Efficacy and safety of once-yearly zoledronic acid in Japanese patients with primary osteoporosis: two-year results from a randomized placebo-controlled double-blind study (ZOledroNate treatment in Efficacy to osteoporosis; ZONE study).

In a 2-year randomized, placebo-controlled study of 665 Japanese patients with primary osteoporosis, once-yearly administration of zoledronic acid (5 mg) reduced the risk of new morphometric vertebral fractures. INTRODUCTION: The purpose of this study was to determine the efficacy and safety of once-yearly intravenous infusion of ZOL in Japanese patients with primary osteoporosis. METHODS: This was a two-year multicenter, randomized, placebo-controlled, double-blind, parallel-group comparative study (ZONE Study). Subjects were 665 Japanese patients between the ages of 65 and 89 years who had prevalent vertebral fracture. Subjects were randomly assigned to receive once-yearly intravenous infusion of 5 mg of ZOL or placebo at baseline and 12 months. RESULTS: The 2-year incidence of new morphometric vertebral fracture was 3.0 % (10/330 subjects) in the ZOL group and 8.9 % (29/327) in the placebo group (p = 0.0016). The 24-month cumulative incidence of new morphometric vertebral fracture was 3.3 % in the ZOL group versus 9.7 % in the placebo group (log-rank test: p = 0.0029; hazard ratio: 0.35; 95 % confidence interval: 0.17-0.72). The cumulative incidence of any clinical fracture, clinical vertebral fracture, and non-vertebral fracture was significantly reduced in the ZOL group by 54, 70, and 45 %, respectively, compared to the placebo group. At 24 months, ZOL administration increased bone mineral density in the lumbar spine, femoral neck, and total hip (t test: p < 0.0001). No new adverse events or osteonecrosis of the jaw were observed in this study. CONCLUSIONS: Once-yearly administration of ZOL 5 mg to Japanese patients with primary osteoporosis reduced the risk of new morphometric vertebral fractures and was found to be safe.

Three-year denosumab treatment in postmenopausal Japanese women and men with osteoporosis: results from a 1-year open-label extension of the Denosumab Fracture Intervention Randomized Placebo Controlled Trial (DIRECT).

SUMMARY: A 12-month extension phase of DIRECT in Japanese subjects with osteoporosis showed that total 3 years of denosumab treatment in Japanese postmenopausal women and men with osteoporosis was associated with low fracture rates, persistent bone turnover marker (BTM) reductions, continuous bone mineral density (BMD) increases, and a favorable overall benefit/risk profile. INTRODUCTION: The DIRECT trial demonstrated that 2 years of treatment with denosumab 60 mg subcutaneously every 6 months significantly reduced the incidence of vertebral fracture compared to placebo in Japanese postmenopausal women and men with osteoporosis. The purpose of this study is to evaluate the efficacy and safety of denosumab treatment for up to 3 years. METHODS: This study includes a 2-year randomized, double-blind, placebo-controlled phase and a 1-year open-label extension phase in which all subjects received denosumab. The data correspond to 3 years of denosumab treatment in subjects who received denosumab (long-term group) and 1 year of denosumab treatment in subjects who received placebo (cross-over group) in the double-blind phase. RESULTS: Eight hundred and ten subjects who completed the double-blind phase enrolled into the extension phase, and 775 subjects completed the study. All subjects received denosumab with daily supplements of calcium and vitamin D. The cumulative 36-month incidences of new or worsening vertebral fractures and new vertebral fractures were 3.8 and 2.5 %, respectively, in the long-term group. In this group, the BMD continued to increase, and the reduction in BTMs was maintained. In the cross-over group, comparable BMD increases and BTMs reductions to those of in their first year of the long-term group were confirmed. Adverse events did not show a notable increase with long-term denosumab administration. One event of osteonecrosis of the jaw occurred in the cross-over group. CONCLUSIONS: Three-year denosumab treatment in Japanese subjects with osteoporosis showed a favorable benefit/risk profile.

Orthodontic miniscrew failure rate and root proximity, insertion angle, bone contact length, and bone density.

OBJECTIVES: To test the hypothesis that there is no significant correlation between miniscrew failure rate and root proximity, insertion angle, bone contact length, and bone density. SETTING AND SAMPLE POPULATION: This study included 107 patients in whom 190 miniscrews had been placed from April 2008 to October 2009 in Tohoku University Hospital (Sendai, Japan). MATERIALS AND METHODS: Cone beam computed tomography scans (CBCT) and periapical radiographs were taken before and after miniscrew placement. Differences in root proximity, screw insertion angle, bone contact length, and bone density were statistically compared; comparisons were also made between the CBCT images and periapical radiographs. RESULTS: A significantly higher success rate was observed in the maxilla than in the mandible. The distance between the miniscrew and the root surface was significantly smaller in the failure group. There were no significant differences in the insertion angle, bone contact length, or bone density between the success group and the failure group. The concordance rate between the periapical dental radiographs and CBCT images was 46.5%. CONCLUSION: While bone contact length, miniscrew angle, and bone density did not exert major effects on miniscrew failure, root proximity was the factor that most affected miniscrew failure, especially for miniscrews placed in the mandible. CBCT was superior to periapical dental X-rays for evaluating the proximity of miniscrews to the root. Correction of the X-ray attenuation coefficient value was necessary for measuring bone density using CBCT.

Hematogenous apoptotic mechanism in salivary glands in chronic periodontitis.

OBJECTIVE: The aim of the study is to investigate the apoptotic mechanism in salivary glands in the rat experimental periodontitis model. DESIGN: A rat periodontitis model was prepared by using a ligature around the second upper molar. In the salivary (parotid and submandibular) glands and blood samples, putative apoptotic factors and pathway molecules were investigated in vivo and in vitro. RESULTS: Four weeks of ligation (chronic periodontitis) demonstrated significant apoptotic atrophy of the salivary gland, but one week of ligation (initial periodontitis) did not. In the blood plasma, tumor necrosis factor-α (TNF-α) was increased in the periodontitis model, but interleukin-1ß and -6 were not. TNF-α receptor type 1, which has an intracellular apoptotic pathway, was expressed in the salivary glands of rats. Western blot analysis of cultured rat primary salivary gland cells demonstrated that TNF-α induced cleavage of poly (ADP-ribose) polymerase (PARP) and caspase-3 in a dose-dependent manner, indicating apoptosis induction. Additionally, we found increment of circulating lymphocytes in the model. Expression of mRNA and immunoreactive cells for the B lymphocyte marker CD19 were increased in the salivary gland in the model. Western blotting showed that coculture with extracted B cells from the periodontitis model increased cleaved PARP in salivary gland cells. CONCLUSIONS: Chronic periodontitis status leads to an increase in circulating TNF-α and B lymphocyte infiltration, resulting in apoptotic atrophy of the salivary gland as a periodontitis-induced systemic response.

Oral medicine psychiatric liaison clinic: study of 1202 patients attending over an 18-year period.

Patients with orofacial pain and discomfort often suffer from psychiatric disorders. However, few studies involving a large sample have examined the diagnostic results of patients with orofacial pain or discomfort in relation to psychiatric disorders. The purpose of this study was to summarize and clarify the characteristics and demographic data of 1202 patients attending the psychiatric liaison clinic at Aichi Gakuin University Hospital. Psychiatric diagnosis was performed by psychiatrists for all patients, based on the Diagnostic and Statistical Manual of Mental Disorders, fifth edition. Among the 1202 patients, 992 (82.5%) were female. The average age of the patients was 57.2±15.0years. The predominant broad categories of orofacial pain and discomfort seen were burning mouth syndrome (n=484, 40.3%), persistent idiopathic facial pain (n=258, 21.5%), and oral dysesthesia (n=215, 17.9%). The predominant broad categories of psychiatric diagnoses seen were somatic symptoms and related disorders (n=934, 77.7%) and depressive disorders (n=76, 6.3%). Among the 934 patients with somatic symptoms and related disorders, 678 had a somatic symptom disorder with predominant pain. The results confirmed that most patients with orofacial pain and discomfort were middle-aged and elderly women suffering from a somatic symptom disorder with predominant pain.

Reverse hydrolysis reaction of a recombinant alkaline ceramidase of Pseudomonas aeruginosa.

Recently, we purified an alkaline ceramidase (CDase) of Pseudomonas aeruginosa and found that the enzyme catalyzed a reversible reaction in which the N-acyl linkage of ceramide was hydrolyzed or synthesized [J. Biol. Chem. 273 (1998) 14368-14373]. Here, we report the characterization of the reverse hydrolysis reaction of the CDase using a recombinant enzyme. The reverse hydrolysis reaction of the CDase was clearly distinguishable from the reaction of an acyl-coenzyme A (CoA) dependent N-acyltransferase, because the CDase catalyzed the condensation of a free fatty acid to sphingosine (Sph) without cofactors but did not catalyze the transfer of a fatty acid from acyl-CoA to Sph. The reverse hydrolysis reaction proceeded most efficiently in the presence of 0.05% Triton X-100 at neutral pH, while the hydrolysis reaction tended to be favored with an increase in the concentration of the detergent at alkaline pH. The specificity of the reverse reaction for fatty acids is quite broad; saturated and unsaturated fatty acids were efficiently condensed to Sph. In contrast, the stereo-specificity of the reverse reaction for the sphingoid bases is very strict; the D-erythro form of Sph, not the L-erythro or D/L-threo one, was only acceptable for the reverse reaction. Chemical modification of the enzyme protein affected or did not affect both the hydrolysis and reverse reactions to the same extent, suggesting that the two reactions are catalyzed at the same catalytic domain.

Acetoacetyl-CoA synthetase specific activity and concentration in rat tissues.

An antibody against acetoacetyl-CoA synthetase purified from rat liver was raised in rabbits. Utilizing the binding of antibody-antigen complexes to a nitrocellulose membrane, a sensitive enzyme-linked immunosorbent assay was developed to estimate the enzyme concentration in rat tissues. The enzyme concentration (microgram immunoreactive protein/mg protein) in rat liver cytosol was increased about 3-, 1.8- and 7-fold by feeding rats diets containing 5% cholestyramine, 0.2% ML-236B (compactin), and 5% cholestyramine plus 0.2% ML-236B for 4 days, respectively, and decreased about 1.8-fold by fasting the animals or 1.3-fold by feeding them a diet containing 5% cholesterol. Changes in the enzyme activity were almost parallel to those in the enzyme concentration, suggesting the physiological role of this enzyme in cholesterol biosynthesis. Immunoblotting of the hepatic cytosol also confirmed that the increase in enzyme concentration on cholestyramine and/or ML-236B feeding was due to an increase in an enzyme protein the same as the purified enzyme and not the isozymic protein. Among various rat tissues examined, the concentrations of immunologically crossreactive enzyme were higher in lipogenic tissues, such as brain, adipose tissue and liver, than in other tissues. The enzymes in these three tissues were identical in molecular weight determined by gel filtration and immunoblotting.

Purification and characterization of NADP-linked acetoacetyl-CoA reductase from Zoogloea ramigera I-16-M.

An NADP-linked acetoacetyl-CoA reductase was purified to electrophoretic homogeneity from Zoogloea ramigera I-16-M, a poly(3-hydroxybutyrate)-accumulating bacterium. The purified enzyme showed specific activity of 412 mumol acetoacetyl-CoA reduced per min per mg protein, which constituted an 880-fold purification compared to the crude extract, with a 32% yield. Electrophoretic analysis of the purified enzyme which had been cross-linked with dimethylsuberimidate showed that the native enzyme (Mr 92,000) is a tetramer of four identical subunits (Mr 25,500). Among the various D-(-)- and L-(+)-3-hydroxyacyl-CoAs tested, the purified enzyme oxidized only D-(-)-3-hydroxybutyryl-CoA and to a lesser extent D-(-)-3-hydroxyvaleryl-CoA in the presence of NADP+. The antiserum prepared against the purified enzyme completely inhibited poly(3-hydroxybutyrate) synthesis from acetyl-CoA by a crude extract of Z. ramigera I-16-M cells. These findings indicate that this enzyme plays an indispensable role as the supplier of D-(-)-3-hydroxybutyryl-CoA in poly(3-hydroxybutyrate) synthesis in this bacterium.

Purification and characterization of acetoacetyl-CoA synthetase from rat liver.

Acetoacetyl-CoA synthetase (acetoacetate:CoA ligase) was purified to electrophoretic homogeneity from a rat liver supernatant by ammonium sulfate fractionation and successive chromatographies on DEAE-Sepharose, Blue-Sepharose, Red-Sepharose, CoA-Sepharose, Ultrogel AcA-44, and DEAE-Sepharose once again. The purified enzyme had a specific activity of 2.3 mumol acetoacetyl-CoA formed per min per mg protein, which constituted a 960-fold purification compared to the crude extract, with a 7.4% yield. The enzyme absolutely required ATP, CoA, a monovalent cation (K+, Rb+, Cs+ or NH4+) and a divalent cation (Mg2+, Mn2+ or Ni2+) for its activity, yielding acetoacetyl-CoA, AMP and PPi in equimolar amounts. The molecular weight of the enzyme was approximately 80 000 as determined by Ultrogel AcA-34 gel filtration, and 71 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme was active only on acetoacetate and to a lesser extent on L-(+)-3-hydroxybutyrate, and the Km values for acetoacetate, L-(+)-3-hydroxybutyrate, ATP and CoA were 8 microM, 75 microM, 60 microM and 10 microM, respectively.

A novel gene, GliH1, with homology to the Gli zinc finger domain not required for mouse development.

The Sonic hedgehog (Shh)-Gli signaling pathway regulates development of many organs, including teeth. We cloned a novel gene encoding a transcription factor that contains a zinc finger domain with highest homology to the Gli family of proteins (61-64% amino acid sequence identity) from incisor pulp. Consistent with this sequence conservation, gel mobility shift assays demonstrated that this new Gli homologous protein, GliH1, could bind previously characterized Gli DNA binding sites. Furthermore, transfection assays in dental pulp cells showed that whereas Gli1 induces a nearly 50-fold increase in activity of a luciferase reporter containing Gli DNA binding sites, coexpression of Gli1 with Gli3 and/or GliH1 results in inhibition of the Gli1-stimulated luciferase activity. In situ hybridization analysis of mouse embryos demonstrated that GliH1 expression is initiated later than the three Gli genes and has a more restricted expression pattern. GliH1 is first detected diffusely in the limb buds at 10.0 days post coitus and later is expressed in the branchial arches, craniofacial interface, ventral part of the tail, whisker follicles and hair, intervertebral discs, teeth, eyes and kidney. LacZ was inserted into the GliH1 allele in embryonic stem cells to produce mice lacking GliH1 function. While this produced indicator mice for GliH1-expression, analysis of mutant mice revealed no discernible phenotype or required function for GliH1. A search of the Celera Genomics and associated databases identified possible gene sequences encoding a zinc finger domain with approximately 90% homology to that of GliH1, indicating there is a family of GliH genes and raising the possibility of overlapping functions during development.

Effects of coupling methods on galvanic corrosion behavior of commercially pure titanium with dental precious alloys.

BACKGROUND: When a dissimilar couple is exposed to corrosive environment, it will normally exhibit a galvanic corrosion. The galvanic corrosion might be influenced by various factors, including type and concentration of electrolyte, surface area ratio between anode and cathode, type of coupling material, and coupling manner. PURPOSE: The purpose of this study was to investigate and compare the galvanic corrosion behavior of commercially pure titanium when coupled with type IV Au alloy, Au-Ag-Pt alloy, and Ag-Au-Pd alloy by different coupling methods. MATERIALS AND METHODS: Couples were prepared by a laser welding or a mechanical adhering method. Electrochemical corrosion studies were conducted in a Ringer's solution at a scanning rate of 0.1 mV/sec in a range from -250 mV to +250 mV with respect to E(OCP). Corrosion parameters (E(OCP), I(CORR), E(CORR)) were obtained. RESULTS: It was found that (i) there was a significant difference between LWC and AJC for three couples (p<0.05), (ii) the crevice line caused all three couples more corrosive than weld joint line, (iii) for both joint, it was found that type (IV) Au alloy exhibited discoloration to some extent. CONCLUSIONS: It is concluded that among the three couples with two different coupling methods, Ti/Ag-Au-Pd couple exhibited best corrosion resistance in a room temperature Ringer's solution.

Eradication of breast cancer xenografts by hyperthermic suicide gene therapy under the control of the heat shock protein promoter.

To investigate the usefulness of heat shock protein (HSP) promoter for breast cancer gene therapy, hyperthermia and HSV thymidine kinase (tk) suicide gene combination therapy was examined with mouse mammary cancer cell line FM3A. HSP promoter activity was markedly increased after heat shock (41-45 degrees C), with maximum activation (about 400-fold) at 3 hr. An in vitro cytotoxic assay showed that HSP-tk-transduced FM3A cells became more sensitive (more than 50,000 times) to ganciclovir (GCV) with heat shock, but untreated cells showed no increased cytotoxic sensitivity to GCV compared with control FM3A cells. In addition to promoter-oriented selective cell killing, a "chemosensitization effect" as a bystander effect was demonstrated by hyperthermia and suicide gene combination therapy, using a non-heat-inducible promoter. Immunohistochemical analysis revealed that this synergistic killing effect was dependent on apoptotic cell death with upregulation of both Fas and FasL (Fas ligand) expression. We also examined the efficacy of HSP-tk gene therapy in vivo by implanting breast cancer in subcutaneous and intraperitoneal models of BALB/c nude mice targeted by the HVJ-anionic liposome method. Significant tumor regression was observed in HSP-tk-transduced tumors followed by hyperthermia therapy, but no such inhibition was noted in either the mock vector transfection or hyperthermia group compared with control tumor-bearing mice. Our results demonstrate that this combination system is synergistically effective in mediating Fas-dependent apoptosis for a specific gene therapy targeting HSP-expressing mammary carcinomas, even in advanced and heat-resistant breast cancer.

Charge-selective permeability of dermo-epidermal junction: tracer studies with cationic and anionic ferritins.

To investigate quantitatively the charge-selective permeability of the basement membrane (BM) of the dermo-epidermal junction (DEJ), tracer experiments using anionic and cationic ferritins were performed on an epidermal sheet, whose lamina densa was exposed on the dermal surface; its dermis was removed with forceps after the treatment of newborn rat skin with 10 mM dithiothreitol. The lamina densa and epidermal components of the sheets were electron microscopically well preserved, and anionic sites were ultrastructurally demonstrated on both the dermal and epidermal aspects of the lamina densa in the DEJ as clusters of cationic ferritins (CF) [isoelectric point (pI) greater than 9.5] or polyethyleneimine particles, indicating that the epidermal sheets were suitable for study of permeability. In tracer experiments, a large number of CF (pI 8.0-9.4) passed the lamina densa and formed clusters on both aspects of the lamina densa and in the intercellular space. The number of native anionic ferritins (NF) (pI 4.1-4.6) passing it was apparently much smaller than that of CF. When the epidermal sheets were pre-treated with protamine sulfate to neutralize the negative charges in the tissue, the number of NF penetrating the lamina densa was significantly larger than the number of those in the untreated sheet. These results indicate that the BM of the DEJ plays a role in a charge-selective filtration, although it is not as selective a barrier as the glomerular basement membrane.

Cholesterol uptake of isolated rat hepatocytes is accelerated by several kinds of phosphatidylcholine.

Uptake of cholesterol by isolated rat hepatocytes in a serum-free medium was remarkably enhanced by dispersion with several kinds of phosphatidylcholine. Of the various phosphatidylcholines tested, dilinoylphosphatidylcholine had the strongest accelerating effect, while dipalmitoylphosphatidylcholine was the weakest. The abilities to accelerate cholesterol uptake were in proportion to the content of unsaturated fatty acid in the phosphatidylcholine used. It was confirmed by electron microscopy that there is no relation between the size of the cholesterol-phosphatidylcholine complex and uptake. These data suggest that recognition of unsaturated fatty acids in phosphatidylcholine by isolated cells enhances uptake of cholesterol.

Mandibular condyle bone mineral density measurement by quantitative computed tomography: a gender-related difference in correlation to spinal bone mineral density.

We conducted volunteer studies to assess age-related changes of mandibular condyle bone mineral density (BMD) and its correlation to the spinal BMD. Quantitative computed tomography was performed on the condyles and spines (L1-3) of 210 healthy subjects (114 men and 96 women, aged 5-85 years). A separate study was performed on 73 young student subjects (39 men and 34 women, aged 23-25 years). The mandibular condyle BMD showed a decrement rate similar to spinal BMD in men, but in women the decrement rate of the mandibular condyle BMD was lower than that of the L1-3 BMD. On the other hand, correlation coefficients in BMD between the mandibular condyle and spine were similar in women and men. Gender-related differences were found to be dramatic when assessed in the young student group; the mandibular condyle and spinal BMDs were highly correlated in women (r = 0.82, p < 0.0001), but no correlation was found in men (r = 0.22). Taken together, these results suggest that the same regulatory mechanisms exist in the mandibular condyle and spine BMDs. However, aside from the spine BMD, additional undefined factor(s), including mechanical stress from the occlusion, may be involved in maintaining mandibular BMD.

Pravastatin alone and in combination with low-dose cholestyramine in patients with primary hypercholesterolemia and coronary artery disease.

This randomized, open-label study compared the cost efficiency of low-dose pravastatin combined with low-dose cholestyramine with high-dose pravastatin monotherapy in 59 patients with moderate hypercholesterolemia and coronary disease. Both regimes were effective in improving lipid profiles in these patients; however, low-dose combination therapy enhanced achievement in therapeutic goals and cost efficiency.

A new X-linked multiple congenital anomalies/mental retardation syndrome.

We report on 2 boys, the sons of sisters, and their mother's brother who have a new, X-linked multiple congenital anomalies/mental retardation (MCA/MR) syndrome. The propositus was a 16-month-old caucasian male with 1) mental retardation, 2) congenital microcephaly, 3) postnatal growth deficiency, 4) ridged metopic suture with narrow bifrontal diameter, 5) upslanted palpebral fissures with persistent epicanthal folds, strabismus, and lacrimal duct obstruction, 6) narrow palate, 7) macrodontia, 8) anteverted ears, 9) atrial septal defect, 10) dry brittle scalp hair and 11) cutis marmorata. His chromosomes were normal. His cousin and uncle were similarly affected. This distinctive MCA/MR syndrome is added to the list of X-linked malformation syndromes known at the present time.

In vitro properties of a chitosan-bonded hydroxyapatite bone-filling paste.

A possible bone substitute material for dental treatment was developed and tested. The material is composed of powdered hydroxyapatite (HA), ZnO and CaO, kneaded into a chitosan sol to make a quick-hardening paste. A composition was found which showed neutral pH, short setting time, and relatively high compressive strength. The use of such a paste for the treatment of periodontal defects or the augmentation of edentulous alveolar ridges may alleviate problems now associated with the implantation of particulate HA, such as early migration of particles and recontouring of the implant.

Experimental study on osteoconductive properties of a chitosan-bonded hydroxyapatite self-hardening paste.

A developmental bone substitute, composed of a chitosan-bonded hydroxyapatite paste and having various possible applications in medical and dental treatments, was evaluated with regard to its osteoconductive properties. Radiographic examination revealed that a bone-like irregular radiopacity appeared in the region of the embedded paste. This was judged histopathologically as the formation of bone tissue with chondral tissue. These data suggest that the paste has osteoconductive properties, and may, therefore, prove clinically useful as a bioactive bone substitute.

Dentin regeneration by dental pulp stem cell therapy with recombinant human bone morphogenetic protein 2.

Regenerative medicine is based on stem cells, signals, and scaffolds. Dental pulp tissue has the potential to regenerate dentin in response to noxious stimuli, such as caries. The progenitor/stem cells are responsible for this regeneration. Thus, stem cell therapy has considerable promise in dentin regeneration. Culture of porcine pulp cells, as a three-dimensional pellet, promoted odontoblast differentiation compared with monolayers. The expression of dentin sialophosphoprotein (Dspp) and enamelysin/matrix metalloproteinase 20 (MMP20) mRNA confirmed the differentiation of pulp cells into odontoblasts and was stimulated by the morphogenetic signal, bone morphogenetic protein 2 (BMP2). Based on the in vitro experiments, an in vivo evaluation of pulp progenitor/stem cells in the dog was performed. The autogenous transplantation of the BMP2-treated pellet culture onto the amputated pulp stimulated reparative dentin formation. In conclusion, BMP2 can direct pulp progenitor/stem cell differentiation into odontoblasts and result in dentin formation.