Prevention of bone loss and improvement of pain-related behavior in hind limb-unloaded mice by administration of teriparatide and bisphosphonate.

OBJECTIVES: There are few reports on the comparison between teriparatide (PTH) and bisphosphonate (BP) in terms of osteoporosis pain-related behavior and immunohistochemical findings. The aims of this study were to evaluate skeletal pain associated with osteoporosis and to examine the inhibitory effect of PTH and BP on pain and bone loss in hind limb-unloaded (HU) mice. The mechanism of osteoporotic pain in HU mice was evaluated by examining pain-related behavior and immunohistochemical findings. The effects of PTH and alendronate (ALN), a potent osteoclast inhibitor, on these parameters were also assessed. METHODS: Eight-week-old male ddY mice were tail-suspended for 2 weeks and assigned to four groups: hind limb-loaded (HL) mice with only tail suspension treated with vehicle; HU mice with tail suspension treated with vehicle; HU mice treated with PTH; and HU mice treated with ALN. Starting immediately after reloading, vehicle, PTH, or ALN was injected subcutaneously. After a 2-week treatment, mechanical sensitivity was examined using von Frey filaments. Bilateral hind limbs were removed for micro-computed tomography, immunohistochemical analysis, and messenger RNA (mRNA) expression analysis. RESULTS: HU mice with tail suspension developed bone loss and mechanical hyperalgesia in the hind limbs. The HU mice showed an increased osteoclasts and sclerostin-positive cells in the hind limb bone. Furthermore, PTH and ALN both prevented HU-induced bone loss and mechanical hyperalgesia in the osteoporotic animal models. Histological examination of the hind limb bone revealed that, similar to ALN, PTH inhibited the osteoclasts and sclerostin-positive cells. The mRNA levels of TNFα and IL-6 tended to decrease with ALN or PTH treatment compared with those without any treatment. CONCLUSIONS: Treatment with PTH as well as BP prevented bone loss, mechanical hyperalgesia, osteoclast increase, and osteocyte increase. Similar to BP, the inhibitory effect of PTH on osteoclasts might contribute to the improvement of skeletal pain.

Teriparatide improves pain-related behavior and prevents bone loss in ovariectomized mice.

PURPOSE: The aim of this study was to examine the inhibitory effect of teriparatide (TPTD) on pain and on bone loss in ovariectomized (OVX) mice. The mechanism of osteoporotic pain in OVX mice was evaluated through an examination of pain-related behavior as well as immunohistochemical examinations. METHODS: Eight-week-old female ddY mice were OVX and assigned to one of three groups: (1) OVX mice treated with vehicle (OVX), (2) OVX mice treated with teriparatide (OVX-TPTD), or (3) SHAM-operated mice treated with vehicle (SHAM). Starting immediately after surgery, vehicle or TPTD was injected subcutaneously. After a 4-week treatment, mechanical sensitivity was tested using von Frey filaments. The proximal tibial metaphyses were analyzed three-dimensionally by microcomputed tomography (µCT). Calcitonin gene-related peptide (CGRP) and transient receptor potential channel vanilloid 1 (TRPV1) expressions in L3-5 dorsal root ganglion (DRG) neurons were examined using immunohistochemistry. RESULTS: Ovariectomy induced bone loss and mechanical hyperalgesia in the hind limbs with upregulation of CGRP and TRPV1 expressions in DRG neurons innervating the hind limbs. Bone loss was prevented more effectively in the OVX-TPTD mice than in the OVX mice. Furthermore, mechanical hyperalgesia and upregulation of CGRP and TRPV1 expressions were significantly lower in the OVX-TPTD mice than in the OVX mice. CONCLUSION: TPTD treatment prevented ovariectomy-induced bone loss and ovariectomy-induced mechanical hyperalgesia in hind limbs, and it suppressed CGRP and TRPV1 expressions in DRG neurons. These results suggest that TPTD is useful for the treatment of osteoporotic pain in postmenopausal women.

Tracing whale myoglobin evolution by resurrecting ancient proteins.

Extant cetaceans, such as sperm whale, acquired the great ability to dive into the ocean depths during the evolution from their terrestrial ancestor that lived about 50 million years ago. Myoglobin (Mb) is highly concentrated in the myocytes of diving animals, in comparison with those of land animals, and is thought to play a crucial role in their adaptation as the molecular aqualung. Here, we resurrected ancestral whale Mbs, which are from the common ancestor between toothed and baleen whales (Basilosaurus), and from a further common quadrupedal ancestor between whale and hippopotamus (Pakicetus). The experimental and theoretical analyses demonstrated that whale Mb adopted two distinguished strategies to increase the protein concentration in vivo along the evolutionary history of deep sea adaptation; gaining precipitant tolerance in the early phase of the evolution, and increase of folding stability in the late phase.

The effects of bisphosphonate on pain-related behavior and immunohistochemical analyses in hindlimb-unloaded mice.

OBJECTIVE: The aim of this study was to evaluate skeletal pain associated with immobility-induced osteoporosis and to examine the inhibitory effect of bisphosphonate (BP) administration on pain in hindlimb-unloaded (HU) mice. METHODS: The mechanism of osteoporotic pain in HU mice was evaluated through an examination of pain-related behavior, as well as immunohistochemical findings. In addition, the effects of alendronate (ALN), a potent osteoclast inhibitor, on these parameters were assessed. RESULTS: HU mice with tail suspension developed bone loss and mechanical hyperalgesia in the hindlimbs. The HU mice showed an increase in the number of calcitonin gene-related peptide (CGRP)-immunoreactive neurons and in transient receptor potential channel vanilloid subfamily member 1 (TRPV1)-immunoreactive neurons in the dorsal root ganglions (DRGs) innervating the hindlimbs. Furthermore, administration of ALN prevented HU-induced bone loss, mechanical hyperalgesia, and upregulation of CGRP and TRPV1 expressions in DRG neurons of immobility-induced osteoporotic animal models. CONCLUSIONS: HU mice appear to be a useful model for immobility-induced osteoporotic pain and hindlimb-unloading-induced bone loss, as well as upregulation of CGRP and TRPV1 expressions in DRG neurons, and BP treatment prevented bone loss and mechanical hyperalgesia. The inhibitory effect of BP on osteoclast function might contribute to improving osteoporosis-related pain.

Plastidial Folate Prevents Starch Biosynthesis Triggered by Sugar Influx into Non-Photosynthetic Plastids of Arabidopsis.

Regulation of sucrose-starch interconversion in plants is important to maintain energy supplies necessary for viability and growth. Arabidopsis mutants were screened for aberrant responses to sucrose to identify candidates with a defect in the regulation of starch biosynthesis. One such mutant, fpgs1-4, accumulated substantial amounts of starch in non-photosynthetic cells. Dark-grown mutant seedlings exhibited shortened hypocotyls and accumulated starch in etioplasts when supplied with exogenous sucrose/glucose. Similar starch accumulation from exogenous sucrose was observed in mutant chloroplasts, when photosynthesis was prevented by organ culture in darkness. Molecular genetic analyses revealed that the mutant was defective in plastidial folylpolyglutamate synthetase, one of the enzymes engaged in folate biosynthesis. Active folate derivatives are important biomolecules that function as cofactors for a variety of enzymes. Exogenously supplied 5-formyl-tetrahydrofolate abrogated the mutant phenotypes, indicating that the fpgs1-4 mutant produced insufficient folate derivative levels. In addition, the antifolate agents methotrexate and 5-fluorouracil induced starch accumulation from exogenously supplied sucrose in dark-grown seedlings of wild-type Arabidopsis. These results indicate that plastidial folate suppresses starch biosynthesis triggered by sugar influx into non-photosynthetic cells, demonstrating a hitherto unsuspected link between plastidial folate and starch metabolism.

Alendronate inhibits hyperalgesia and suppresses neuropeptide markers of pain in a mouse model of osteoporosis.

BACKGROUND: Chronic back pain is one of the most important complications of postmenopausal osteoporosis. The aim of this study was to evaluate skeletal pain associated with osteoporosis and to examine the inhibitory effect of bisphosphonates (BPs) on pain in ovariectomized (OVX) mice. The mechanism of osteoporotic pain in OVX mice was evaluated through an examination of pain-related behavior, as well as immunohistochemical findings. In addition, the effects of alendronate (ALN), a potent osteoclast inhibitor, on these parameters were assessed. METHODS: 8-week-old female ddY mice were ovariectomized and assigned to 3 groups: SHAM-operated mice treated with vehicle (SHAM; n = 8); OVX mice treated with vehicle (OVX-V; n = 8); and OVX mice treated with ALN (OVX-ALN; n = 8). Starting immediately after surgery, vehicle or 40 µg/kg ALN was injected subcutaneously twice a week for 4 weeks. The bilateral distal femoral metaphyses and proximal tibial metaphyses were analyzed three-dimensionally by µCT. Mechanical sensitivity was tested using von Frey filaments. Transient receptor potential channel vanilloid 1 (TRPV1) and calcitonin gene-related peptide (CGRP) expressions in L3-5 dorsal root ganglion (DRG) neurons were examined immunohistochemically. RESULTS: Ovariectomy induced bone loss and mechanical hyperalgesia in hindlimbs with upregulation of TRPV1 and CGRP expressions in DRG neurons innervating hindlimbs. ALN prevented bone loss and mechanical hyperalgesia in ovariectomized mouse hindlimbs, and it suppressed upregulation of pain markers. CONCLUSIONS: ALN prevented ovariectomy-induced bone loss and mechanical hyperalgesia in hindlimbs, and it suppressed TRPV1 and CGRP expressions in DRG neurons. The results suggest that bone resorption with upregulation of TRPV1 and CGRP expressions is one of the causes of postmenopausal osteoporotic pain.

Anti-tumor necrosis factor therapy inhibits lung metastasis in an osteosarcoma cell line.

BACKGROUND: Osteosarcoma is the most common primary malignancy of bone, and patients often develop pulmonary metastases. In a previous study, tumor necrosis factor (TNF)-α treatment of human osteosarcoma cells increases their metastatic ability in an animal model. TNF-α can act as a tumor necrosis factor and also as a tumor-promoting factor. In the present study, the effect of a TNF-α inhibitor on osteosarcoma aggressiveness and pulmonary metastases was investigated in vitro and in vivo. METHODS: The effect of infliximab, a TNF-α inhibitor, on a metastatic osteosarcoma 143B cell growth and motility was investigated in vitro. An orthotopic xenograft model of 143B cell growth and spontaneous metastasis in SCID mice was used to assess the in vivo effect of infliximab. RESULTS: Infliximab greatly reduced cell motility and pulmonary metastases in 143B cells. The mechanism of pulmonary metastasis inhibition involved decreased expression of CXC chemokine receptor 4 (CXCR4), Rho (small GTPase protein), and its effector. CONCLUSIONS: These results suggest a novel role for TNF-α inhibition in the reduction or prevention of pulmonary metastases of osteosarcoma in this animal model. TNF-α inhibition may become a preventive therapeutic option for the pulmonary metastases of osteosarcoma.

The structure of a deoxygenated 400 kDa haemoglobin reveals ternary- and quaternary-structural changes of giant haemoglobins.

The quaternary structures of invertebrate haemoglobins (Hbs) are quite different from those of vertebrate Hbs. The extracellular giant Hbs of molecular masses of about 400 and 3600 kDa are composed of a dome-shaped dodecameric subassembly which consists of four individual globin subunits. Several crystal structures of 400 kDa Hbs from annelids have been reported, including structures in oxygenated and partially unliganded states, but the structure of the fully deoxygenated state has not been reported. In the present study, crystal structures of V2Hb from the tube worm Lamellibrachia satsuma have been determined in both the fully oxygenated and deoxygenated states. A glycosylation site and novel metal-binding sites for divalent cations were clearly observed with no intersubunit interactions in V2Hb. A comparison of the oxygenated and the deoxygenated forms of V2Hb reveals that the ternary- and quaternary-structural changes occur in a manner that maintains the molecular D3 symmetry. These structures suggest that the mechanisms of quaternary-structural changes between the oxy and deoxy states for the giant Hbs are identical across species.

Purification and characterization of coacervate-forming cuticular proteins from Papilio xuthus pupae.

The Papilio xuthus (Lepidoptera: Papilionidae) pupa expresses novel soluble proteins that undergo reversible temperature-dependent coacervate-formation. We purified two coacervate-forming proteins, PX-1 and PX-4, from the wings of pharate adults. PX-1 and PX-4 form coacervates upon warming. Transmission electron microscopy analysis revealed that these proteins assemble ordered bead-like ultrastructures. We cloned and sequenced PX-1 and PX-4 cDNAs. The PX-1 and PX-4 amino acid sequences contain many hydrophobic residues and show homologies to insect cuticular proteins. Moreover, when recombinant PX-1 and PX-4 were overexpressed in Escherichia coli, both recombinant proteins exhibited temperature-dependent coacervation. Furthermore, analyses of truncated mutants of PX-1 suggest that both the Val/Pro-rich region and Gly/lle-rich regions of PX-1 are involved in such coacervation.

Structural basis for the heterotropic and homotropic interactions of invertebrate giant hemoglobin.

The oxygen binding properties of extracellular giant hemoglobins (Hbs) in some annelids exhibit features significantly different from those of vertebrate tetrameric Hbs. Annelid giant Hbs show cooperative oxygen binding properties in the presence of inorganic cations, while the cooperativities of vertebrate Hbs are enhanced by small organic anions or chloride ions. To elucidate the structural basis for the cation-mediated cooperative mechanisms of these giant Hbs, we determined the crystal structures of Ca2+- and Mg2+-bound Hbs from Oligobrachia mashikoi at 1.6 and 1.7 A resolution, respectively. Both of the metal-bound structures were determined in the oxygenated state. Four Ca2+-binding sites and one Mg2+-binding site were identified in each tetramer subassembly. These cations are considered to stabilize the oxygenated form and increase affinity and cooperativity for oxygen binding, as almost all of the Ca2+ and Mg2+ cations were bound at the interface regions, forming either direct or hydrogen bond-mediated interactions with the neighboring subunits. A comparison of the structures of the oxygenated form and the partially unliganded form provides structural insight into proton-coupled cooperativity (Bohr effect) and ligand-induced transitions. Two histidine residues are assumed to be primarily associated with the Bohr effect. With regard to the ligand-induced cooperativity, a novel quaternary rotation mechanism is proposed to exist at the interface region of the dimer subassembly. Interactions among conserved residues Arg E10, His F3, Gln F7, and Val E11, together with the bending motion of the heme molecules, appear to be essential for quaternary rearrangement.

Structure of the partially unliganded met state of 400 kDa hemoglobin: insights into ligand-induced structural changes of giant hemoglobins.

Recent crystallographic studies have revealed the structures of some invertebrate extracellular giant hemoglobins of 3,600 kDa or 400 kDa and their common quaternary structure of dodecameric subassembly composed of four kinds of globin subunits (A1, A2, B1, and B2). These results have provided insight into the mechanisms of their unique functional properties of oxygen binding and sulfide binding. All of these structures were solved with oxygenated or CO-liganded forms at low or moderate resolutions. We have determined the crystal structure of 400 kDa Hb from a polychaete Oligobrachia mashikoi at 1.95 A resolution. The electron densities at higher resolution confirm the existence of an isoform of the B1 subunit because of the inconsistency with the model that was built from the formerly known amino acid sequence. The brownish color of the crystals used in this study and the absorption spectrum from the dissolved crystals strongly indicated that the obtained structure was a ferric met state, whereas complete absence of electron density around the distal heme pockets were observed at the A2, B1, and B2 subunits. We concluded that the obtained structure was in unliganded met forms at three of four globin subunits in the 24mer assembly and in oxygenated forms at the remaining A1 subunits. The partially unliganded structure showed remarkable structural changes at the AB loop regions causing quaternary rearrangements of the EF-dimer structure. In contrast, few changes occurred at the interface regions composed of the E and F helices. These results suggest that the ligand-induced structural changes of Oligobrachia Hb are quite different from those of the well-studied mollusk Hb having the same EF-dimer structure. The structural rearrangements make the dodecameric subassembly form a tighter conformation than those of fully oxygenated or CO-liganded dodecamer structure.

Direct evidence that extracellular giant hemoglobin is produced in chloragogen tissues in a beard worm, Oligobrachia mashikoi (Frenulata, Siboglinidae, Annelida).

In Oligobrachia mashikoi, a mouthless and gutless polychaete known as a beard worm, sites of production of extra-cellular giant hemoglobin were examined with whole-mount in-situ hybridization and semi-quantitative RT-PCR. An RNA probe was prepared from mRNA of the A2-globin subunit. Clear signals were obtained from a peritoneal membrane covering the trophosome in the posterior body in all seven individuals examined in this study. In addition, weak signals were observed in the peritoneal membrane covering tissues in the middle part of the body in some individuals. Furthermore, in one individual, signals were obtained in complicated bodies invaginated into the dorsal vessel from a peritoneal membrane that also released signals. The results of RT-PCR regarding the expression levels of four kinds of globin-subunit genes suggest that the main site of hemoglobin production is the peritoneal membrane in the posterior body.

Oxygenation properties of extracellular giant hemoglobin from Oligobrachia mashikoi.

Oxygenation properties of hemoglobin (Hb) from Oligobrachia mashikoi were extensively investigated. Compared to human Hb, Oligobrachia Hb showed a high oxygen affinity (P(50)=1.4 mmHg), low cooperativity (n =1.4), and a small Bohr effect (deltaH(+)=-0.28) at pH 7.4 in the presence of minimum salts. Addition of NaCl caused no change in the oxygenation properties of Oligobrachia Hb, indicating that Na(+) and Cl(-) had no effect. Mg(2+) and Ca(2+) remarkably increased the oxygen affinity and cooperativity. The dependence of the oxygen affinity on Ca(2+) concentration indicated that ca. 0.6 Ca(2+) per heme is bound to the protein moiety upon oxygen binding. CO(2) and a polyanion, inositol hexaphosphate, showed a null effect on the oxygenation properties. Thus, unlike the vertebrate Hbs, but like the annelid extracellular Hbs, the oxygen binding properties of Oligobrachia Hb are regulated by divalent cations which preferentially bind to the oxy form.

Structure of an extracellular giant hemoglobin of the gutless beard worm Oligobrachia mashikoi.

Mouthless and gutless marine animals, pogonophorans and vestimentiferans, obtain their nutrition solely from their symbiotic chemoautotrophic sulfur-oxidizing bacteria. These animals have sulfide-binding 400-kDa and/or 3,500-kDa Hb, which transports oxygen and sulfide simultaneously. The symbiotic bacteria are supplied with sulfide by Hb of the host animal and use it to provide carbon compounds. Here, we report the crystal structure of a 400-kDa Hb from pogonophoran Oligobrachia mashikoi at 2.85-A resolution. The structure is hollow-spherical, composed of a total of 24 globins as a dimer of dodecamer. This dodecameric assemblage would be a fundamental structural unit of both 400-kDa and 3,500-kDa Hbs. The structure of the mercury derivative used for phasing provides insights into the sulfide-binding mechanism. The mercury compounds bound to all free Cys residues that have been expected as sulfide-binding sites. Some of the free Cys residues are surrounded by Phe aromatic rings, and mercury atoms come into contact with these residues in the derivative structure. It is strongly suggested that sulfur atoms bound to these sites could be stabilized by aromatic-electrostatic interactions by the surrounding Phe residues.

Crystallization and preliminary X-ray crystallographic analysis of extracellular giant hemoglobin from pogonophoran Oligobrachia mashikoi.

An extracellular giant hemoglobin of Oligobrachia mashikoi, composed of 24 globins with the molecular mass of approximately 400 kDa was crystallized in its intact form. Two crystal forms were obtained by the vapor-diffusion method. Form I crystals obtained using sodium acetate as a precipitant belong to the space group P6(1)22 or P6(5)22, with unit-cell parameters a=112.41, c=621.25 A, and diffracted X-rays beyond 3.0 A resolution. Form II crystals obtained using PEG 10000 as a precipitant belong to the space group R32, with unit-cell parameters a=111.50, c=276.84 A, and diffracted X-rays beyond 2.9 A resolution. The crystals are suitable for X-ray crystallography to determine the supramacromolecular assembly of this giant hemoglobin.

Purification, characterization and sequence analyses of the extracellular giant hemoglobin from Oligobrachia mashikoi.

We purified an extracellular hemoglobin with the molecular mass of ca. 440 kDa from the whole homogenates of Oligobrachia mashikoi (phylum Pogonophora) by a one-step gel-filtration. The preparation was pure to be crystallized. The P50 values of the hemoglobin and the fresh blood prepared from O. mashikoi were about 0.82 Torr and 0.9 Torr, respectively, which were much lower than the P50 value of human hemoglobin. However, the n values of the hemoglobin and the blood were about 1.2 and 1.1, respectively. Using the improved tricine SDS-PAGE, we could separate O. mashikoi hemoglobin into four kinds of the globin chains, A1, A2, B1 and B2, and succeeded for the first time in cloning and sequencing of the complete cDNA encoding B1 globin gene, in addition to A1, A2 and B2 globin genes in full length. We found that all globin genes have the extracellular signal sequences in each molecule and the distal His of the B1 globin chain is replaced to Gln. Finally, we constructed phylogenetic trees of the hemoglobins from Pogonophora, Vestimentifera and Annelida.

External morphology of the posterior end, the "opisthosoma", of the beard worm Oligobrachia mashikoi (Pogonophora).

The entire length of the beard worm, Oligobrachia mashikoi (Pogonophora), including the posterior end, the "opisthosoma" was collected successfully. This species is exclusive to Tsukumo Bay in Ishikawa Prefecture, Japan. Although the portion preceding the opisthosoma was similar to a fine filament, it abruptly assumed a shape similar to a shovel and appeared to be composed of many segmental structures. The number of segments exceeded 50. The dorsal side of the opisthosoma differed from that of the ventral side in morphology. The opisthosoma was equipped with 4 lines of setae arranged longitudinally and a sucker on the tip. When considering the fact that the Family Oligobrachiidae is the most primitive group of pogonophores, the external morphology of the opisthosoma is interesting as it may be reminiscent of the ancestral condition. This is the first report of the opisthosoma in Oligobrachiidae.