RESUMO
2-[4'-Maleimidylanilino]naphthalene 6-sulfonic acid (MIANS) irreversibly inactivates Na,K-ATPase in a time- and concentration-dependent manner. Inactivation is prevented by 3 mM ATP or low K(+) (<1 mM); the protective effect K(+) is reversed at higher concentrations. This biphasic effect was also observed with K(+) congeners. In contrast, Na(+) ions did not protect. MIANS inactivation disrupted high affinity ATP binding. Tryptic fragments of MIANS-labeled protein were analyzed by reversed phase high performance liquid chromatography. ATP clearly protected one major labeled peptide peak. This observation was confirmed by separation of tryptic peptides in SDS-polyacrylamide gel electrophoresis revealing a single fluorescently-labeled peptide of approximately 5 kDa. N-terminal amino acid sequencing identified the peptide (V(545)LGFCH...). This hydrophobic peptide contains only two Cys residues in all sodium pump alpha-subunit sequences and is found in the major cytoplasmic loop between M4 and M5, a region previously associated with ATP binding. Subsequent digestion of the tryptic peptide with V8 protease and N-terminal amino acid sequencing identified the modified residue as Cys(577). The cation-dependent change in reactivity of Cys(577) implies structural alterations in the ATP-binding domain following cation binding and occlusion in the intramembrane domain of Na,K-ATPase and expands our knowledge of the extent to which cation binding and occlusion are sensed in the ATP hydrolysis domain.
Assuntos
Trifosfato de Adenosina/metabolismo , Cisteína/química , ATPase Trocadora de Sódio-Potássio/química , Naftalenossulfonato de Anilina/farmacologia , Sítios de Ligação , Cátions/farmacologia , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Cinética , Fragmentos de Peptídeos/química , Ligação Proteica , Análise de Sequência , TripsinaRESUMO
Most of the residues associated with cation coordination seem to reside within transmembrane segments of the alpha-subunit of the Na,K-ATPase, whereas amino acids which appear to be involved in the coordination of ATP are found in the major cytoplasmic loop between transmembrane segments M4 and M5 (see Lingrel & Kuntzweiler, 1994; Lutsenko & Kaplan, 1995). The coupling of the two functions of cation transport and ATP hydrolysis involved in the active transport of Na and K ions must involve interactions between these two structural units. This paper summarizes recent experimental results and conclusions of studies on the renal Na,K-ATPase which have employed controlled proteolysis in the presence of physiological ligands, chemical modification with a range of reagents and a variety of functional assays. The data provide evidence for movements between specific transmembrane segments associated with cation-binding conformations and coupled changes which take place in the ATP binding domain. The binding of different cations in the cation-binding domain is sensed in the ATP binding domain and manifested as a change in reactivity. This occurs at amino acid residues which are widely spaced in primary structure. It is apparent that structural changes are transmitted through much of the ATP-binding domain as a consequence of the occupancy of the cation-binding domain. We also provide evidence that both the number and identity of cations bound are also sensed in the ATP-binding domain.
Assuntos
Trifosfato de Adenosina/metabolismo , Cátions/metabolismo , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Transporte Biológico/fisiologia , Humanos , Hidrólise , Rim/enzimologia , Relação Estrutura-AtividadeRESUMO
Patellar tendon (PT) force was measured during activity with an implantable force transducer (IFT) in adult goats. PT force, vertical ground reaction force (VGRF) and the animal's speed were recorded for standing, walking and trotting. Following data collection, animals were euthanized and the IFT calibrated in vitro. Standing PT force averaged 207 N. Maximum PT force was approximately 800 N for walking and 1000 N for trotting and occurred at mid-stance. PT force dropped from 200 N at toe-off to 0 N by mid-swing. For each activity, the PT force increased with increases in VGRF. Maximum in vivo PT stress occurred during trotting and measured 29 MPa. This study demonstrates the IFT's usefulness in measuring tendon force directly.
Assuntos
Patela , Próteses e Implantes , Tendões/fisiologia , Transdutores de Pressão , Animais , Fenômenos Biomecânicos , Marcha , CabrasRESUMO
OBJECTIVE: To establish the relation of insulin-like growth factor-I (IGF-I) and IGF-binding protein-1 (IGFBP-1) with 24-hour metabolic excursions in normal healthy women and in response to acute interruption of metabolic homeostasis by hypoinsulinemia. METHODS: Hourly blood samples during the 24-hour metabolic clock were obtained from seven normally cycling women. Uniform dietary composition (50% carbohydrate, 35% fat, and 15% protein) and timing of meals (8 AM, 12 PM, and 6 PM) were prescribed. Daytime hypoinsulinemia was induced by omitting meals and by Sandostatin (100 micrograms) administration. Changes in serum levels of glucose, insulin, cortisol, IGF-I, and IGFBP-1 were measured. RESULTS: The diurnal pattern of serum IGFBP-1 levels during the 24-hour metabolic clock was characterized by a rapid fall during the feeding phase of the day and a progressive 3.5-fold rise during nocturnal fasting; IGF-I levels were unchanged. Changes in IGFBP-1 levels were in parallel to those of cortisol and were inversely related to increases in glucose (80%) and insulin (tenfold) levels after each meal and to their decline during nocturnal fasting. Daytime fasting and administration of Sandostatin were accompanied by rapid and sustained increases in IGFBP-1 when insulin levels declined to 54 +/- 20 pmol/L. CONCLUSIONS: With constant levels of IGF-I, the diurnal rhythm of IGFBP-1 may subserve a physiologic function by coordinating insulin and IGF-I action with substrate availability. Fluctuations of insulin levels during the 24-hour metabolic clock in normal women appear to serve as a signal, with an inhibitory effect on IGFBP-1 production when levels are above 70 pmol/L and a stimulatory effect at levels below 70 pmol/L. These findings provide a basis for future investigations in women with nutritionally related reproductive disorders.
Assuntos
Ritmo Circadiano/fisiologia , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/sangue , Insulina/sangue , Octreotida/farmacologia , Adulto , Glicemia/metabolismo , Ritmo Circadiano/efeitos dos fármacos , Jejum , Feminino , Hormônios/farmacologia , Humanos , Insulina/metabolismo , Secreção de Insulina , Fator de Crescimento Insulin-Like I/metabolismoRESUMO
In order to determine the measurements and calibration methods necessary to accurately measure in vivo forces in the anterior cruciate ligament (ACL) of the goat, an in vitro study was conducted to evaluate the effect of several factors that could influence the sensitivity of a transducer implanted within the ligament. Four factors were studied in six specimens: flexion angle [0 degrees, 10 degrees, 30 degrees, 50 degrees, and 70 degrees from full extension (FFE)]; tibial rotation (0 degrees and 10 degrees of internal rotation at 30 degrees, 50 degrees, and 70 degrees flexion FFE); loading rate (cycling frequencies of 0.2, 0.5, 1.0, and 2.0 Hz); and temperature (22 degrees C and 37 degrees C). Anteroposterior tibial displacements were applied to the specimens following tissue resection to isolate the ACL. The resultant ACL force magnitude was measured with a multi-component load cell, and transducer sensitivity was calculated as the slope of the output vs force curve in the linear response region. Transducer sensitivity varied with joint position in each specimen, but there was no consistent trend from specimen to specimen in how the sensitivity changed. As a result, there were no statistically significant mean differences (p > 0.05). There were no significant differences and little variation in sensitivity due to changes in either loading rate or tissue temperature, although the latter produced a voltage offset. The results show that the transducer output with zero force on the ligament must be determined in vivo, after which in vitro calibrations may be conducted at room temperature.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Ligamento Cruzado Anterior/fisiologia , Transdutores de Pressão , Animais , Ligamento Cruzado Anterior/cirurgia , Temperatura Corporal , Calibragem , Feminino , Fêmur/fisiologia , Cabras , Membro Posterior , Próteses e Implantes , Rotação , Sensibilidade e Especificidade , Estresse Mecânico , Tíbia/fisiologia , Suporte de Carga/fisiologiaRESUMO
In vivo forces in the anterior cruciate ligament (ACL) were measured in three adult goats during quiet standing and during gait (walking or trotting). A modified pressure transducer (MPT) was implanted within the anteromedial band of the ligament to make direct measurements of ACL force. One or two days following implantation, measurements were made of ACL force, knee joint flexion angle, ground reaction forces, and speed of locomotion. MPT calibration was performed in vitro using anteroposterior displacement tests at six flexion angles. The ACL was loaded during quiet standing (30-61 N) and during the stance phase of gait. Peak ACL forces were achieved within the first 40% of stance, with magnitudes ranging from 63 to 124 N during walking and from 102 to 150 N during trotting. The average ACL force during the stance phase ranged from 34 to 68 N while walking and from 46 to 69 N while trotting. The partial correlations between peak ACL force and speed, and between average ACL force and speed, were both statistically different from zero (p < 0.01). ACL forces dropped to zero during the swing phase in all trials. ACL forces were less than 15 N throughout swing in two of the animals, both of which did not extend their knees during gait beyond 20 degrees from full extension. In the animal which did show knee extension beyond 40 degrees (20 degrees from full extension), ACL loading occurred during late swing. The magnitude of the peak ACL force during late swing was significantly correlated with the extent of knee extension in this animal.
Assuntos
Ligamento Cruzado Anterior/fisiologia , Cabras/fisiologia , Corrida/fisiologia , Caminhada/fisiologia , Animais , Feminino , Fêmur/anatomia & histologia , Fêmur/fisiologia , Marcha/fisiologia , Articulação do Joelho/anatomia & histologia , Articulação do Joelho/fisiologia , Amplitude de Movimento Articular/fisiologia , Rotação , Processamento de Sinais Assistido por Computador , Estresse Mecânico , Tíbia/anatomia & histologia , Tíbia/fisiologia , Transdutores de Pressão , Suporte de Carga/fisiologiaRESUMO
A technique to quantify the restraining action of specific knee structures during anterior/posterior (A/P) tibial displacement tests in the goat knee that has a reconstructed anterior cruciate ligament (ACL) is described. Joint specimens were mounted in an instrumented test stand to measure both the forces on, and resulting translations of, the tibia in the A/P direction. The intact grafted knee was tested first, after which structures were sequentially cut or removed, or both, and the test was repeated. The ACL graft remained intact in the joint during this process; therefore, subsequent axial failure testing was possible. Analysis of the resulting force-displacement curves allowed the percentage contribution and anterior joint stiffness to be determined for each structure at a fixed anterior displacement in 12 goat knees 6 months after ACL allografting. The allografts were found to provide about 56% of the total restraining force, more than any other structure examined. Studies of this kind will be important in the documentation of the changing role of an autograft or allograft in a reconstructed knee over time.
Assuntos
Ligamento Cruzado Anterior/transplante , Articulação do Joelho/fisiologia , Articulação do Joelho/cirurgia , Animais , Fenômenos Biomecânicos , Elasticidade , Cabras , Meniscos Tibiais/fisiologiaRESUMO
Best described as a transformation from the infantile state to an accentuated dimorphic adult state, puberty is a sequence of events characterized by the secretion of gonadal hormones leading to the development of secondary sexual characteristics, gametogenesis, and reproductive function. In girls, the first signs of puberty may be evident at age 8, with the process largely completed by age 16; in boys, puberty commonly begins between ages 10 and 12 and is largely completed by age 18. Adrenarche, the secretion of adrenal androgens, starts between ages 6 and 8 and is clinically accompanied by pubarche. Premature pubarche should be diagnosed as either typical or atypical. In atypical premature pubarche, corticotropin testing is recommended to determine nonclassical adrenal enzyme deficiency of steroidogenesis. Children with either type of premature pubarche should be under continued follow-up throughout puberty. The trigger of the onset of puberty is still unknown. The presence of gonadotropin activity and possible circadian rhythm in the prepubertal years allows for new understanding in possible triggering mechanisms of puberty. Precocious puberty, which is associated with significant psychologic implications and possible pathology, must be categorized as complete precocious puberty with activation of the hypothalamic-pituitary axis or incomplete precocious puberty without activation of the central axis as effective therapies are so different. The categorization does not yield diagnoses, as there are multiple etiologies within each category. The treatment of central precocious puberty with gonadotropin-releasing hormone agonists will postpone pubertal progression to a more appropriate age.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Glândulas Suprarrenais/metabolismo , Puberdade Precoce/fisiopatologia , Puberdade/fisiologia , Adolescente , Criança , Feminino , Hormônio Foliculoestimulante/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Hormônio Liberador de Gonadotropina/uso terapêutico , Humanos , Hormônio Luteinizante/metabolismo , Masculino , Puberdade Precoce/tratamento farmacológico , Puberdade Precoce/etiologiaRESUMO
Total anterior-posterior translation is commonly used to assess the integrity of the cruciate ligaments and the success of reconstructive surgery. The purpose of this study was to determine, after surgical reconstruction of the anterior cruciate ligament with a biological graft, if total anterior-posterior translation correlated with graft length, cross-sectional area, or mechanical properties. These factors were investigated by analyzing data from three previous studies. These studies involved replacement of the anterior cruciate ligament in cynomolgus monkeys and goats, with free and vascularized patellar tendon autografts and both patellar tendon and anterior cruciate ligament allografts. Data were available at time periods of 6 and 12 months after surgery. We found statistically significant inverse correlations between the amount of anterior-posterior translation and cross-sectional area of a graft at the time of sacrifice. The Pearson correlation coefficients ranged from -0.966 (p < 0.002) to -0.830 (p < 0.05). We hypothesize that these correlations result from the following mechanism: the increased anterior translation reflects a slack graft; a slack graft is stress shielded by other structures about the knee; the reduced in vivo stresses on the graft modulate cellular metabolism in a way that over time produces a small cross-sectional area.
Assuntos
Ligamento Cruzado Anterior/fisiopatologia , Ligamento Cruzado Anterior/cirurgia , Transferência Tendinosa/métodos , Tendões/transplante , Análise de Variância , Animais , Ligamento Cruzado Anterior/transplante , Feminino , Cabras , Macaca fascicularis , Masculino , Movimento , Análise de Regressão , Estresse Mecânico , Resistência à Tração , Transplante Autólogo/métodos , Transplante Homólogo/métodos , Suporte de CargaRESUMO
Twenty-eight goats underwent ACL reconstruction with freeze-dried bone-patellar tendon-bone allografts in one knee, the opposite knee serving as a control. One group of 16 knees was evaluated, in groups of four, at 6, 12, 26, and 52 weeks by histologic and vascular injection techniques. The other group of 12 knees was evaluated in two groups of six at 26 and 52 weeks by morphological and biomechanical techniques of analysis. Within the first 12 weeks these allografts were revascularized; in the first 26 weeks they had matured to resemble normal connective tissue. Graft stiffness was 29% of the control value and maximum force to failure was 43% of the control value. The results of this study indicated that freeze-dried bone-patellar tendon-bone allografts are biomechanically and biologically similar to patellar tendon autografts.
Assuntos
Ligamento Cruzado Anterior/cirurgia , Transplante Ósseo , Patela/transplante , Tendões/transplante , Transplante de Tecidos , Animais , Ligamento Cruzado Anterior/anatomia & histologia , Fenômenos Biomecânicos , Óxido de Etileno , Liofilização , Cabras , Neovascularização Patológica , Patela/irrigação sanguínea , Tendões/irrigação sanguíneaRESUMO
Many running injuries are successfully treated with footwear modifications designed to reduce pronation, but the underlying mechanism of treatment is not well understood. Previous attempts to correlate reduction in pronation with changes in ground reaction parameters have been unsuccessful. In this study, the free moment of ground reaction (Mz') was measured for 10 rearfoot strikers running at 4.5 m s-1 in each of three different pairs of running shoes designed to vary the extent of pronation during ground contact. Mz' patterns were highly variable between feet, but were repeatable within a given foot/footwear combination. Mz' was greatest in magnitude during the first half of support, when it acted in a direction resisting foot abduction, a component of pronation. It was opposite in sign and smaller in magnitude during the last 30% of support. The peak magnitude and the net angular impulse of Mz' were both increased significantly with increases in pronation. A net ground reaction moment was also calculated about a vertical axis fixed in the shoe, and was used in a first approximation model of the shoe/ground interface to predict when the foot is most likely to ab/adduct during running. In conclusion, this study characterized the Mz' pattern for a well-defined group of runners, and found that Mz' is sensitive to relatively large within-subject changes in pronation.
Assuntos
Pé/fisiologia , Pronação/fisiologia , Corrida , Sapatos , Adulto , Desenho de Equipamento , Humanos , Masculino , Rotação , Estresse Mecânico , Propriedades de SuperfícieRESUMO
We examined three surgical variables that affect the ability of an anterior cruciate ligament replacement to restore the limit of anterior tibial translation. These were the placement site of the substitute on the femur, the initial tension applied to the replacement, and the flexion angle of the knee at the time of tensioning. An anterior load of 100 N was applied to the tibia. As the knee was flexed, we measured the tensile force in the substitute and the anteroposterior position of the femur relative to the tibia. Placement largely determined whether the force in the replacement increased or decreased with flexion. Placement also largely determined whether the tibia moved anteriorly or posteriorly with flexion compared to its position in the intact knee. The initial tension and the flexion angle at tensioning affected the magnitude of force in the substitute and the magnitude of the change in AP position. They did not affect how force and AP position changed with flexion. Greater increases in force and greater posterior shifts in tibial position were produced by changing the flexion angle at tensioning from 0 degrees to 30 degrees than by increasing the initial tension from 22 to 44 N.
Assuntos
Articulação do Joelho/cirurgia , Ligamentos Articulares/cirurgia , Adulto , Feminino , Fêmur/anatomia & histologia , Fêmur/fisiologia , Humanos , Articulação do Joelho/anatomia & histologia , Articulação do Joelho/fisiologia , Ligamentos Articulares/anatomia & histologia , Ligamentos Articulares/fisiologia , Masculino , Pessoa de Meia-Idade , Estresse Mecânico , Tíbia/anatomia & histologia , Tíbia/fisiologiaAssuntos
Política de Saúde , Aposentadoria , Voluntários , Idoso , Envelhecimento , Humanos , Estados UnidosRESUMO
There has been a growing interest in the use of allografts as ACL substitutes. Allografts are often freeze dried to increase shelf storage time and sterilized with ethylene oxide. This study was conducted to determine the effect of a specific ethylene oxide sterilization procedure and freeze drying process on the initial mechanical properties of femur-ACL-tibia preparations. Twelve knees (stifle joints) from six mature goats were divided into two groups (one knee of pair to each group). Knees were cleaned of all soft tissue except for the anterior cruciate, posterior cruciate, and collateral ligaments. Group 1 was sterilized with ethylene oxide (simulating clean procurement) then freeze dried. Group 2 was freeze dried only (simulating sterile procurement). The knees were rehydrated and then tested in tension to failure to determine their structural mechanical properties. The ethylene oxide-freeze dried specimens (Group 1) had a mean maximum load before failure of 2059 +/- 273 newtons (N) (+/- SE) which was not statistically different than the maximum load of the freeze dried specimens (Group 2) of 2023 +/- 214 N. The average strength of Group 1 and 2 combined was not significantly different than a third group of 12 normal femur-ACL-tibia controls which had an average maximum force of 2403 +/- 133 N. No significant differences between the groups were observed for stiffness, energy to maximum strength, or elongation to maximum force. It appears the freeze dry processing, with or without ethylene oxide sterilization, using the procedure we used, has, at most, a small effect on the initial mechanical properties of the preparations.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Fíbula/transplante , Liofilização , Articulação do Joelho/cirurgia , Ligamentos Articulares/transplante , Esterilização , Tíbia/transplante , Preservação de Tecido/métodos , Animais , Fenômenos Biomecânicos , Óxido de Etileno , Cabras , Articulação do Joelho/fisiologia , Esterilização/métodos , Resistência à TraçãoRESUMO
Mechanical properties of fascia lata autografts used to replace the anterior cruciate ligament (ACL) in the goat were measured at 0, 2, 4, and 8 weeks after surgery. The ACL was replaced in the right knee of 50 animals divided equally into two groups according to graft fixation technique: (a) two smooth staples at each end, with the tissue pulled back toward the joint over the first staple and (b) reinforced fixation with a spiked bushing placed through the tissue and a 3-cm-long flat polypropylene braid sutured to each end of the graft. Eleven unoperated contralateral knees were tested as controls. All statistically significant effects of the reinforced versus staple fixation were observed at 0 weeks, with the reinforced group showing less anteroposterior (AP) translation of the joint and greater maximum force and stiffness of the femur-graft-tibia units. The reinforced group had increased AP translation and decreased strength and stiffness by 2 weeks after surgery. Increased AP translation resulted primarily from increases in the low-stiffness region of the force-displacement curve (primary AP translation) and to a lesser extent from increased translation in the high-stiffness region (secondary anterior translation). Failures at 0 weeks with the reinforced fixation occurred at the bushing or end of the reinforcing braid, while all but one of the later failures occurred in the tissue mid-substance. In the staple group, maximum force was greater at 8 weeks than at 0 weeks, as the failure locations changed from the fixation to the tissue mid-substance.(ABSTRACT TRUNCATED AT 250 WORDS)