Inhibition of GSK-3β Rescues the Impairments in Bone Formation and Mechanical Properties Associated with Fracture Healing in Osteoblast Selective Connexin 43 Deficient Mice

Connexin 43 (Cx43) is the most abundant gap junction protein in bone and is required for osteoblastic differentiation and bone homeostasis. During fracture healing, Cx43 is abundantly expressed in osteoblasts and osteocytes, while Cx43 deficiency impairs bone formation and healing. In the present study we selectively deleted Cx43 in the osteoblastic lineage from immature osteoblasts through osteocytes and tested the hypothesis that Cx43 deficiency results in delayed osteoblastic differentiation and impaired restoration of biomechanical properties due to attenuated β-catenin expression relative to wild type littermates. Here we show that Cx43 deficiency results in alterations in the mineralization and remodeling phases of healing. In Cx43 deficient fractures the mineralization phase is marked by delayed expression of osteogenic genes. Additionally, the decrease in the RankL/ Opg ratio, osteoclast number and osteoclast size suggest decreased osteoclast bone resorption and remodeling. These changes in healing result in functional deficits as shown by a decrease in ultimate torque at failure. Consistent with these impairments in healing, β-catenin expression is attenuated in Cx43 deficient fractures at 14 and 21 days, while Sclerostin (Sost) expression, a negative regulator of bone formation is increased in Cx43cKO fractures at 21 days, as is GSK-3β, a key component of the β-catenin proteasomal degradation complex. Furthermore, we show that alterations in healing in Cx43 deficient fractures can be rescued by inhibiting GSK-3β activity using Lithium Chloride (LiCl). Treatment of Cx43 deficient mice with LiCl restores both normal bone formation and mechanical properties relative to LiCl treated WT fractures. This study suggests that Cx43 is a potential therapeutic target to enhance fracture healing and identifies a previously unknown role for Cx43 in regulating β-catenin expression and thus bone formation during fracture repair.     

Impact on energy metabolism of quantitative and functional cyclosporine-induced damage of kidney mitochondria

In this study we have measured, under experimental conditions which maintained efficient coupling, respiratory intensity, respiratory control, oxidative phosphorylation capacity and protonmotive force. Succinate cytochrome-c reductase and cytochrome-c oxidase activities were also studied. These investigations were carried out using kidney mitochondria from cyclosporine-treated rats (in vivo studies) and from untreated rats in the presence of cyclosporine (in vitro studies). Inhibition of respiratory intensity by cyclosporine did not exceed 21.1% in vitro and 15.9% in vivo. Since there was no in vitro inhibition of succinate cytochrome-c reductase and cytochrome-c oxidase activities, the slowing of electron flow observed can be interpreted as a consequence of an effect produced by cyclosporine between cytochromes b and c₁. Cyclosporine had no effect on respiratory control either in vitro or in vivo. Statistically significant inhibition of the oxidative phosphorylation was observed both in vitro (6.6%) and in vivo (12.1%). Moreover, cyclosporine did not induce any change of membrane potential either in vivo or in vitro. Our findings show that cyclosporine is neither a protonophore, nor a potassium ionophore. In cyclosporine-treated rats we noticed a decrease of protein in subcellular fraction, including the mitochondrial fraction. The role of the inhibition respiratory characteristics by cyclosporine in nephrotoxicity in vivo must take account of these two parameters: inhibition of the respiratory characteristics measured in vitro and diminution of mitochondrial protein in cyclosporine-treated rats.     

Epidermal-growth-factor-stimulated phosphorylation of calpactin II in membrane vesicles shed from cultured A-431 cells.

Membrane vesicles shed from intact A-431 epidermoid carcinoma cells and harvested in the presence of Ca2+ contained epidermal-growth-factor (EGF) receptor/kinase substrates of apparent molecular masses 185, 85, 70, 55, 38 and 27 kDa. The 38 kDa substrate (p38) was recognized by an antibody that had been raised against the human placental EGF receptor/kinase substrate calpactin II (lipocortin I). The A-431 and placental substrates, isolated by immunoprecipitation after phosphorylation in situ, yielded identical phosphopeptide maps upon limited proteolytic digestion with each of five different enzymes. The A-431-cell vesicular p38 is therefore calpactin II. EGF treatment of the intact A-431 cells before inducing vesiculation was not necessary for the substrate to be present within the vesicles. Our data thus indicate that receptor internalization is not a prerequisite for receptor-mediated phosphorylation of calpactin II. The ability of the protein to function as a substrate for the receptor/kinase depended upon the continued presence of Ca2+ during the vesicle-isolation procedure. EGF-stimulated phosphorylation of calpactin II was much less pronounced in vesicles prepared from A-431 cells in the absence of Ca2+, although comparable amounts of the protein were detectable by immunoblotting. Calpactin II therefore appears to be sequestered in a Ca2+-modulated manner within shed vesicles, along with at least four other major targets for the EGF receptor/kinase. The vesicle preparation may be a useful model system in which to study the phosphorylation and function of potentially important membrane-associated substrates for the receptor.     

Functional and phenotypic modifications induced by IL-4, as single agent or in combination with IL-2, on PBMC preactivated in vivo by alpha-interferon + interleukin-2 therapy

The effect of human IL-4, used as a single agent or in combination with low or high dose IL-2, upon LAK-cell proliferation and activation has been tested on PBMC from patients treated with alpha 2-IFN and IL-2. Four days in vitro culture with IL-4 did not induce any LAK-cell activation; IL-4 induced the proliferation of CD3+ CD4+ T-cells, but decreased the percentage of NK cells in culture samples. When combined with high dose IL-2, IL-4 improved the recovery of MN cell without modification of T-cell subsets; however, IL-4 had no major effect on IL-2-induced NK or LAK cell activity. The combination of IL-4 and low dose IL-2 still significantly improved the total MN cell recovery but did not modify the distribution of T and NK lymphocytes; IL-4 inhibited low dose IL-2-induced NK and LAK cell activity, and increased the BL-esterase activity induced by high or low dose IL-2. The combination of IL-4 and IL-2 did not induce any large variation in the percentage of IL-2R (p55) expressing cells. In all tested conditions, IL-2R (p55) was mainly expressed on CD4+ T cells; less than 2% of the cells coexpressed the NK cell marker CD56 and IL-2R (p55). The effect of IL-4 upon IL-2-induced LAK cell expansion is thus very different on PBMC pre-activated in vivo by alpha IFN + IL-2 therapy than on PBMC pre-treated in vitro with IL-2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thidiazuron-induced adventitious shoot regeneration of sweetpotato (Ipomoea batatas)

Adventitious shoots of sweetpotato (Ipomoea batatas L. Lam.) were produced in vitro using a two-stage culture method. Petiole explants were incubated on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxy acetic acid (0.2 mg·liter^−1) for 3 d, and transferred to MS medium with thidiazuron (0 to 0.4 mg·liter^−1). Shoot regeneration was observed in most explants (78.2%) of genotype PI 318846-3 within 28 days when cultured on thidiazuron at 0.2 mg·liter^−1. Histological studies of cultured petiole explants showed meristematic activity within cells of vascular bundles and throughout the ground tissue. Explants isolated from apical leaves exhibited higher shoot regeneration frequency than those isolated from the basal portion of the shoot. Leaf lamina explants exhibited lower frequency of regeneration than petiole explants. In contrast to thidiazuron, the use of zeatin riboside, and kinetin resulted in a lower frequency of shoot regeneration although more sweetpotato genotypes could be regenerated using either of these two cytokinins. The sweetpotato plants regenerated using thidiazuron grew vigorously and rooted easily when transferred to the greenhouse.     

A Novel Photoaffinity Ligand for Kainate Sites Labels Two Polypeptides with Molecular Mass 45 and 33.5 kDa in Chick Cerebellum

Abstract: The synthesis of (2 S ,3 S ,4 S )-4-[1-(4-azidobenzamidomethyl)ethenyl]-2-carboxy-3-pyrrolidineacetic acid (ABCPA) is described. This novel kainic acid analogue, bearing a photolabile functionality on the isopropenyl side chain, was proven to be a good inhibitor of [³H]CNQX and [³H]kainic acid binding on chick cerebellar membranes. [³H]ABCPA was photoaffinity cross-linked on the membrane fraction of chick cerebellum. Electrophoretic analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two major radioactive bands with apparent molecular masses of 45 and 33.5 kDa. [³H]ABCPA incorporation in both bands was completely blocked by 2 m M CNQX. When photoaffinity labeling was performed in the presence of 2 m M kainic acid, incorporation of [³H]ABCPA was blocked by ∼70% in the 45-kDa band and by 18% in the 33.5-kDa band. Incorporation of radioactivity in both bands was blocked by ∼30% with 10 m M glutamate.     

Transfection of the mouse ICAM-1 gene into murine neuroblastoma enhances susceptibility to lysis,reduces in vivo tumorigenicity and decreases ICAM-2-dependent killing

We determined the expression of intercellular adhesion molecules (ICAM) on neuro-2a cells in order to evaluate whether they were involved in cytolysis of murine neuroblastoma. Fluorescence-activated cell sorting analysis revealed that the control neomycin-resistance-genetransduced line (neuro-2a/LN) had poor expression of ICAM-1 (mean channel fluorescence, MCF=3.7). An ICAM-1-positive transfectant of neuro-2a (neuro-2a/ICAM-1⁺) (CMF=64.3) was generated to evaluate directly the role of this adhesion molecule in cytolysis. Neuro-2a/ICAM-1⁺ was more sensitive to LAK killing (69.7% at an effector-to-target ratio of 1001) compared to neuro-2a/LN (48.6%) (P<0.001). Blocking of neuro-2a/LN and neuro-2a/ICAM-1⁺ lysis with anti-ICAM-1 monoclonal antibodies (mAbs) did not account for all the LFA-1-dependent killing. These data indicate that even in neuro-2a/ICAM-1⁺ cells, other LFA-1 ligands participated in the effector-target interaction. Therefore, we examined these cell lines for ICAM-2 expression. Both neuro-2a/LN and neuro-2a/ICAM-1⁺ lines expressed ICAM-2 (MCF=16.4 and 16.5). ICAM-2 accounted for the majority of the LFA-1-dependent killing in the ICAM-1-negative target, neuro-2a/LN, while ICAM-1 played a primary role in the cytolysis of the ICAM-1⁺ transfectant. Inhibition of lysis in the presence of anti-ICAM-1 and ICAM-2 mAbs was comparable to that seen with the addition of anti-LFA-1 mAb, indicating that other LFA-1 ligands were not involved in this system. ICAM-1 expression was associated with decreased in vivo tumorigenicity; mice inoculated with neuro-2a/ICAM-1⁺ cells had a significantly longer survival compared to those receiving neuro-2a/LN cells (median survival time 35.5 versus 24.5 days) (P<0.001). It is important to note that ICAM-1 transfection of murine neuroblastoma did not alter its metastatic potential. We conclude that transfection of mouse neuroblastome with ICAM-1 increases its sensitivity to in vitro lysis and reduces its in vivo tumorgenicity. In ICAM-1-negative murine neuroblastoma cells, ICAM-2 plays a primary role in cell-mediated lysis.This work was supported in part by the Children's Cancer Research Fund, the Minnesota Medical Foundation, the Viking Children's Fund and NIH grants PO1-CA-21737, NO1-AI-85002. E. K. is a recipient of the Irvine McQuarrie Research Scholar Award and B. R. B. a recipient of the Edward Mallinkrodt Foundation Scholar Award     

Environmental responses of plants and ecosystems as predictors of the impact of global change

An understanding of plant responses to fluctuations in environment is critical to predictions of plant and ecosystem responses to climate change. In the northern hemisphere, the northern limits of distribution of major biomes are probably determined by the tolerance of their dominant physiognomic types (e.g., deciduous hardwood trees) to minimum winter temperatures and can thus be predicted from long-term patterns of temperature fluctuations. At a more detailed level, the responses of functional groups of plants to altered climate can be predicted from their known responses to fluctuations in soil resources (nutrients and water) and the expected effect of climatic change on these soil resources. Laboratory and field experiments demonstrate the feasibility of this approach.     

Molecular characterization of mouse-virulent poliovirus type 1 Mahoney mutants: involvement of residues of polypeptides VP1 and VP2 located on the inner surface of the capsid protein shell.

Most poliovirus (PV) strains, including PV PV-1/Mahoney, are unable to cause paralysis in mice. Determinants for restriction of PV-1/Mahoney in mice have been identified by manipulating PV-1 cDNA and located on the viral capsid protein VP1. These determinants consist of a highly exposed amino acid sequence on the capsid surface corresponding to the B-C loop (M. Murray, J. Bradley, X. Yang, E. Wimmer, E. Moss, and V. Racaniello, Science 241:213-215, 1988; A. Martin, C. Wychowski, T. Couderc, R. Crainic, J. Hogle, and M. Girard, EMBO J. 7:2839-2847, 1988) and of residues belonging to the N-terminal sequence located on the inner surface of the protein shell (E. Moss and V. Racaniello, EMBO J. 10:1067-1074, 1991). Using an in vivo approach, we isolated two mouse-neurovirulent PV-1 mutants in the mouse central nervous system after a single passage of PV-1/Mahoney inoculated by the intracerebral route. Both mutants were subjected to two additional passages in mice, plaque purified, and subsequently characterized. The two cloned mutants, Mah-NK13 and Mah-NL32, retained phenotypic characteristics of the parental PV-1/Mahoney, including epitope map, heat lability, and temperature sensitivity. Mah-NK13 exhibited slightly smaller plaques than did the parental virus. The nucleotide sequences of the mutant genomes were determined, and mutations were identified. Mutations were independently introduced into the parental PV-1/Mahoney genome by single-site mutagenesis. Mutated PV-1/Mahoney viruses were then tested for their neurovirulence in mice. A single amino acid substitution in the capsid proteins VP1 (Thr-22-->Ile) and VP2 (Ser-31-->Thr) identified in the Mah-NK13 and Mah-NL32 genomes, respectively, conferred the mouse-virulent phenotype to the mouse-avirulent PV-1/Mahoney. Ile-22 in VP1 was responsible for the small-plaque phenotype of Mah-NK13. Both mutations arose during the first passage in the mouse central nervous system. We thus identified a new mouse adaptation determinant on capsid protein VP1, and we showed that at least one other capsid protein, VP2, could also express a mouse adaptation determinant. Both determinants are located in the inside of the three-dimensional structure of the viral capsid. They may be involved in the early steps of mouse nerve cell infection subsequent to receptor attachment.