Alendronate does not prevent long bone fragility in an inactive rat model.

The lack of estrogen and inactivity are both important in the pathogenesis of osteoporosis in elderly women, and there have been no appropriate rodent studies to examine the effects of common bisphosphonates on these two components separately. We compared the efficacy of alendronate (ALN) on the long bones of aged female rats, which were sedentary, estrogen deficient, or both. The rats were either forced to remain in a sitting position or allowed to walk in standard cages with or without ALN administration. The 8-week experimental period began 5 weeks after ovariectomy or sham surgery. Parameters of the hindlimb bones were determined by a three-point bending test, peripheral quantitative computed tomography, microfocus computed tomography, confocal laser Raman microspectroscopy, and dynamic histomorphometry. Regardless of ovariectomy, ALN was ineffective against the deterioration of breaking stress caused by sitting even though the trabecular bone mineral density was significantly higher in the sitting-ALN groups. Toughness was significantly deficient in the ovariectomy sitting-ALN group. This was in agreement with the bone geometry with a greater marrow space. Sitting also increased the mineral-to-matrix ratio and the carbonate-to-phosphate ratio, both indicative of aged bone. A greater loss of proteinaceous amide intensity compared with mineral intensity resulted in an increased mineral-to-matrix ratio in the presence of ALN. Sitting resulted in deficits in the quality and the geometry of cortical bone, resulting in fragility. The use of bisphosphonates, such as ALN, may provide a therapy best suited for osteoporotic individuals whose daily activity is not limited.

Andreev reflection in graphene nanoribbons induced byd-wave superconductors.

Honeycomb and square lattices are combined as a tight-binding model to examine the Andreev reflection in graphene nanoribbons induced by a superconductor. The superconducting symmetry is assumed to be thed-wave. The zero-bias tunneling conductance peak, which is generally produced by thed-wave superconductor, is absent for the nanoribbons under conditions similar to those when a quantum wire is the normal conductor. For the anisotropic superconductivity, propagating modes appear in the superconductor even for biases below the top of the superconducting energy gap. Features appear in the conductance at the subgap population thresholds of these propagating modes as a finite-size effect of the lattice system. The surface Andreev bound states responsible for the zero-bias anomaly also cause transport resonances in the vicinity of the zero bias despite the aforementioned destruction of the anomaly. The conductance spectra revealing these excitation behaviors are fairly unchanged regardless of the presence of a hopping barrier at the interface with the superconductor. The insensitivity to the interface scattering highlights the fact that barrier-less situation cannot be realized for the model due to the heterogeneous lattice. Concerning specular Andreev reflection, the wavefunction parity gives rise to its blocking for single-mode zigzag-edged nanoribbons. The blocking is suppressed when the anisotropic superconductivity is asymmetric for the nanoribbons.

Role of symmetry in quantum blocking of Andreev reflection in graphene nanoribbons side-terminated by superconductors.

Andreev reflection can be forbidden by means of quantum interference when superconductors are attached at the side edges of graphene nanoribbons (GNRs). The blocking is restricted to single-mode nanoribbons having symmetric zigzag edges and is destroyed by the application of a magnetic field. These characteristics are shown to be the effects of the wavefunction parity on the Andreev retro and specular reflections. Not only the mirror symmetry of the GNRs but also symmetric coupling of the superconductors is required for the quantum blocking. The quasi-flat-band states around the Dirac point energy induced for armchair nanoribbons by adding carbon atoms at the nanoribbon edges do not cause the quantum blocking due to the lack of the mirror symmetry. Furthermore, the phase modulation by the superconductors is shown to be able to convert the quasi-flat dispersion for the edge states of zigzag nanoribbons to a quasi-vertical dispersion.

Mechanotransduction activates α5ß1 integrin and PI3K/Akt signaling pathways in mandibular osteoblasts.

It is unclear how bone cells at different sites detect mechanical loading and how site-specific mechanotransduction affects bone homeostasis. To differentiate the anabolic mechanical responses of mandibular cells from those of calvarial and long bone cells, we isolated osteoblasts from C57B6J mouse bones, cultured them for 1week, and subjected them to therapeutic low intensity pulsed ultrasound (LIPUS). While the expression of the marker proteins of osteoblasts and osteocytes such as alkaline phosphatase and FGF23, as well as Wnt1 and ß-catenin, was equally upregulated, the expression of mandibular osteoblast messages related to bone remodeling and apoptosis differed from that of messages of other osteoblasts, in that the messages encoding the pro-remodeling protein RANKL and the anti-apoptotic protein Bcl-2 were markedly upregulated from the very low baseline levels. Blockage of the PI3K and α(5)ß(1) integrin pathways showed that the mandibular osteoblast required mechanotransduction downstream of α(5)ß(1) integrin to upregulate expression of the proteins ß-catenin, p-Akt, Bcl-2, and RANKL. Mandibular osteoblasts thus must be mechanically loaded to preserve their capability to promote remodeling and to insure osteoblast survival, both of which maintain intact mandibular bone tissue. In contrast, calvarial Bcl-2 is fully expressed, together with ILK and phosphorylated mTOR, in the absence of LIPUS. The antibody blocking α(5)ß(1) integrin suppressed both the baseline expression of all calvarial proteins examined and the LIPUS-induced expression of all mandibular proteins examined. These findings indicate that the cellular environment, in addition to the tridermic origin, determines site-specific bone homeostasis through the remodeling and survival of osteoblastic cells. Differentiated cells of the osteoblastic lineage at different sites transmit signals through transmembrane integrins such as α(5)ß(1) integrin in mandibular osteoblasts, whose signaling may play a major role in controlling bone homeostasis.

Magnetotransport in graphene nanoribbons sandwiched by superconductors at side edges.

Magnetotransport properties of the graphene nanoribbons (GNR) that are in contact with superconductors at side edges are investigated numerically with respect to oscillations caused by the cyclotron motion. In terms of the modelling, the superconductors are incorporated as superconducting GNRs to make the Andreev reflection at the graphene-superconductor interface almost perfect. The classical commensurability oscillation appears at low magnetic fields where the cyclotron radius is larger than the width of the nanoribbons. A transition to the circumstance dominated by the quantum interference between Andreev- and normal-reflected components takes place when the Andreev reflection probability is reduced by introducing a barrier at the interface. The near perfection of the Andreev reflection enlarges the period of the oscillation associated with skipping orbits a few orders of magnitude in the quantum limit. Chaotic fluctuations emerge furthermore in the regime of Hofstadter's butterfly. The periodicity of a transmission modulation at the onset of the chaos is revealed to change continuously over eight orders of magnitude of the magnetic-field variation. The commensurability and edge-state oscillations are examined additionally for the situations with specular Andreev reflection.

Quantum magnetotransport oscillations in graphene nanoribbons coupled to superconductors.

Magnetotransport properties of zigzag and armchair graphene nanoribbons that are in contact with superconductors are investigated using a tight-binding model. The cyclotron orbital motion together with the quantum interference under the coexistence of Andreev and normal reflections gives rise to a number of oscillations in characteristic magnetic-field regimes when the superconducting coupling is weak. The oscillations become irregular and/or suppressed as the coupling is made strong. The period of the oscillations differs from that when a nonrelativistic two-dimensional electron gas is employed rather than the graphene sheet. The modifications of the oscillations are attributed to the phase shift associated with the reflection from the graphene-superconductor interface. The presence of a magnetic field suppresses the quantum blocking of Andreev transmission, which occurs for the edge mode of zigzag nanoribbons, in the same way regardless of it being induced by the Andreev retro- or specular reflection.

Prior treatment with vitamin K(2) significantly improves the efficacy of risedronate.

UNLABELLED: Prior 8-week treatment with menatetrenone, MK-4, followed by 8-week risedronate prevented the shortcomings of individual drugs and significantly increased the strength of ovariectomized ICR mouse femur compared to the ovariectomized (OVX) controls. Neither MK-4 following risedronate nor the concomitant administration may be recommended because they brought the least beneficial effect. INTRODUCTION: The objective of this study was to determine the best combinatory administration of risedronate at 0.25 mg/kg/day (R) with vitamin K(2) at approximately 100 microg MK-4/kg/day (K) to improve strength of osteoporotic mouse bone. METHODS: Thirteen-week-old ICR mice, ovariectomized at 9-week, were treated for 8 weeks with R, K, or R plus K (R/K), and then, either the treatment was withdrawn (WO) or switched to K or R in the case of R and K. After another 8 weeks, the mice were killed, and mechanical tests and analyses of femur properties by peripheral quantitative computed tomography, microfocus X-ray tube computed tomography, and confocal laser Raman microspectroscopy were carried out. RESULTS: The K to R femur turned out superior in parameters tested such as material properties, bone mineral density, BMC, trabecular structure, and geometry of the cortex. The increased cross-sectional moment of inertia, which occurred after K withdrawal, was prevented by risedronate in K to R. In addition to K to R, some properties of R to WO diaphysis and K to WO epiphysis were significantly better than OVX controls. CONCLUSION: Prior treatment with MK-4 followed by risedronate significantly increased femur strength in comparison to the OVX controls.

Cell-substratum attachment and cell surface hyaluronate of Rous sarcoma virus-transformed chondrocytes.

Hyaluronate is associated with the cell surface of cultured Rous sarcoma virus-transformed chondrocytes. Detachment of these cells from their substratum by a variety of reagents is accompanied by release of 75-100% of this hyaluronate into solution. Treatment of the cells with 200 U/ml protease-free Streptomyces hyaluronidase at 37 degrees C cause release of greater than 90% of the cell surface hyaluronate and complete cell detachment. Treatment with a lower concentration of Streptomyces hyaluronidase (30 U/ml) at 25 degrees C or a corresponding activity of testicular hyaluronidase gives similar results, but only in the presence of mM EGTA. Treatment with the lower activities of either hyaluronidase or with 1 mM EGTA alone release only approximately 45% of the cell surface hyaluronate and does not cause significant cell detachment. It is concluded that there are two populations of cell surface hyaluronate differing in their accessibility or their resistance to dissociation from other components of the cell surface. It is proposed that the less readily released fraction is located between the transformed chondrocyte surface and substratum and is necessary for their interaction.

Commensurability and quantum interference magnetotransport oscillations in a two-dimensional electron gas sandwiched by superconductors.

Magnetic-field dependence of the transport properties of ballistic two-dimensional electrons in a planar superconductor-normal-conductor-superconductor structure is numerically investigated. In the circumstance where the Andreev reflection from the normal-conductor-superconductor interfaces is almost perfect, two oscillatory behaviors occur for magnetic fields higher and lower than that for the coincidence of the cyclotron diameter with the separation between the superconductors. The oscillation period for the former and latter cases is proportional to the magnetic field and inverse of the magnetic field, respectively. The low-field oscillation originates from commensurability-driven guiding of Andreev-reflected trajectories along the interfaces between the normal conductor and the superconductors. If the Andreev reflection probability is considerably less than unity, the commensurability oscillation is suppressed in amplitude and is dwarfed by additionally emerged oscillations originating from the quantum interference between the Andreev- and normal-reflected components.

Low-intensity pulsed ultrasound accelerates periodontal wound healing after flap surgery.

BACKGROUND AND OBJECTIVE: A study was conducted to evaluate the effects of low-intensity pulsed ultrasound on wound healing in periodontal tissues after mucoperiosteal flap surgery. MATERIAL AND METHODS: Bony defects were surgically produced bilaterally at the mesial roots of the mandibular fourth premolars in four beagle dogs. The flaps were repositioned to cover the defects and sutured after scaling and planing of the root surface to remove cementum. The affected area in the experimental group was exposed to low-intensity pulsed ultrasound, daily for 20 min, for a period of 4 wk from postoperative day 1 using a probe, 13 mm in diameter. On the control side, no ultrasound was emitted from the probe placed contralaterally. After the experiment, tissue samples were dissected out and fixed in 10% formalin for histological and immunohistochemical analyses. RESULTS: The experimental group showed that the processes in regeneration of both cementum and mandibular bone were accelerated by low-intensity pulsed ultrasound compared with the control group. In addition, the expression level of heat shock protein 70 was higher in the gingival epithelial cells of the low-intensity pulsed ultrasound-treated tooth. CONCLUSION: Our results suggest that osteoblasts, as well as cells in periodontal ligament and gingival epithelium, respond to mechanical stress loaded by low-intensity pulsed ultrasound, and that ultrasound accelerates periodontal wound healing and bone repair.

RNA recognition by the human polyadenylation factor CstF.

Polyadenylation of mammalian mRNA precursors requires at least two signal sequences in the RNA: the nearly invariant AAUAAA, situated 5' to the site of polyadenylation, and a much more variable GU- or U-rich downstream element. At least some downstream sequences are recognized by the heterotrimeric polyadenylation factor CstF, although how, and indeed if, all variations of this diffuse element are bound by a single factor is unknown. Here we show that the RNP-type RNA binding domain of the 64-kDa subunit of CstF (CstF-64) (64K RBD) is sufficient to define a functional downstream element. Selection-amplification (SELEX) experiments employing a glutathione S-transferase (GST)-64K RBD fusion protein selected GU-rich sequences that defined consensus recognition motifs closely matching those present in natural poly(A) sites. Selected sequences were bound specifically, and with surprisingly high affinities, by intact CstF and were functional in reconstituted, CstF-dependent cleavage assays. Our results also indicate that GU- and U-rich sequences are variants of a single CstF recognition motif. For comparison, SELEX was performed with a GST fusion containing the RBD from the apparent yeast homolog of CstF-64, RNA15. Strikingly, although the two RBDs are almost 50% identical and yeast poly(A) signals are at least as degenerate as the mammalian downstream element, a nearly invariant 12-base U-rich sequence distinct from the CstF-64 consensus was identified. We discuss these results in terms of the function and evolution of mRNA 3'-end signals.

Complex protein interactions within the human polyadenylation machinery identify a novel component.

Polyadenylation of mRNA precursors is a two-step reaction requiring multiple protein factors. Cleavage stimulation factor (CstF) is a heterotrimer necessary for the first step, endonucleolytic cleavage, and it plays an important role in determining the efficiency of polyadenylation. Although a considerable amount is known about the RNA binding properties of CstF, the protein-protein interactions required for its assembly and function are poorly understood. We therefore first identified regions of the CstF subunits, CstF-77, CstF-64, and CstF-50, required for interaction with each other. Unexpectedly, small regions of two of the subunits participate in multiple interactions. In CstF-77, a proline-rich domain is necessary not only for binding both other subunits but also for self-association, an interaction consistent with genetic studies in Drosophila. In CstF-64, a small region, highly conserved in metazoa, is responsible for interactions with two proteins, CstF-77 and symplekin, a nuclear protein of previously unknown function. Intriguingly, symplekin has significant similarity to a yeast protein, PTA1, that is a component of the yeast polyadenylation machinery. We show that multiple factors, including CstF, cleavage-polyadenylation specificity factor, and symplekin, can be isolated from cells as part of a large complex. These and other data suggest that symplekin may function in assembly of the polyadenylation machinery.

Sequences downstream of AAUAAA signals affect pre-mRNA cleavage and polyadenylation in vitro both directly and indirectly.

To investigate the role of sequences lying downstream of the conserved AAUAAA hexanucleotide in pre-mRNA cleavage and polyadenylation, deletions or substitutions were constructed in polyadenylation signals from simian virus 40 and adenovirus, and their effects were assayed in both crude and fractionated HeLa cell nuclear extracts. As expected, these sequences influenced the efficiency of both cleavage and polyadenylation as well as the accuracy of the cleavage reaction. Sequences near or upstream of the actual site of poly(A) addition appeared to specify a unique cleavage site, since their deletion resulted, in some cases, in heterogeneous cleavage. Furthermore, the sequences that allowed the simian virus 40 late pre-RNA to be cleaved preferentially by partially purified cleavage activity were also those at the cleavage site itself. Interestingly, sequences downstream of the cleavage site interacted with factors not directly involved in catalyzing cleavage and polyadenylation, since the effects of deletions were substantially diminished when partially purified components were used in assays. In addition, these sequences contained elements that could affect 3'-end formation both positively and negatively.

Multiple forms of poly(A) polymerases purified from HeLa cells function in specific mRNA 3'-end formation.

Poly(A) polymerases (PAPs) from HeLa cell cytoplasmic and nuclear fractions were extensively purified by using a combination of fast protein liquid chromatography and standard chromatographic methods. Several forms of the enzyme were identified, two from the nuclear fraction (NE PAPs I and II) and one from the cytoplasmic fraction (S100 PAP). NE PAP I had chromatographic properties similar to those of S100 PAP, and both enzymes displayed higher activities in the presence of Mn2+ than in the presence of Mg2+, whereas NE PAP II was chromatographically distinct and had approximately equal levels of activity in the presence of Mn2+ and Mg2+. Each of the enzymes, when mixed with other nuclear fractions containing cleavage or specificity factors, was able to reconstitute efficient cleavage and polyadenylation of pre-mRNAs containing an AAUAAA sequence element. The PAPs alone, however, showed no preference for precursors containing an intact AAUAAA sequence over a mutated one, providing further evidence that the PAPs have no intrinsic ability to recognize poly(A) addition sites. Two additional properties of the three enzymes suggest that they are related: sedimentation in glycerol density gradients indicated that the native size of each enzyme is approximately 50 to 60 kilodaltons, and antibodies against a rat hepatoma PAP inhibited the ability of each enzyme to function in AAUAAA-dependent polyadenylation.

A multicomponent complex is required for the AAUAAA-dependent cross-linking of a 64-kilodalton protein to polyadenylation substrates.

A 64-kilodalton (kDa) polypeptide that is cross-linked by UV light specifically to polyadenylation substrate RNAs containing a functional AAUAAA element has been identified previously. Fractionated HeLa nuclear components that can be combined to regenerate efficient and accurate polyadenylation in vitro have now been screened for the presence of the 64-kDa protein. None of the individual components contained an activity which could generate the 64-kDa species upon UV cross-linking in the presence of substrate RNA. It was necessary to mix two components, cleavage stimulation factor and specificity factor, to reconstitute 64-kDa protein-RNA cross-linking. The addition of cleavage factors to this mixture very efficiently reconstituted the AAUAAA-specific 64-kDa protein-RNA interaction. The 64-kDa protein, therefore, is present in highly purified, reconstituted polyadenylation reactions. However, it is necessary to form a multicomponent complex to efficiently cross-link the protein to a substrate RNA.

The Drosophila homologue of the 64 kDa subunit of cleavage stimulation factor interacts with the 77 kDa subunit encoded by the suppressor of forked gene.

During mRNA 3' end formation, cleavage stimulation factor (CstF) binds to a GU-rich sequence downstream from the polyadenylation site and helps to stabilise the binding of cleavage-polyadenylation specificity factor (CPSF) to the upstream poly-adenylation sequence (AAUAAA). The 64 kDa subunit of CstF (CstF-64) contains an RNA binding domain and is responsible for the RNA binding activity of CstF. It interacts with CstF-77, which in turn interacts with CPSF. The Drosophila suppressor of forked gene encodes a homologue of CstF-77, and mutations in it affect mRNA 3' end formation in vivo. A Drosophila homologue for CstF-64 has now been isolated, both through homology with the human protein and through protein-protein interaction in yeast with the suppressor of forked gene product. Alignment of CstF-64 homologues shows that the proteins have a conserved N-terminal 200 amino acids, the first half of which is the RNA binding domain with the second half likely to contain the CstF-77 interaction domain; a central region variable in length and rich in glycine, proline and glutamine residues and containing an unusual degenerate repeat motif; and then a conserved C-terminal 50 amino acids. In Drosophila, the CstF-64 gene has a single 63 bp intron, is transcribed throughout development and probably corresponds to l(3)91Cd.

Antitumor and immunosuppressive effects of mycophenolic acid derivatives.

Thirth-three derivatives of mycophenolic acid obtained by modifying phenolic hydroxyl and/or carboxyl groups were examined for antitumor and immunosuppressive activities. Some compounds showed more potent antitumor activities to L1210 leukemia and Ehrlich solid tumor than did mycophenolic acid. Most of these suppressed the production of antibody against sheep red blood cells in mice as strongly as did the parent substance. Correlation between antitumor and immunosuppressive activities was generally observed. However, a few compounds possessed a potent antitumor activity with less or no immunosuppressive activity.

In vivo generation of tumor-specific cytotoxic T-cells in the regional lymph node: effect of subcutaneous inoculation of mls-disparate spleen cells around implanted tumors in mice.

Generation of immunological resistance to a B-cell tumor was attempted by inoculating histoincompatible spleen cells around the sites of implanted tumor cells. Syngeneic hybridomas developed in the regional area of 96% of the control mice but in 4% of the mice that had received s.c. inoculation of H-2-matched, mls-disparate spleen cells. The regional lymph node cells of the mice in which tumors did not develop showed direct cytotoxicity against the hybridoma cells. This cytotoxicity was sensitive to treatment with the monoclonal anti-Thy-1.2 and complement and was specific for the tumor cells. After s.c. inoculation of mls-disparate spleen cells, cells of the regional lymph node were shown to produce interleukin 2. Primary cultures of the recipient lymph node cells and the donor spleen cells in the presence of T-cell growth factors showed that the tumor-specific cytotoxic T-lymphocytes were derived from the donor spleen cells. These results strongly suggest that on injection of mls antigen-disparate spleen cells, injected splenic T-cells specific for the tumor developed into functional cytotoxic T-lymphocytes with the help of interleukin 2 which was produced by the mixed lymphocyte reaction in vivo and thus prevented tumor growth. The possibility of clinical application of this procedure in the immunotherapy of neoplasms is discussed.

Klein tunneling of helical edge states in narrow strips of a two-dimensional topological insulator.

The quantum transmission of helical edge states across a square potential barrier is numerically investigated in narrow channels of a two-dimensional topological insulator. Although the transmission probability in general decreases when a potential offset is introduced in the middle of the channels, the transmission remains almost perfect regardless of the amplitude and length of the potential offset when the hybridization energy gap is closed by tuning the off-diagonal spin-orbit terms in the effective four-band Hamiltonian. The approximate absence of scattering resembling the Klein tunneling, where the transmission is unimpeded as an electron propagates relativistically as a hole in the barrier without decay, improves further when an interference condition is satisfied within the barrier. The dependence of the residual reflection on the Fermi level reveals anomalous characteristics in the Klein tunneling regime.

Primary structure of human hepatocellular carcinoma-associated aldehyde dehydrogenase.

Tumor-associated aldehyde dehydrogenase (T-ALDH) is strongly expressed in hepatocellular carcinoma (HCC) but undetectable in normal liver. In the present study, this enzyme from human HCC, HCC T-ALDH, was purified and the partial amino acid sequences (384 residues) determined by direct protein sequencing matched the amino acid sequence (453 residues) deduced from cloned HCC T-ALDH cDNAs with an open reading frame. The coding sequences of HCC T-ALDH cDNA, human stomach ALDH3A1 cDNA [Hsu et al., J. Biol. Chem. 267 (1992) 3030-3037] and human squamous cell carcinoma (SCC) T-ALDH cDNA (Schuuring et al., GenBank I.D. M74542) matched one another except for discrepancies at four positions, with consequent P12R, I27F and S134A substitutions. R and A were found in HCC and SCC T-ALDHs, whereas P and S were present in stomach ALDH3A1. To confirm that these discrepancies would have general occurrence, coding sequences of HCC T-ALDH cDNAs from six patients and stomach ALDH3A1 cDNAs from two individuals were examined and all were found to encode ALDH3A1 having R, I and A at protein positions 12, 27 and 134, respectively, indicating HCC T-ALDH to be variant ALDH3A1 which is common in human stomach tissues.