RESUMO
Soft tissue reconstruction often requires multiple surgical procedures that can result in scars and disfiguration. Facial soft tissue reconstruction represents a clinical challenge because even subtle deformities can severely affect an individual's social and psychological function. We therefore developed a biosynthetic soft tissue replacement composed of poly(ethylene glycol) (PEG) and hyaluronic acid (HA) that can be injected and photocrosslinked in situ with transdermal light exposure. Modulating the ratio of synthetic to biological polymer allowed us to tune implant elasticity and volume persistence. In a small-animal model, implanted photocrosslinked PEG-HA showed a dose-dependent relationship between increasing PEG concentration and enhanced implant volume persistence. In direct comparison with commercial HA injections, the PEG-HA implants maintained significantly greater average volumes and heights. Reversibility of the implant volume was achieved with hyaluronidase injection. Pilot clinical testing in human patients confirmed the feasibility of the transdermal photocrosslinking approach for implantation in abdomen soft tissue, although an inflammatory response was observed surrounding some of the materials.
Assuntos
Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/farmacologia , Reagentes de Ligações Cruzadas/farmacologia , Luz , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Colágeno Tipo I/metabolismo , Derme/efeitos dos fármacos , Derme/efeitos da radiação , Humanos , Ácido Hialurônico/química , Implantes Experimentais , Teste de Materiais , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/efeitos da radiação , Projetos Piloto , Polietilenoglicóis/química , Ratos , Reologia/efeitos dos fármacosRESUMO
Centromere-associated protein-E (CENP-E) is a kinetochore-associated mitotic kinesin that is thought to function as the key receptor responsible for mitotic checkpoint signal transduction after interaction with spindle microtubules. We have identified GSK923295, an allosteric inhibitor of CENP-E kinesin motor ATPase activity, and mapped the inhibitor binding site to a region similar to that bound by loop-5 inhibitors of the kinesin KSP/Eg5. Unlike these KSP inhibitors, which block release of ADP and destabilize motor-microtubule interaction, GSK923295 inhibited release of inorganic phosphate and stabilized CENP-E motor domain interaction with microtubules. Inhibition of CENP-E motor activity in cultured cells and tumor xenografts caused failure of metaphase chromosome alignment and induced mitotic arrest, indicating that tight binding of CENP-E to microtubules is insufficient to satisfy the mitotic checkpoint. Consistent with genetic studies in mice suggesting that decreased CENP-E function can have a tumor-suppressive effect, inhibition of CENP-E induced tumor cell apoptosis and tumor regression.
Assuntos
Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Sarcosina/análogos & derivados , Sítio Alostérico , Animais , Antineoplásicos/química , Sítios de Ligação , Compostos Bicíclicos Heterocíclicos com Pontes/química , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Cães , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Técnicas In Vitro , Cinesinas/antagonistas & inibidores , Cinesinas/química , Cinesinas/metabolismo , Camundongos , Microtúbulos/metabolismo , Mitose/efeitos dos fármacos , Modelos Moleculares , Estrutura Molecular , Sarcosina/química , Sarcosina/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Structural changes in the mitotic arrest deficient protein 2 (Mad2) have been proposed to be essential for spindle checkpoint function. Current models for checkpoint activation propose that a C-Mad2-Mad1 core complex at unattached kinetochores is required for the structural activation through a process involving the interaction of two Mad2 conformers: a closed conformer bound to Mad1 or Cdc20 and an open conformer unbound to these ligands. To gain a molecular understanding of the mechanisms that accelerate the structural transition between the open and closed Mad2 conformations, we constructed a unique in vitro homogeneous Mad2 activity assay that specifically reports C-Mad2-Cdc20 formation. Using this assay we were are able to directly establish that (a) O-Mad2 transforms into a C-Mad2-Cdc20 complex >300-fold slower than unliganded C-Mad2, (b) a stable C-Mad2-Mad1 core complex catalyzes the transformation of O-Mad2 into a Cdc20-bound C-Mad2 complex, (c) a C-Mad2-Cdc20 complex can promote its own transformation of O-Mad2 into a Cdc20-bound C-Mad2 complex, and (d) the binding interaction between unliganded C-Mad2 and Cdc20 cannot be catalyzed by a C-Mad2-Mad1 core complex. Our data are consistent with the "Mad2 template" catalytic model in which a C-Mad2 template facilitates the binding of O-Mad2 to Cdc20 and supports a mechanism of C-Mad2-Cdc20 formation away from Mad1 containing kinetochores. Furthermore, our unique homogeneous Mad2 assay could be translated into a screening platform to identify small molecule drug-like compounds that directly modulate C-Mad2-Cdc20 formation.