RESUMEN
The purpose of this article is to quantify changes in thickness of the ligamentum flavum (LF) associated with motion of the cervical spine and to compare the thickness of the LF at each cervical level using kinetic magnetic resonance imaging (kMRI). Two hundred fifty-seven symptomatic patients (129 men; 128 women) underwent kMRI in neutral, flexion, and extension positions. Midsagittal images were digitally marked and electronically analyzed by spine surgeons. Thickness of LF in the cervical region from C2-3 to C7-T1 was measured in all three positions. LF at C7-T1 was significantly thicker than C2-3 to C6-7 in neutral, flexion, and extension positions (p < 0.05). LF was significantly thicker in extension than in flexion at C3-4 to C6-7. LF thickness increases with extension and decreases with flexion. LF is uniquely thick at C6-7 and at C7-T1 in the extension position, which may predispose these levels to cord compression syndromes and associated neuropathies.
RESUMEN
BACKGROUND AND PURPOSE: Autologous Iliac Crest Bone Grafting (ICBG) is considered the gold-standard graft choice for spinal arthrodesis; however, it is associated with donor site morbidity and a limited graft supply. Bone graft alternatives to replace autograft and augment arthrodesis are a topic of ongoing research. This article will review properties of Demineralized Bone Matrix (DBM) and review the evidence for its use, including animal models and human clinical trials. METHODS: A systematic and critical review of the English-language literature was conducted on Pubmed, Cochrane, CINAHL, and Google Scholar using search key terms such as 'Demineralized Bone Matrix', 'Spine' and 'Fusion'. Papers that were included were original research articles in peer-reviewed journals that investigated fusion outcomes. Scientific validity of articles was appraised using the PRISMA methodology. Articles were critically examined and compared according to study design, DBM type, outcomes, and results. Primary outcome of interest was fusion rate. Secondary outcomes included Oswestry Disability Index; Short Form-36 survey; Odom's criteria; Visual Analog Scale neurologic pain score; Japanese Orthopedic Association myelopathy score; Neck Disability and Ishihara Curvature Indices; and pseudarthrosis and surgical failure rates. RESULTS: Demineralized Bone Matrix has been evaluated in animal models and human clinical trials of spine fusion. Results of animal studies indicate variation in performance within and among DBM products. The majority of human clinical trials report high fusion rates when DBM is employed as a graft extender or a graft enhancer. Few prospective randomized controlled trials have been performed comparing DBM to autologous iliac crest bone graft in spine fusion. CONCLUSIONS: Although many animal and human studies demonstrate comparable efficacy of DBM when combined with autograft or compared to autograft alone, additional high level of evidence studies are required to clearly define the indications for its use in spine fusion surgeries and the appropriate patient population that will benefit from DBM.
Asunto(s)
Matriz Ósea/trasplante , Trasplante Óseo/métodos , Ilion/trasplante , Enfermedades de la Columna Vertebral/cirugía , Fusión Vertebral/métodos , Humanos , Ilion/citología , Resultado del TratamientoRESUMEN
FKBP12, the 12-kDa FK506-binding protein, is a ubiquitous abundant protein that acts as a receptor for the immunosuppressant drug FK506, binds tightly to intracellular calcium release channels and to the transforming growth factor beta (TGF-beta) type I receptor. We now demonstrate that cells from FKBP12-deficient (FKBP12(-/-)) mice manifest cell cycle arrest in G(1) phase and that these cells can be rescued by FKBP12 transfection. This arrest is mediated by marked augmentation of p21(WAF1/CIP1) levels, which cannot be further augmented by TGF-beta1. The p21 up-regulation and cell cycle arrest derive from the overactivity of TGF-beta receptor signaling, which is normally inhibited by FKBP12. Cell cycle arrest is prevented by transfection with a dominant-negative TGF-beta receptor construct. TGF-beta receptor signaling to gene expression can be mediated by SMAD, p38, and ERK/MAP kinase (extracellular signal-regulated kinase/mitogen-activated protein kinase) pathways. SMAD signaling is down-regulated in FKBP12(-/-) cells. Inhibition of ERK/MAP kinase fails to affect p21 up-regulation. By contrast, activated phosphorylated p38 is markedly augmented in FKBP12(-/-) cells and the p21 up-regulation is prevented by an inhibitor of p38. Thus, FKBP12 is a physiologic regulator of cell cycle acting by normally down-regulating TGF-beta receptor signaling.
Asunto(s)
Ciclo Celular/fisiología , Proteína 1A de Unión a Tacrolimus/fisiología , Animales , Secuencia de Bases , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Cartilla de ADN , Ratones , Ratones Noqueados , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Reacción en Cadena de la Polimerasa , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Proteína 1A de Unión a Tacrolimus/genética , Regulación hacia Arriba , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
FKBP12, a cis-trans prolyl isomerase that binds the immunosuppressants FK506 and rapamycin, is ubiquitously expressed and interacts with proteins in several intracellular signal transduction systems. Although FKBP12 interacts with the cytoplasmic domains of type I receptors of the transforming growth factor-beta (TGF-beta) superfamily in vitro, the function of FKBP12 in TGF-beta superfamily signalling is controversial. FKBP12 also physically interacts stoichiometrically with multiple intracellular calcium release channels including the tetrameric skeletal muscle ryanodine receptor (RyR1). In contrast, the cardiac ryanodine receptor, RyR2, appears to bind selectively the FKBP12 homologue, FKBP12.6. To define the functions of FKBP12 in vivo, we generated mutant mice deficient in FKBP12 using embryonic stem (ES) cell technology. FKBP12-deficient mice have normal skeletal muscle but have severe dilated cardiomyopathy and ventricular septal defects that mimic a human congenital heart disorder, noncompaction of left ventricular myocardium. About 9% of the mutants exhibit exencephaly secondary to a defect in neural tube closure. Physiological studies demonstrate that FKBP12 is dispensable for TGF-beta-mediated signalling, but modulates the calcium release activity of both skeletal and cardiac ryanodine receptors.
Asunto(s)
Isomerasas de Aminoácido/fisiología , Proteínas Portadoras/fisiología , Proteínas de Unión al ADN/fisiología , Cardiopatías Congénitas/etiología , Proteínas de Choque Térmico/fisiología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Anomalías Múltiples/embriología , Anomalías Múltiples/etiología , Anomalías Múltiples/genética , Activinas , Isomerasas de Aminoácido/deficiencia , Isomerasas de Aminoácido/genética , Animales , Encéfalo/anomalías , Encéfalo/embriología , Cardiomiopatía Dilatada/embriología , Cardiomiopatía Dilatada/etiología , Cardiomiopatía Dilatada/genética , Proteínas Portadoras/genética , Proteínas de Unión al ADN/genética , Femenino , Muerte Fetal , Eliminación de Gen , Cardiopatías Congénitas/embriología , Cardiopatías Congénitas/genética , Defectos de los Tabiques Cardíacos/embriología , Defectos de los Tabiques Cardíacos/etiología , Defectos de los Tabiques Cardíacos/genética , Proteínas de Choque Térmico/genética , Inhibinas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Transducción de Señal , Proteínas de Unión a Tacrolimus , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Reactive oxygen intermediates and nitric oxide modulate the contractile function of skeletal muscle fibers, possibly via direct interaction with the Ca2+ release channel. Oxidants produce disulfide bonds between subunits of the Ca2+ release channel tetramer, and this is accompanied by an increase in channel activity. The sulfhydryl alkylating agent N-ethylmaleimide has three distinct effects on Ca2+ release channel activity: first, channel activity is decreased (phase 1); then with continued exposure the activity is dramatically increased (phase 2); and finally, the channel is again inhibited (phase 3) (Aghdasi, B., Zhang, J. Z., Wu, Y., Reid, M. B., and Hamilton, S. L., (1997) J. Biol. Chem. 272, 3739-3749). Both H2O2 and nitric oxide (NO) block the phase 1 inhibitory effect of N-ethylmaleimide. NO donors, at concentrations that have no detectable effect on channel activity, block intersubunit cross-linking and prevent activation of the channel by the disulfide inducing agent, diamide. These findings support a model in which NO modulates the activity of the Ca2+ release channel by preventing oxidation of regulatory sulfhydryls. However, higher concentrations of NO donors activate the channel and produce intersubunit cross-links, supporting a bifunctional effect of NO on channel activity. Low NO concentrations prevent oxidation of the Ca2+ release channel whereas higher concentrations oxidize it.
Asunto(s)
Músculo Esquelético/metabolismo , Óxido Nítrico/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Alquilación , Animales , Etilmaleimida/metabolismo , Peróxido de Hidrógeno/farmacología , Membrana Dobles de Lípidos/metabolismo , Contracción Muscular , Oxidación-Reducción , Conformación Proteica , Conejos , Especies Reactivas de Oxígeno/metabolismo , Compuestos de Sulfhidrilo/metabolismoRESUMEN
The skeletal muscle Ca2+ release channel (RYR1), which plays a critical role in excitation-contraction coupling, is a homotetramer with a subunit molecular mass of 565 kDa. Oxidation of the channel increases its activity and produces intersubunit cross-links within the RYR1 tetramer (Aghdasi, B., Zhang, J., Wu, Y., Reid, M. B., and Hamilton, S. L. (1997) J. Biol. Chem. 272, 3739-3748). Alkylation of hyperreactive sulfhydryls on RYR1 with N-ethylmaleimide (NEM) inhibits channel function and blocks the intersubunit cross-linking. We used calpain and tryptic cleavage, two-dimensional SDS-polyacrylamide gel electrophoresis, N-terminal sequencing, sequence-specific antibody Western blotting, and [14C]NEM labeling to identify the domains involved in these effects. Our data are consistent with a model in which 1) diamide, an oxidizing agent, simultaneously produces an intermolecular cross-link between adjacent subunits within the RYR1 tetramer and an intramolecular cross-link within a single subunit; 2) all of the cysteines involved in both cross-links are in either the region between amino acids approximately 2100 and 2843 or the region between amino acids 2844 and 4685; 3) oxidation exposes a new calpain cleavage site in the central domain of the RYR1 (in the region around amino acid 2100); 4) sulfhydryls that react most rapidly with NEM are located in the N-terminal domain (between amino acids 426 and 1396); 5) alkylation of the N-terminal cysteines completely inhibits the formation of both inter- and intrasubunit cross-links. In summary, we present evidence for interactions between the N-terminal region and the putatively cytoplasmic central domains of RYR1 that appear to influence subunit-subunit interactions and channel activity.
Asunto(s)
Canales de Calcio/química , Canales de Calcio/metabolismo , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Estructura Secundaria de Proteína , Animales , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/aislamiento & purificación , Calpaína/metabolismo , Reactivos de Enlaces Cruzados , Diamida , Etilmaleimida/metabolismo , Etilmaleimida/farmacología , Membranas Intracelulares/metabolismo , Sustancias Macromoleculares , Modelos Estructurales , Peso Molecular , Proteínas Musculares/aislamiento & purificación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Conejos , Rianodina/metabolismo , Canal Liberador de Calcio Receptor de Rianodina , Retículo Sarcoplasmático/metabolismoRESUMEN
The effect of D-erythro-C18-sphingosine (sphingosine) and related compounds on the Ca(2+)-release channel (ryanodine binding protein) was examined on rabbit skeletal muscle membranes, on the purified ryanodine binding protein, and on the channel reconstituted into planar lipid bilayers. Sphingosine inhibited [3H]ryanodine binding to sarcoplasmic reticulum (SR) membranes in a dose-dependent manner similar to published results (R. A. Sabbadini, R. Betto, A. Teresi, G. Fachechi-Cassano, and G. Salviati. J. Biol. Chem. 267: 15475-15484, 1992). The sphingolipid also inhibited [3H]ryanodine binding to the purified ryanodine binding protein. Our results demonstrate that the inhibition of [3H]ryanodine binding by sphingosine is due to an increased rate of dissociation of bound [3H]ryanodine from SR membranes and a decreased rate of association of [3H]ryanodine to the high-affinity site. Unlike other modulators of the Ca(2+)-release channel, sphingosine can remove bound [3H]ryanodine from the high-affinity site within minutes. Sphingosine increased the rate of dissociation of [3H]ryanodine bound to a solubilized proteolytic fragment derived from the carboxy terminus of the ryanodine binding protein (cleavage at Arg4475). Sphingosine also inhibited the activity of the Ca(2+)-release channel incorporated into planar lipid bilayers. Taken together, the data provide evidence for a direct effect of sphingosine on the Ca(2+)-release channel. Sphingosine is a noncompetitive inhibitor at the high-affinity ryanodine binding site, and it interacts with a site between Arg4475 and the carboxy terminus of the Ca(2+)-release channel.
Asunto(s)
Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Músculo Esquelético/metabolismo , Esfingosina/farmacología , Animales , Membrana Dobles de Lípidos/metabolismo , Conejos , Rianodina/antagonistas & inhibidores , Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Esfingolípidos/farmacologíaRESUMEN
Excitation-contraction coupling in skeletal muscle is thought to involve a physical interaction between the alpha 1-subunit of the dihydropyridine receptor (DHPR) and the sarcoplasmic reticulum (SR) Ca(2+)-release channel (also known as the ryanodine receptor). Considerable evidence has accumulated to suggest that the cytoplasmic loop between domains II and III of the DHPR alpha 1-subunit is at least partially responsible for this interaction. Other parts of this subunit or other subunits may, however, contribute to the functional and/or structural coupling between these two proteins. A synthetic peptide corresponding to a conserved sequence located between amino acids 1487 and 1506 in the carboxy terminus of the alpha 1-subunit inhibits both [3H]ryanodine binding to skeletal and cardiac SR membranes and the activity of skeletal SR Ca(2+)-release channels reconstituted into planar lipid bilayers. A second, multiantigenic peptide synthesized to correspond to the same sequence inhibits both binding and channel activity at lower concentrations than the linear peptide. These peptides slow the rate at which [3H]ryanodine binds to its high-affinity binding site and decrease the rate at which [3H]ryanodine dissociates from this site. A third polypeptide synthesized in Escherichia coli and corresponding to amino acids 1381-1627 and encompassing the above sequence has similar effects. This portion of the alpha 1-subunit of the transverse tubule DHPR is therefore a candidate for contributing to the interaction of this protein with the Ca(2+)-release channel.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/química , Canales de Calcio/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Animales , Canales de Calcio/metabolismo , Canales de Calcio Tipo L , Membrana Dobles de Lípidos/metabolismo , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Conejos , Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMEN
Two sulfhydryl reagents, N-ethylmaleimide (NEM), an alkylating agent, and diamide, an oxidizing agent, were examined for effects on the skeletal muscle Ca2+ release channel. NEM incubated with the channel for increasing periods of time displays three distinct phases in its functional effects on the channel reconstituted into planar lipid bilayers; first it inhibits, then it activates, and finally it again inhibits channel activity. NEM also shows a three-phase effect on the binding of [3H]ryanodine by first decreasing binding (phase 1), followed by a recovery of the binding (phase 2), and then a final phase of inhibition (phase 3). In contrast, diamide 1) activates the channel, 2) enhances [3H]ryanodine binding, 3) cross-links subunits within the Ca2+ release channel tetramer, and 4) protects against phase 1 inhibition by NEM. All diamide effects can be reversed by the reducing agent, dithiothreitol. Diamide induces intersubunit dimer formation of both the full-length 565-kDa subunit of the channel and the 400-kDa generated by endogenous calpain digestion, suggesting that the cross-link does not involve sulfhydryls within the N-terminal 170-kDa fragment of the protein. NEM under phase 1 conditions blocks the formation of the intersubunit cross-links by diamide. In addition, single channels activated by diamide are further activated by the addition of NEM. Diamide either cross-links phase 1 sulfhydryls or causes a conformational change in the Ca2+ release channel which leads to inaccessibility of phase 1 sulfhydryls to NEM alkylation. The data presented here lay the groundwork for mapping the location of one of the sites of subunit-subunit contact in the Ca2+ release channel tetramer and for identifying the functionally important sulfhydryls of this protein.
Asunto(s)
Canales de Calcio/efectos de los fármacos , Diamida/farmacología , Etilmaleimida/farmacología , Animales , Canales de Calcio/metabolismo , Membrana Dobles de Lípidos/metabolismo , Peso Molecular , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Conejos , Rianodina/metabolismoRESUMEN
Neomycin is a potent inhibitor of skeletal muscle sarcoplasmic reticulum (SR) calcium release. To elucidate the mechanism of inhibition, the effects of neomycin on the binding of [3H]ryanodine to the Ca2+ release channel and on its channel activity when reconstituted into planar lipid bilayer were examined. Equilibrium binding of [3H]ryanodine was partially inhibited by neomycin. Inhibition was incomplete at high neomycin concentrations, indicating noncompetitive inhibition rather than direct competitive inhibition. Neomycin and [3H]ryanodine can bind to the channel simultaneously and, if [3H]ryanodine is bound first, the addition of neomycin will slow the dissociation of [3H]ryanodine from the high affinity site. Neomycin also slows the association of [3H]ryanodine with the high affinity binding site. The neomycin binding site, therefore, appears to be distinct from the ryanodine binding site. Dissociation of [3H]ryanodine from trypsin-treated membranes or from a solubilized 14 S complex is also slowed by neomycin. This complex is composed of polypeptides derived from the carboxyl terminus of the Ca2+ release channel after Arg-4475 (Callaway, C., Seryshev, A., Wang, J. P., Slavik, K., Needleman, D. H., Cantu, C., Wu, Y., Jayaraman, T., Marks, A. R., and Hamilton, S. L. (1994) J. Biol. Chem. 269, 15876-15884). The proteolytic 14 S complex isolated with ryanodine bound produces a channel upon reconstitution into planar lipid bilayers, and its activity is inhibited by neomycin. Our data are consistent with a model in which the ryanodine binding sites, the neomycin binding sites, and the channel-forming portion of the Ca2+ release channel are located between Arg-4475 and the carboxyl terminus.