RESUMO
OBJECTIVE: To describe a modification of the tibial tuberosity advancement (TTA) procedure that required tuberosity advancement in excess of 12 mm for the stabilization of cranial cruciate ligament (CrCL) deficient stifle joints. METHODS: Four large breed dogs with CrCL deficient stifle joints (one bilateral) underwent a modified TTA of 15 or 16 mm in order to obtain a patellar tendon angle of 90 degrees to the tibial plateau slope or common tangent between femur and tibia in the extended limb position. The desired TTA was achieved by displacing a 12-mm cage distally; this displacement distance was calculated from two similar triangles formed within the planned osteotomy site. An allogenous cancellous bone block placed proximal to the cage provided buttress support; a corticocancellous allograft filled the remainder of the gap. Tibial tuberosity fixation was performed as previously described. RESULTS: Healing of the osteotomy defects with incorporation of the cancellous block was observed at a mean of 8.6 weeks postoperatively. Normal return of limb function was reported in all of the dogs except for one dog that underwent revision surgery four months postoperatively for a continued lameness. Technical errors at the time of the original surgical procedure in this dog resulted in insufficient tuberosity advancement; additional advancement was performed, which resolved the lameness. CLINICAL SIGNIFICANCE: Results in this series suggest that our modification of the TTA, in order to advance the tuberosity in excess of 12 mm, could be successfully obtained using the currently available implants.
Assuntos
Lesões do Ligamento Cruzado Anterior , Ligamento Cruzado Anterior/cirurgia , Doenças do Cão/cirurgia , Marcha/fisiologia , Joelho de Quadrúpedes/cirurgia , Animais , Fenômenos Biomecânicos , Peso Corporal/fisiologia , Placas Ósseas/veterinária , Cruzamento , Cães/lesões , Cães/cirurgia , Seguimentos , Masculino , Osteotomia/métodos , Osteotomia/veterinária , Complicações Pós-Operatórias/veterinária , Estudos Prospectivos , Joelho de Quadrúpedes/patologia , Tíbia/patologia , Tíbia/cirurgia , Resultado do TratamentoRESUMO
Calcium entry into myocytes drives contraction of the embryonic heart. To prepare for the next contraction, myocytes must extrude calcium from intracellular space via the Na+/Ca2+ exchanger (NCX1) or sequester it into the sarcoplasmic reticulum, via the sarcoplasmic reticulum Ca2+-ATPase2 (SERCA2). In mammals, defective calcium extrusion correlates with increased intracellular calcium levels and may be relevant to heart failure and sarcoplasmic dysfunction in adults. We report here that mutation of the cardiac-specific NCX1 (NCX1h) gene causes embryonic lethal cardiac arrhythmia in zebrafish tremblor (tre) embryos. The tre ventricle is nearly silent, whereas the atrium manifests a variety of arrhythmias including fibrillation. Calcium extrusion defects in tre mutants correlate with severe disruptions in sarcomere assembly, whereas mutations in the L-type calcium channel that abort calcium entry do not produce this phenotype. Knockdown of SERCA2 activity by morpholino-mediated translational inhibition or pharmacological inhibition causes embryonic lethality due to defects in cardiac contractility and morphology but, in contrast to tre mutation, does not produce arrhythmia. Analysis of intracellular calcium levels indicates that homozygous tre embryos develop calcium overload, which may contribute to the degeneration of cardiac function in this mutant. Thus, the inhibition of NCX1h versus SERCA2 activity differentially affects the pathophysiology of rhythm in the developing heart and suggests that relative levels of NCX1 and SERCA2 function are essential for normal development.
Assuntos
Cálcio/metabolismo , Coração/embriologia , Coração/fisiopatologia , Morfogênese/fisiologia , Contração Miocárdica/fisiologia , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Cálcio/farmacologia , ATPases Transportadoras de Cálcio/genética , ATPases Transportadoras de Cálcio/metabolismo , Coração/efeitos dos fármacos , Humanos , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Mutação/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Trocador de Sódio e Cálcio/química , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/metabolismoRESUMO
The conserved CDC5 family of Myb-related proteins performs an essential function in cell cycle control at G(2)/M. Although c-Myb and many Myb-related proteins act as transcription factors, herein, we implicate CDC5 proteins in pre-mRNA splicing. Mammalian CDC5 colocalizes with pre-mRNA splicing factors in the nuclei of mammalian cells, associates with core components of the splicing machinery in nuclear extracts, and interacts with the spliceosome throughout the splicing reaction in vitro. Furthermore, genetic depletion of the homolog of CDC5 in Saccharomyces cerevisiae, CEF1, blocks the first step of pre-mRNA processing in vivo. These data provide evidence that eukaryotic cells require CDC5 proteins for pre-mRNA splicing.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-myb , Precursores de RNA , Splicing de RNA , Células 3T3 , Animais , Proteínas de Ciclo Celular/genética , Núcleo Celular/metabolismo , Proteínas Fúngicas/genética , Humanos , Camundongos , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas , Proteínas de Ligação a RNA , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe , Spliceossomos , Frações Subcelulares , Quinase 1 Polo-LikeRESUMO
A mutation within the Schizosaccharomyces pombe cdc24(+) gene was identified previously in a screen for cell division cycle mutants and the cdc24(+) gene was determined to be essential for S phase in this yeast. We have isolated the cdc24(+) gene by complementation of a new temperature-sensitive allele of the gene, cdc24-G1. The DNA sequence predicts the presence of an open reading frame punctuated by six introns which encodes a pioneer protein of 58 kD. A cdc24 null mutant was generated by homologous recombination. Haploid cells lacking cdc24(+) are inviable, indicating that cdc24(+) is an essential gene. The transcript of cdc24(+) is present at constant levels throughout the cell cycle. Cells lacking cdc24(+) function show a checkpoint-dependent arrest with a 2N DNA content, indicating a block late in S phase. Arrest is accompanied by a rapid loss of viability and chromosome breakage. An S. pombe homolog of the replicative DNA helicase DNA2 of S. cerevisiae suppresses cdc24. These results suggest that Cdc24p plays a role in the progression of normal DNA replication and is required to maintain genomic integrity.
Assuntos
Proteínas de Ciclo Celular/genética , Fatores de Troca do Nucleotídeo Guanina , Proteínas Proto-Oncogênicas/genética , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Alelos , Sequência de Aminoácidos , Sequência de Bases , Ciclo Celular/genética , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/química , Divisão Celular/genética , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Teste de Complementação Genética , Genótipo , Íntrons , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/química , Recombinação Genética , Proteínas Repressoras/química , Proteínas Repressoras/genética , Mapeamento por Restrição , Schizosaccharomyces/crescimento & desenvolvimento , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , TemperaturaRESUMO
Conventional myosin II is an essential protein for cytokinesis, capping of cell surface receptors, and development of Dictyostelium cells. Myosin II also plays an important role in the polarization and movement of cells. All conventional myosins are double-headed molecules but the significance of this structure is not understood since single-headed myosin II can produce movement and force in vitro. We found that expression of the tail portion of myosin II in Dictyostelium led to the formation of single-headed myosin II in vivo. The resultant cells contain an approximately equal ratio of double- and single-headed myosin II molecules. Surprisingly, these cells were completely blocked in cytokinesis and capping of concanavalin A receptors although development into fruiting bodies was not impaired. We found that this phenotype is not due to defects in myosin light chain phosphorylation. These results show that single-headed myosin II cannot function properly in vivo and that it acts as a dominant negative mutation for myosin II function. These results suggest the possibility that cooperativity of myosin II heads is critical for force production in vivo.
Assuntos
Dictyostelium/genética , Genes Dominantes , Mutação , Miosinas/genética , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Miosinas/metabolismo , Fenótipo , FosforilaçãoRESUMO
The ability of myosin II to form filaments is essential for its function in vivo. This property of self association is localized in the light meromyosin (LMM) region of the myosin II molecules. To explore this property in more detail within the context of living cells, we expressed the LMM portion of the Dictyostelium myosin II heavy chain gene in wild-type Dictyostelium cells. We found that the LMM protein was expressed at high levels and that it folded properly into alpha-helical coiled-coiled molecules. The expressed LMM formed large cytoplasmic inclusions composed of entangled short filaments surrounded by networks of long tubular structures. Importantly, these abnormal structures sequestered the cell's native myosin II, completely removing it from its normal cytoplasmic distribution. As a result the cells expressing LMM displayed a myosin-null phenotype: they failed to undergo cytokinesis and became multinucleate, failed to form caps after treatment with Con A, and failed to complete their normal developmental cycle. Thus, expression of the LMM fragment in Dictyostelium completely abrogates myosin II function in vivo. The dominant-negative character of this phenotype holds promise as a general method to disrupt myosin II function in many cell types without the necessity of gene targeting.