1.707) in the 25-to 49-nmol/l subgroup and 1.759 (1.267-2.443) in the <25-nmol/l subgroup. Compared with the serum ≥ 75-nmol/l 25-hydroxyvitamin D subgroup, serum insulin and homeostasis model assessment 2%B, a marker of insulin secretory capacity, were significantly higher, and homeostasis model assessment 2%S, a marker of insulin sensitivity, was significantly lower in the <25- and 25- to 49-nmol/l serum 25-hydroxyvitamin D subgroups than those in the other subgroups (P<0.001). CONCLUSIONS: The findings suggest that vitamin D deficiency, possibly involving altered insulin sensitivity, is associated with an increased risk for diabetes mellitus in the Korean population.

A novel PRKAR1A mutation in Korean Carney complex family.

Carney complex (CNC) is an autosomal dominant hereditary or sporadic multiple neoplastic syndrome that shows variable clinical symptoms. Generally, CNC appears as skin pigmentation, cardiac or cutaneous myxomas, and multiple endocrine tumours. We performed an extensive evaluation of 9 individuals within 1 family in whom CNC was suspected. Among them, 5 had CNC with various clinical manifestations. We also performed mutational analysis of suspected genes in these patients. Although all patients were members of the same family, variable CNC-related manifestations were observed in each patient. An analysis showed a novel deletion mutation (c.537delA) in exon 6 of the PRKAR1A gene in the patients. Based on our results, the patients were determined to have CNC type I. This is the first such mutational report in Korea.

Effects of insulin glulisine as mono- or add-on therapy in patients with type 2 diabetes mellitus.

AIM: To evaluate the safety and efficacy of insulin glulisine (glulisine) with and without oral antidiabetic drugs (OAD; sulphonylurea or sulphonylurea + biguanide) relative to that of OAD alone in Japanese and Korean patients with inadequately controlled type 2 diabetes mellitus (T2DM). METHODS: In an open, randomized, parallel-group, comparative, controlled trial, 387 patients were randomized and treated with glulisine + OAD (n = 130), glulisine monotherapy (n = 127) or OAD only (n = 130) for 16 weeks. Glulisine was self-injected subcutaneously three times daily (0-15 minutes before meals) at a starting dose of >or=0.2 U/kg/day. Patients titrated the glulisine dose to achieve a 2-h postprandial plasma glucose (2h-PPG) level of 7.1-9.5 mmol/l (128-172 mg/dl) by administering at least one additional unit at each appropriate meal time if the 2h-PPG level was > 9.5 and < 11.1 mmol/l (> 172 and < 200 mg/dl) and by administering at least two additional units if the 2h-PPG level was >or= 11.1 mmol/l (>or= 200 mg/dl). Therapy with OAD was continued at the stable baseline regimen. The primary efficacy endpoint was change in haemoglobin A(1c) (HbA(1c)) from baseline to endpoint in the intention-to-treat population. RESULTS: At baseline, therapy with OAD was a sulphonylurea only and a sulphonylurea + a biguanide in approximately 24 and 76% of patients respectively. Both glulisine groups had larger reductions in adjusted mean HbA(1c) than the OAD-only group (glulisine + OAD, -2.07%; glulisine monotherapy, -1.25%; OAD only, -0.61%). Superiority of glulisine + OAD and glulisine monotherapy vs. OAD only was shown by differences in adjusted mean HbA(1c) change from baseline values of -1.46% (p < 0.0001) and -0.64% (p < 0.0001) respectively. Both glulisine groups had better 2h-PPG control than the OAD-only group. Mean daily glulisine doses increased from baseline to endpoint (glulisine + OAD, 13.3-22.5 U; glulisine monotherapy, 14.2-38.0 U). The rate of all symptomatic hypoglycaemia events per patient-year in the entire treatment phase was 11.9 in the glulisine + OAD group, 8.8 in the glulisine monotherapy group and 1.7 in the OAD-only group. There was only one event of severe hypoglycaemia, which occurred in the glulisine + OAD group. Efficacy and safety were similar in Japanese and Korean subpopulations. CONCLUSIONS: Both glulisine + OAD and glulisine monotherapy were well tolerated and effective for Japanese and Korean patients with T2DM mellitus inadequately controlled by OAD therapy alone.

Cyclosporine A and FK506 induce osteoclast apoptosis in mouse bone marrow cell cultures.

Studies were carried out to characterize the effects of cyclosporines and FK506 on the formation and survival of osteoclasts deriving from mouse bone marrow cultures. Cyclosporin A (CsA), cyclosporin B (CsB), cyclosporin H (CsH), and FK506 all inhibited receptor activator of NFkappaB ligand (RANKL)-stimulated tartrate-resistant acid phosphatase (TRAP) activity and generation of TRAP+ multinucleated cells in the cultures. CsA and CsG were approximately equipotent, CsH was approximately one order of magnitude less potent than the other cyclosporines, and FK506 was approximately two orders of magnitude more potent than CsA and CsG. All of the inhibitors demonstrated greater potency and efficacy on decreasing the number of TRAP+ multinucleated cells than on decreasing total TRAP activity. Further evidence that late stages were more sensitive to inhibition was obtained in experiments in which CsA was present for different segments of the RANKL-stimulated culture period. CsA was as efficacious when added for the final 2 days of a 4-day culture as when added for the entire culture period, whereas it was less effective if added for only the first 2 days of the culture. When CsA or FK506 were added for 1 day to cultures in which osteoclasts had already formed, the numbers of TRAP+ osteoclasts decreased. Treatment with CsA or FK506 produced nuclear fragmentation and disruption of the multinucleated osteoclasts and an increase in caspase-3 activity. The apoptosis inhibitor z-VAD partially prevented the inhibitory effects of CsA and FK506 on the survival of TRAP+ multinucleated cells in the cultures and also preserved the normal osteoclast morphology. The data indicate that an important component of the inhibitory effects of CsA and FK506 on marrow-derived osteoclasts is the induction of apoptosis.

Destruxins, cyclodepsipeptides, block the formation of actin rings and prominent clear zones and ruffled borders in osteoclasts.

Bone-resorbing osteoclasts exhibit polarized morphological structures such as actin rings, clear zones, and ruffled borders. To gain insight into the mechanism of bone-resorbing activity of osteoclast and to discover new types of anti-resorptive agents, we have screened for natural compounds that inhibit the bone-resorbing activity of osteoclast-like multinucleated cells (OCLs). Destruxin B (DestB) and E (DestE), cyclodepsipeptides, were found to inhibit pit formation without affecting osteoclast differentiation and survival. Destruxins reversibly induced morphological changes in OCLs in a dose-dependent manner (DestB, 0.2-1 microM; DestE, 0.01-0.05 microM) and inhibited pit formation. Destruxin-induced morphological changes were accompanied by disruption of the actin rings in OCLs. The formation of actin rings in OCLs after adhesion was also inhibited by destruxins. Electron microscopical analysis revealed that destruxin-treated OCLs on dentine slices have no prominent clear zones and ruffled borders. The effective concentrations of destruxins on the morphological changes were almost the same as those that inhibited bone resorption in organ culture system. These results suggest that the anti-resorptive effects of destruxins result from induction of a disorder of the morphological structures in polarized OCLs.

The effects of retinoic acid on reversing the adipocyte differentiation into an osteoblastic tendency in ST2 cells, a murine bone marrow-derived stromal cell line.

Although the mouse bone marrow stromal cell line ST2 has been known to be differentiated into osteoblasts, the differentiation characteristics of the cell into adipocyte and the concerned relationship between its adipogenesis and osteogenesis remains unknown. The adipogenic induction medium which is made up of insulin, dexamethasone (DEX) and 3-isobutyl-1-methylxanthine(IBMX), stimulated the expression of n early adipogenic marker PPAR gamma and a late marker GPDH in ST2 cells. The triglyceride accumulation and lipid stain level generated by the induction medium in ST2 cells was inhibited by RA with IC(50) at about 1 nM. The induction medium up-regulated expression of PPARgamma and GPDH was also inhibited by RA whereas RA (30 nM) exterted no effect on the cell growth. Interestingly, treatment of the cells with induction medium in the presense of RA caused a 3- or 10-fold higher in ALP activity respectively as compared to those treated with RA or the induction medium alone. RT-PCR analysis showed that such a synergistic effect of RA and the induction medium paralleled the process of inhibition on adipogenesis. Additional experiments showed that IBMX played a key role in increasing the effect of RA and ALP activity. Our results suggested that the relationship between adipogenesis and osteogenesis in ST2 cells was reciprocally interrelated and the process of adipogenesis could be potentially reversed into an osteoblastogenic tendency. This is the first report demonstrating that RA transforms adipogenic potential into an osteoblastic tendency.

Intracellular calcium and protein kinase C mediate expression of receptor activator of nuclear factor-kappaB ligand and osteoprotegerin in osteoblasts.

Receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) produced by osteoblasts/stromal cells are involved as positive and negative regulators in osteoclast formation. Three independent signals have been proposed to induce RANKL expression in osteoblasts/stromal cells: vitamin D receptor-, cAMP-, and gp130-mediated signals. We previously reported that intracellular calcium-elevating compounds such as ionomycin, cyclopiazonic acid, and thapsigargin induced osteoclast formation in cocultures of mouse bone marrow cells and primary osteoblasts. Increases in calcium concentration in culture medium also induced osteoclast formation in cocultures. Treatment of primary osteoblasts with these compounds or with high calcium medium stimulated the expression of both RANKL and OPG messenger RNAs (mRNAs). 1,2-Bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)-tetra(acetoxymethyl)ester, an intracellular calcium chelator, suppressed both ionomycin-induced osteoclast formation in cocultures and expression of RANKL and OPG mRNAs in primary osteoblasts. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, also stimulated osteoclast formation in these cocultures and the expression of RANKL and OPG mRNAs in primary osteoblasts. Protein kinase C inhibitors such as calphostin and staurosporin suppressed ionomycin- and PMA-induced osteoclast formation in cocultures and expression of RANKL and OPG mRNAs in primary osteoblasts. Ionomycin stimulated RANKL mRNA expression in ST2 and MC3T3-G2/PA6 cells, but not in MC3T3-E1 or NIH-3T3 cells. These effects were closely correlated with osteoclast formation in response to ionomycin in cocultures with these stromal cell lines. OPG strongly inhibited osteoclast formation induced by calcium-elevating compounds and PMA in cocultures, suggesting that RANKL expression in osteoblasts is a rate-limiting step for osteoclast induction. Forskolin, an activator of cAMP signals, also stimulated osteoclast formation in cocultures. Forskolin enhanced RANKL mRNA expression but suppressed OPG mRNA expression in primary osteoblasts. These results suggest that the calcium/protein kinase C signal in osteoblasts/stromal cells is the fourth signal for inducing RANKL mRNA expression, which, in turn, stimulates osteoclast formation.

Compactin suppresses bone resorption by inhibiting the fusion of prefusion osteoclasts and disrupting the actin ring in osteoclasts.

Compactin (mevastatin), which inhibits 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, and thus biosynthesis of cholesterol and the prenylation of proteins, inhibits osteoclastic bone resorption. Although it has been suggested that compactin inhibits bone resorption by inducing apoptosis of osteoclasts, the pathway by which compactin inhibits resorption has not been established. We investigated the effect of compactin on the differentiation of osteoclasts and the relationship between the morphological changes elicited by compactin and its inhibitory effect on bone resorption. Compactin inhibited the differentiation of osteoclasts, interfering with the fusion process by which prefusion osteoclasts (pOCs) develop into multinucleated osteoclast-like cells (OCLs), and also disrupted the actin ring of OCLs. The potency of compactin to inhibit fusion of pOCs and to disrupt the actin ring of OCLs corresponded to that of compactin to inhibit bone resorption. The effects of compactin were prevented by the addition of MVA lactone or its downstream products farnesylpyrophosphate (FPP) and geranylgeranyl-pyrophosphate (GGPP) but not by squalene. Apoptosis of OCLs was not induced by the concentration of compactin that inhibited fusion of pOCs and disrupted the actin ring. The normal process of pOC fusion and the integrity of the actin ring were restored by the withdrawal of compactin from the cultures after they had been treated with compactin for 24 h, but they were not restored by the addition of zVAD-fmk, a caspase inhibitor. Compactin also reversibly inhibited interleukin-1beta (IL-1beta)-, 1alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3)-, and parathyroid hormone (PTH)-stimulated 45Ca release in bone organ cultures. Our results indicate that the inhibitory effects of compactin on bone resorption result from the inhibition of fusion of pOCs into OCLs and disruption of actin ring in OCLs and that apoptosis of OCLs is not necessary for these inhibitory effects of compactin. These effects of compactin are likely to be a consequence of the inhibition of prenylation of proteins that play an important role in the fusion of pOCs and in maintaining actin ring integrity in OCLs.

Receptor activator of NF-kappaB ligand induces the fusion of mononuclear preosteoclasts into multinucleated osteoclasts.

The osteoclasts are bone-resorbing multinucleatedcells formed by the fusion of mononuclearpreosteoclasts (pOCs) of hematopoietic origin.Although receptor activator of NF-kappaBligand (RANKL) has been shown to regulate osteoclastdifferentiation and function, its effect on the fusionof pOCs into multinucleated osteoclast-like cells(OCLs) has not been known. Using our fusion assaysystem, that is not contaminated with multinucleatedcells (MNCs) and osteoblastic cells, we determined theeffect of RANKL on the fusion of pOCs into MNCs. WhenpOCs were cultured on the plates, most of pOCs diedand disappeared from the plates within 24 h in theabsence of additives, but pOCs were fused to MNCswithin 6 h in the presence of RANKL. RANKL-inducedMNCs showed typical properties of OCL such astartrate-resistant acid phosphatase (TRAP) activity,actin ring formation, and bone-resorbing activity. Thefusion of pOCs into OCLs induced by osteoblastic cellsor RANKL was inhibited by OPG/OCIF, but that inducedby IL-1beta was not. Both RANKL- andIL-1beta-induced OCL formation from pOCs wasinhibited by ZLLL-H, a peptide inhibitor ofproteasome. These findings indicate that RANKLsupports the survival of pOCs and induces the fusionof pOCs into OCLs and suggest that NF-kappaBactivation is involved in these processes induced byRANKL and IL-1beta.

Osteoblastic cells induce fusion and activation of osteoclasts through a mechanism independent of macrophage-colony-stimulating factor production.

Fusion and activation of osteoclasts are the final two events in osteoclastic bone resorption. To investigate the regulatory mechanism of these events, mononuclear osteoclasts (preosteoclasts, pOCs) were isolated from co-cultures of mouse osteoblastic cells and bone marrow cells. Most of the pOCs cultured without any additives died within 12 h. Survival of pOCs was supported by addition of either osteoblastic cells or macrophage-colony-stimulating factor (M-CSF). pOCs began to fuse with each other after culture for 12 h in the presence of osteoblastic cells or M-CSF. However, the properties of multinucleated osteoclast-like cells (OCLs) induced by osteoblastic cells were considerably different from those induced by M-CSF. Fusion of pOCs induced by osteoblastic cells was retarded after culture for 24 h. In contrast, M-CSF-induced fusion of pOCs continued throughout the 48-h culture period, which was not inhibited by addition of calcitonin. When pOCs together with osteoblastic cells were cultured for 48 h on dentine slices, many resorption pits were formed on the slices. Calcitonin completely inhibited the fusion and pit-forming activity of pOCs treated with osteoblastic cells. Resorption pits were hardly detected on dentine slices in pOC cultures treated with M-CSF. Osteoblastic cells prepared from osteopetrotic (op/op) mice, which cannot produce functional M-CSF, stimulated the fusion and pit-forming activity of pOCs. Recombinant RANKL (receptor activator of NF-kappaB ligand), a cytokine which is produced by osteoblastic cells and is responsible for osteoclast differentiation, induced the fusion and pit-forming activity of pOCs. These results suggested that osteoblastic cells are involved in fusion and activation of osteoclasts through a mechanism independent of M-CSF production. RANKL appears to be responsible for fusion and activation of osteoclasts induced by osteoblastic cells.

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 streptozotocin-induced diabetes mellitus in Sprague-Dawley rats. METHODS: Eight weeks after streptozotocin treatment, a pelleted diet containing 0.03% cilostazol (15 mg/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.

Immunosuppressive effects of gallic acid and chebulagic acid on CTL-mediated cytotoxicity.

Gallic acid (GA) and chebulagic acid (CA) were isolated from the extract of a herbal medicine, kashi (myrobalans: the fruit of Terminalia chebula) as active principles that blocked the cytotoxic T lymphocyte (CTL)-mediated cytotoxicity. GA and CA inhibited the killing activity of CD8+ CTL clone at IC50 values of 30 microM and 50 microM, respectively. Granule exocytosis in response to anti-CD3 stimulation was also blocked by GA and CA at the equivalent concentrations.

Ca2+-ATPase inhibitors and Ca2+-ionophore induce osteoclast-like cell formation in the cocultures of mouse bone marrow cells and calvarial cells.

Osteoclasts which derive from hemopoietic cells are multinucleated cells responsible for bone resorption. We found that cyclopiazonic acid (CPA), thapsigargin (TG), and 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ) induced osteoclast-like cell (OCL) formation in cocultures of mouse calvaria-derived stromal cells and hemopoietic cells such as bone marrow cells and spleen cells. OCLs induced by these compounds showed typical characteristics of osteoclasts such as tartrate-resistant acid phosphatase activity and pit forming activity. These compounds are known as endoplasmic reticulum (ER)/sarcoplasmic reticulum (SR) Ca2+-ATPase inhibitors that increase intracellular Ca2+ levels by inhibiting Ca2+-ATPase activity located in the membrane of ER/SR. Ca2+-ionophores such as ionomycin which increase intracellular Ca2+ levels also stimulated OCL formation in the cocultures. Differentiation of hemopoietic cells into OCLs induced by these compounds required the presence of calvarial cells. These results indicate that an increase of intracellular Ca2+ levels may be a part of signaling pathways to induce osteoclast differentiation in the presence of calvarial cells.

Prodigiosin 25-C and metacycloprodigiosin suppress the bone resorption by osteoclasts.

Prodigiosin 25-C and metacycloprodigiosin were found to suppress PTH-stimulated pit formation by cultured osteoclasts on bone slices. They also inhibited the acidification of vacuolar organelles in intact osteoclastic cells. Since the acidic pH in these organelles is generated by the action of proton-pumping ATPases of the organelle, these results indicate that the proton-pumping activity of V-ATPase in osteoclastic cells is essential in bone resorption and that the inhibition of the acidification of vacuolar organelles by prodigiosins results in suppression of PTH-stimulated bone resorption.

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 20 cm 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 1 alpha-hydroxyvitamin D3 or 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 woven 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.

Immunomodulating activities of polysaccharide fractions from dried safflower petals.

In the course of screening for immunomodulators, we found a significant blastogenic activity specific for splenic B cells in the extracts of safflower (Carthamus tinctorius L.). Active fractions termed SF1 and SF2 were purified from dried petals of safflower by boiling water extraction, ethanol precipitation and Sepharose CL-2B column chromatography. The elution profiles of the gel filtration indicated that the molecular weight of SF1 and SF2 was estimated to be more than 100 kD. Major components of SF1 and SF2 seem to be polysaccharides, and structural analysis of alditol acetate derivatives by GC-MS revealed some differences between SF1 and SF2 in the sugar component. Biological activities of SF1 and SF2 on B cells and macrophages were examined in comparison with lipopolysaccharides (LPS). SF1 and SF2 induced both the proliferation and the IgM production of B cells to the equivalent level as those induced by LPS. In macrophages, SF1 and SF2 effectively stimulated the production of NO. However, SF1 stimulated the production of IL-1, IL-6, and TNF as much as LPS, while SF2 induced them only weakly or not at all. Thus, these results suggest that SF1 and SF2 activate B cells and macrophages in different mechanisms.