[Concomitant treatment of the osteoporotic drugs].

Osteoporotic drugs are classified to 3 categories ; anti-resorptives, supplemental and anabolic drugs. A lot of evidence has been made about each osteoporotic drug. Although concomitant drug subscriptions are often used by many physicians, little evidence has been ever made about the concomitant drug treatment. Theoretically different categorized drugs should be combined in concomitant treatment. Fragility fracture risks have been tested compared alendronate (Aln) mono-therapy and Aln + alfacalcidol (VD(3)) for 2 year in JOINT02 of ATOP study. Aln + alfacalcidol (VD(3)) concomitant treatment has significantly reduced vertebral fracture risk compared with Aln mono-therapy in patients with advanced osteoporosis with multiple and high SQ grade vertebral fracture and also reduced weight bearing bone fracture risk.

[Long-term and high-dose bisphosphonate administration causes no bone over-mineralization].

OBJECTIVE: To evaluate the effects of long-term bisphosphonate administration (incadronate) upon the mean degree of secondary bone mineralization. METHODS: Thirty adult beagles were divided randomly into three groups of CNT, YML and YMH based upon their body weights (5 males and 5 females in each group). Animals in CNT were orally given lactose 12 mg x kg(-1) x d(-1) while those in YML and YMH were orally given incadronate disodium at doses of 0.3 mg x kg(-1) x d(-1) and 0.6 mg x kg(-1) x d(-1) respectively. The dosing procedure lasted for three years. Prior to sacrifices, all animals were double-labeled intravenously with oxytetracycline hydrochloride (20 mg x kg(-1)). After necropsy, the left 9th ribs were excised for evaluation. RESULTS: Histomorphometry showed that activation frequency in YML and YMH [(4.93 +/- 0.92)/mm(2) and (2.50 +/- 0.78)/mm(2)] were significantly lower than that in CNT (7.83 +/- 0.96)/mm(2) per year. The mean degree of mineralization in bone in YML and YMH (1.40 +/- 0.12, 1.48 +/- 0.09) were significantly higher than CNT (1.07 +/- 0.06). The increments were 31% and 38% respectively. CONCLUSION: Long-term and high-dose bisphosphonate administration significantly suppresses bone turnover so as to increase the degree of bone mineralization. But there is no resulting bone over-mineralization. (incadronate) administration on the mean degree of secondary mineralization in bone.

The transcription factor IDEF1 regulates the response to and tolerance of iron deficiency in plants.

Iron is essential for most living organisms and is often the major limiting nutrient for normal growth. Plants induce iron utilization systems under conditions of low iron availability, but the molecular mechanisms of gene regulation under iron deficiency remain largely unknown. We identified the rice transcription factor IDEF1, which specifically binds the iron deficiency-responsive cis-acting element IDE1. IDEF1 belongs to an uncharacterized branch of the plant-specific transcription factor family ABI3/VP1 and exhibits the sequence recognition property of efficiently binding to the CATGC sequence within IDE1. IDEF1 transcripts are constitutively present in rice roots and leaves. Transgenic tobacco plants expressing IDEF1 under the control of the constitutive cauliflower mosaic virus 35S promoter transactivate IDE1-mediated expression only in iron-deficient roots. Transgenic rice plants expressing an introduced IDEF1 exhibit substantial tolerance to iron deficiency in both hydroponic culture and calcareous soil. IDEF1 overexpression leads to the enhanced expression of the iron deficiency-induced transcription factor gene OsIRO2, suggesting the presence of a sequential gene regulatory network. These findings reveal cis element/trans factor interactions that are functionally linked to the iron deficiency response. Manipulation of IDEF1 also provides another approach for producing crops tolerant of iron deficiency to enhance food and biomass production in calcareous soils.

Expression and enzyme activity of glutathione reductase is upregulated by Fe-deficiency in graminaceous plants.

Glutathione reductase (GR) plays an important role in the response to biotic and abiotic stresses in plants. We studied the expression patterns and enzyme activities of GR in graminaceous plants under Fe-sufficient and Fe-deficient conditions by isolating cDNA clones for chloroplastic GR (HvGR1) and cytosolic GR (HvGR2) from barley. We found that the sequences of GR1 and GR2 were highly conserved in graminaceous plants. Based on their nucleotide sequences, HvGR1 and HvGR2 were predicted to encode polypeptides of 550 and 497 amino acids, respectively. Both proteins showed in vitro GR activity, and the specific activity for HvGR1 was 3-fold that of HvGR2. Northern blot analyses were performed to examine the expression patterns of GR1 and GR2 in rice (Os), wheat (Ta), barley (Hv), and maize (Zm). HvGR1, HvGR2, and TaGR2 were upregulated in response to Fe-deficiency. Moreover, HvGR1 and TaGR1 were mainly expressed in shoot tissues, whereas HvGR2 and TaGR2 were primarily observed in root tissues. The GR activity increased in roots of barley, wheat, and maize and shoot tissues of rice, barley, and maize in response to Fe-deficiency. Furthermore, it appeared that GR was not post-transcriptionally regulated, at least in rice, wheat, and barley. These results suggest that GR may play a role in the Fe-deficiency response in graminaceous plants.

Human parathyroid hormone (1-34) accelerates natural fracture healing process in the femoral osteotomy model of cynomolgus monkeys.

Several studies in rats have demonstrated that parathyroid hormone accelerates fracture healing by increasing callus formation or stimulating callus remodeling. However the effect of PTH on fracture healing has not been tested using large animals with Haversian remodeling system. Using cynomolgus monkey that has intracortical remodeling similar to humans, we examined whether intermittent treatment with human parathyroid hormone [hPTH(1-34)] accelerates the fracture healing process, especially callus remodeling, and restores geometrical shapes and mechanical properties of osteotomized bone. Seventeen female cynomolgus monkeys aged 18-19 years were allocated into three groups: control (CNT, n=6), low-dose PTH (0.75 microg/kg; PTH-L, n=6), and high-dose PTH (7.5 microg/kg; PTH-H, n=5) groups. In all animals, twice a week subcutaneous injection was given for 3 weeks. Then fracture was produced surgically by transversely cutting the midshaft of the right femur and fixing with stainless plate. After fracture, intermittent PTH treatment was continued until sacrifice at 26 weeks after surgery. The femora were assessed by soft X-ray, three-point bending mechanical test, histomorphometry, and degree of mineralization in bone (DMB) measurement. Soft X-ray showed that complete bone union occurred in all groups, regardless of treatment. Ultimate stress and elastic modulus in fractured femur were significantly higher in PTH-H than in CNT. Total area and percent bone area of the femur were significantly lower in both PTH-L and PTH-H than in CNT. Callus porosity decreased dose-dependently following PTH treatment. Mean DMB of callus was significantly higher in PTH-H than in CNT or PTH-L. These results suggested that PTH decreased callus size and accelerated callus maturation in the fractured femora. PTH accelerates the natural fracture healing process by shrinking callus size and increasing degree of mineralization of the fracture callus, thereby restoring intrinsic material properties of osteotomized femur shaft in cynomolgus monkeys although there were no significant differences among the groups for structural parameters.

OsZIP4, a novel zinc-regulated zinc transporter in rice.

Zinc (Zn) is an essential element for the normal growth of plants but information is scarce on the mechanisms whereby Zn is transported in rice (Oryza sativa L.) plants. Four distinct genes, OsZIP4, OsZIP5, OsZIP6, and OsZIP7 that exhibit sequence similarity to the rice ferrous ion transporter, OsIRT1, were isolated. Microarray and northern blot analysis revealed that OsZIP4 was highly expressed under conditions of Zn deficiency in roots and shoots. Real-time-PCR revealed that the OsZIP4 transcripts were more abundant than those of OsZIP1 or OsZIP3 in Zn-deficient roots and shoots. OsZIP4 complemented a Zn-uptake-deficient yeast (Saccharomyces cerevisiae) mutant, Deltazrt1,Deltazrt2, indicating that OsZIP4 is a functional transporter of Zn. OsZIP4-synthetic green fluorescent protein (sGFP) fusion protein was transiently expressed in onion epidermal cells localized to the plasma membrane. In situ hybridization analysis revealed that OsZIP4 in Zn-deficient rice was expressed in shoots and roots, especially in phloem cells. Furthermore, OsZIP4 transcripts were detected in the meristem of Zn-deficient roots and shoots. These results suggested that OsZIP4 is a Zn transporter that may be responsible for the translocation of Zn within rice plants.

Three nicotianamine synthase genes isolated from maize are differentially regulated by iron nutritional status.

Nicotianamine synthase (NAS) is an enzyme that is critical for the biosynthesis of the mugineic acid family of phytosiderophores in graminaceous plants, and for the homeostasis of metal ions in nongraminaceous plants. We isolated one genomic NAS clone, ZmNAS3, and two cDNA NAS clones, ZmNAS1 and ZmNAS2, from maize (Zea mays cv Alice). In agreement with the increased secretion of phytosiderophores with Fe deficiency, ZmNAS1 and ZmNAS2 were positively expressed only in Fe-deficient roots. In contrast, ZmNAS3 was expressed under Fe-sufficient conditions, and was negatively regulated by Fe deficiency. This is the first report describing down-regulation of NAS gene expression in response to Fe deficiency in plants, shedding light on the role of nicotianamine in graminaceous plants, other than as a precursor in phytosiderophore production. ZmNAS1-green fluorescent protein (sGFP) and ZmNAS2-sGFP were localized at spots in the cytoplasm of onion (Allium cepa) epidermal cells, whereas ZmNAS3-sGFP was distributed throughout the cytoplasm of these cells. ZmNAS1 and ZmNAS3 showed NAS activity in vitro, whereas ZmNAS2 showed none. Due to its duplicated structure, ZmNAS2 was much larger (65.8 kD) than ZmNAS1, ZmNAS3, and previously characterized NAS proteins (30-38 kD) from other plant species. We reveal that maize has two types of NAS proteins based on their expression pattern and subcellular localization.

Evidence that electron-dense bodies in Cyanidium caldarium have an iron-storage role.

The acidophilic and thermophilic unicellular red alga, Cyanidium caldarium (Tilden) Geitler, is widely distributed in acidic hot springs. Observation by transmission electron microscopy (TEM) showed that algae grown in Allen's medium contained electron-dense bodies with diameters from 100 to 200 nm. Electron dispersive x-ray analysis indicated that the electron-dense bodies contained high levels of iron, phosphorous, and oxygen; P/Fe ratios were from 1.3 to 2.0. The electron spin resonance (ESR) spectrum of the intact C. caldarium cells showed an isotropic signal at a g value of 2.00. Density-gradient centrifugation of the cell lysate yielded a fraction that included substances showing the isotropic ESR signal. EDTA treatment of this fraction reduced the ESR signal intensity, whereas it increased a signal that is typical of Fe(III)-EDTA. The fact that the isotropic signal dominates the ESR spectrum, together with a previous finding that iron is confined to the electron-dense bodies, led us to conclude that iron in the electron-dense bodies accounts for the isotropic ESR signal. Since the intensity of the ESR signal depends on the amount of iron in the cells, the electron-dense bodies are probably iron storage sites.

Cloning an iron-regulated metal transporter from rice.

Rice cDNA and genomic libraries were screened in order to clone an Fe(II) transporter gene. A cDNA clone highly homologous to the Arabidopsis Fe(II) transporter gene IRT1 was isolated from Fe-deficient rice roots. The cDNA clone was named OsIRT1. A genomic clone corresponding to the cDNA was also obtained, sequenced and analysed. When expressed in yeast cells, OsIRT1 cDNA reversed the growth defects of the yeast iron-uptake mutant. Northern blot analysis revealed that OsIRT1 mRNA was predominantly expressed in roots and was induced by Fe- and Cu-deficiency. This suggests that OsIRT1 is a functional metal transporter for iron, and is actively engaged in Fe uptake from soils, especially under limiting conditions.

cDNA microarray analysis of gene expression during Fe-deficiency stress in barley suggests that polar transport of vesicles is implicated in phytosiderophore secretion in Fe-deficient barley roots.

To acquire Fe from soil, graminaceous plants secrete mugineic acid family phytosiderophores (MAs) from their roots. The secretion of MAs increases in response to Fe deficiency, and shows a distinct diurnal rhythm. We used a microarray that included 8987 cDNAs of rice EST clones to examine gene expression profiles in barley roots during Fe-deficiency stress. Approximately 200 clones were identified as Fe-deficiency-inducible genes, of which seven had been identified previously. In order to meet the increased demand for methionine to produce MAs, Fe-deficiency enhances the expression of genes that participate in methionine synthesis, as well as recycling methionine through the Yang cycle. Of these 200 genes, approximately 50 exhibited different transcription levels in Fe-deficient roots at noon and at night. Northern blot analysis of time course experiments confirmed that five of these genes exhibited a diurnal change in their level of expression. The diurnal changes in the expression of these genes suggest that polar vesicle transport is involved in the diurnal secretion of MAs.

IDI7, a new iron-regulated ABC transporter from barley roots, localizes to the tonoplast.

A new Fe-deficiency-induced cDNA, IDI7, was isolated from the roots of Fe-deficient barley (Hordeum vulgare L. cv. Ehimehadaka no. 1). The transcript levels of IDI7 in roots strongly correlated with iron nutritional status, and induction by Fe-deficiency was restricted to roots. Excess treatment with heavy metal ions, such as copper, manganese, and zinc, did not cause obvious IDI7 induction in either leaves or roots. IDI7 encodes a 644 amino acid protein, and has features typical of ATP-binding cassette (ABC) transporters. Phylogenetic analysis revealed that IDI7 is closely related to the half-type ABC protein subfamily, which includes mammalian transporters associated with antigen processing (TAPs). A transiently expressed fusion protein of IDI7 to green fluorescent protein (GFP) was localized to tonoplasts in suspension-cultured tobacco (Nicotiana tabacum L.) cells. IDI7 and its orthologues are thought to comprise a new class of ABC transporters, located in the tonoplasts of higher plants. A possible Fe-deficiency adaptation role for IDI7 in barley root cells, involving transport across the tonoplast, is proposed.