RESUMO
The myosin II motors are ATP-powered force-generating machines driving cardiac and muscle contraction. Myosin II heavy chain isoform-beta (ß-MyHC) is primarily expressed in the ventricular myocardium and in slow-twitch muscle fibers, such as M. soleus. M. soleus-derived myosin II (SolM-II) is often used as an alternative to the ventricular ß-cardiac myosin (ßM-II); however, the direct assessment of biochemical and mechanical features of the native myosins is limited. By employing optical trapping, we examined the mechanochemical properties of native myosins isolated from the rabbit heart ventricle and soleus muscles at the single-molecule level. We found purified motors from the two tissue sources, despite expressing the same MyHC isoform, displayed distinct motile and ATPase kinetic properties. We demonstrate ßM-II was approximately threefold faster in the actin filament-gliding assay than SolM-II. The maximum actomyosin (AM) detachment rate derived in single-molecule assays was also approximately threefold higher in ßM-II, while the power stroke size and stiffness of the "AM rigor" crossbridge for both myosins were comparable. Our analysis revealed a higher AM detachment rate for ßM-II, corresponding to the enhanced ADP release rates from the crossbridge, likely responsible for the observed differences in the motility driven by these myosins. Finally, we observed a distinct myosin light chain 1 isoform (MLC1sa) that associates with SolM-II, which might contribute to the observed kinetics differences between ßM-II and SolM-II. These results have important implications for the choice of tissue sources and justify prerequisites for the correct myosin heavy and light chains to study cardiomyopathies.
Assuntos
Miosinas Cardíacas , Cadeias Pesadas de Miosina , Animais , Ventrículos do Coração , Miosina Tipo II , Miosinas , Isoformas de Proteínas , Coelhos , Miosinas VentricularesRESUMO
OBJECTIVE: The aim of this study was to evaluate the survival benefit of sirolimus in patients undergoing liver transplantation (LT) for hepatocellular carcinoma (HCC) (exploratory analysis of the SiLVER-trial). SUMMARY AND BACKGROUND DATA: Patients receiving LT) for HCC are at a high risk for tumor recurrence. Calcineurin inhibitors have shown evidence to promote cancer growth, whereas mammalian target of rapamycin (mTOR) inhibitors like sirolimus have anticancer effects. In the SiLVER-trial (Clinicaltrials.gov: NCT00355862), the effect of sirolimus on the recurrence of HCC after LT was investigated in a prospective randomized trial. Although the primary endpoint of improved disease-free survival (DFS) with sirolimus was not met, outcomes were improved for patients in the sirolimus-treatment arm in the first 3 to 5 years. To learn more about the key variables, a multivariate analysis was performed on the SiLVER-trial data. PATIENTS AND METHODS: Data from 508 patients of the intention-to-treat analysis were included in exploratory univariate and multivariate models for overall survival (OS), DFS and a competing risk analysis for HCC recurrence. RESULTS: Sirolimus use for ≥3 months after LT for HCC independently reduced the hazard for death in the multivariate analysis [hazard ratio (HR): 0.7 (95% confidence interval, CI: 0.52-0.96, P = 0.02). Most strikingly, patients with an alpha-fetoprotein (AFP) ≥10 ng/mL and having used sirolimus for ≥3 months, benefited most with regard to OS, DFS, and HCC-recurrence (HR: 0.49-0.59, P = 0.0079-0.0245). CONCLUSIONS: mTOR-inhibitor treatment with sirolimus for ≥3 months improves outcomes in LT for HCC, especially in patients with AFP-evidence of higher tumor activity, advocating particularly for mTOR inhibitor use in this subgroup of patients. CLINICAL TRIAL REGISTRATION: EudraCT: 2005-005362-36 CLINICALTRIALS.GOV:: NCT00355862.
Assuntos
Carcinoma Hepatocelular/cirurgia , Imunossupressores/uso terapêutico , Neoplasias Hepáticas/cirurgia , Transplante de Fígado , Recidiva Local de Neoplasia/prevenção & controle , Sirolimo/uso terapêutico , Idoso , Carcinoma Hepatocelular/mortalidade , Feminino , Humanos , Análise de Intenção de Tratamento , Neoplasias Hepáticas/mortalidade , Transplante de Fígado/mortalidade , Masculino , Pessoa de Meia-Idade , Taxa de SobrevidaRESUMO
Myosin family motors play diverse cellular roles. Precise insights into how the light chains contribute to the functional variabilities among myosin motors, however, remain unresolved. Here, it is demonstrated that the fast skeletal muscle myosin II isoform myosin heavy chain (MHC-IID) can be transformed into a processive motor, by simply replacing the native regulatory light chain MLC2f with the regulatory light chain variant MLC2v from the slow muscle myosin II. Single molecule kinetic analyses and optical trapping measurements of the hybrid motor reveal marked changes such as increased association rate of myosin toward adenosine triphosphate (ATP) and actin by more than twofold. The direct consequence of high adenosine diphosphate (ADP) affinity and increased actin rebinding is the altered overall actomyosin association time during the cross-bridge cycle. The data indicate that the MLC2v influences the duty ratio in the hybrid motor, suggestive of promoting interhead communication and enabling processive movement. This finding establishes that the regulatory light chain fine-tunes the motor's mechanical output that may have important implications under physiological conditions. Furthermore, the success of this approach paves the way to engineer motors from a known motor protein element to assemble highly specialized biohybrid machines for potential applications in nano-biomedicine and engineering.
Assuntos
Músculo Esquelético/metabolismo , Miosina Tipo II/metabolismo , Citoesqueleto de Actina/metabolismo , Actomiosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Humanos , Cinética , Pinças Ópticas , Coelhos , Imagem Individual de MoléculaRESUMO
Cytoplasmic dynein, a microtubule-based motor protein, is responsible for many cellular functions ranging from cargo transport to cell division. The various functions are carried out by a single isoform of cytoplasmic dynein, thus requiring different forms of motor regulation. A possible pathway to regulate motor function was revealed in optical trap experiments. Switching motor function from single steps to processive runs could be achieved by changing Mg2+ and ATP concentrations. Here, we confirm by single molecule total internal reflection fluorescence microscopy that a native cytoplasmic dynein dimer is able to switch to processive runs of more than 680 consecutive steps or 5.5 µm. We also identified the ratio of Mg2+ -free ATP to Mg.ATP as the regulating factor and propose a model for dynein processive stepping.
Assuntos
Trifosfato de Adenosina/química , Citoplasma/química , Dineínas/química , Pinças Ópticas , Trifosfato de Adenosina/metabolismo , Animais , Citoplasma/metabolismo , Dineínas/metabolismo , SuínosRESUMO
Tonantzitlolone A, a diterpene isolated from the Mexican plant Stillingia sanguinolenta, shows cytostatic activity. Both the natural product tonantzitlolone A and its synthetic enantiomer induce monoastral spindle formation in cell experiments which indicates inhibitory activity on kinesin-5 mitotic motor molecules. These inhibitory effects on kinesin-5 could be verified in in vitro single-molecule motility assays, where both tonantzitlolones interfered with kinesin-5 binding to its cellular interaction partner microtubules in a concentration-dependent manner, yet with a larger effect of the synthetic enantiomer. In contrast to kinesin-5 inhibition, both tonantzitlolone A enantiomers did not affect conventional kinesin-1 function; hence tonantzitlolones are not unspecific kinesin inhibitors. The observed stronger inhibitory effect of the synthetic enantiomer demonstrates the possibility to enhance the overall moderate anti-proliferative effect of the lead compound tonantzitlolon A by chemical modification.
Assuntos
Proliferação de Células/efeitos dos fármacos , Diterpenos/química , Diterpenos/farmacologia , Euphorbiaceae/química , Cinesinas/antagonistas & inibidores , Compostos Macrocíclicos/química , Compostos Macrocíclicos/farmacologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Cinesinas/metabolismo , Camundongos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Mitose/efeitos dos fármacos , Modelos Moleculares , EstereoisomerismoRESUMO
BACKGROUND: We investigated whether sirolimus-based immunosuppression improves outcomes in liver transplantation (LTx) candidates with hepatocellular carcinoma (HCC). METHODS: In a prospective-randomized open-label international trial, 525 LTx recipients with HCC initially receiving mammalian target of rapamycin inhibitor-free immunosuppression were randomized 4 to 6 weeks after transplantation into a group on mammalian target of rapamycin inhibitor-free immunosuppression (group A: 264 patients) or a group incorporating sirolimus (group B: 261). The primary endpoint was recurrence-free survival (RFS); intention-to-treat (ITT) analysis was conducted after 8 years. Overall survival (OS) was a secondary endpoint. RESULTS: Recurrence-free survival was 64.5% in group A and 70.2% in group B at study end, this difference was not significant (P = 0.28; hazard ratio [HR], 0.84; 95% confidence interval [95% CI], 0.62; 1.15). In a planned analysis of RFS rates at yearly intervals, group B showed better outcomes 3 years after transplantation (HR, 0.7; 95% CI, 0.48-1.00). Similarly, OS (P = 0.21; HR, 0.81; 95% CI, 0.58-1.13) was not statistically better in group B at study end, but yearly analyses showed improvement out to 5 years (HR, 0.7; 95% CI, 0.49-1.00). Interestingly, subgroup (Milan Criteria-based) analyses revealed that low-risk, rather than high-risk, patients benefited most from sirolimus; furthermore, younger recipients (age ≤60) also benefited, as well sirolimus monotherapy patients. Serious adverse event numbers were alike in groups A (860) and B (874). CONCLUSIONS: Sirolimus in LTx recipients with HCC does not improve long-term RFS beyond 5 years. However, a RFS and OS benefit is evident in the first 3 to 5 years, especially in low-risk patients. This trial provides the first high-level evidence base for selecting immunosuppression in LTx recipients with HCC.
Assuntos
Carcinoma Hepatocelular/cirurgia , Imunossupressores/uso terapêutico , Neoplasias Hepáticas/cirurgia , Transplante de Fígado , Sirolimo/uso terapêutico , Adulto , Fatores Etários , Idoso , Austrália , Canadá , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Progressão da Doença , Intervalo Livre de Doença , Quimioterapia Combinada , Europa (Continente) , Feminino , Humanos , Análise de Intenção de Tratamento , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Transplante de Fígado/efeitos adversos , Transplante de Fígado/mortalidade , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Estudos Prospectivos , Medição de Risco , Fatores de Risco , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Fatores de Tempo , Resultado do Tratamento , Adulto JovemRESUMO
In highly polarized and elongated cells such as neurons, Tau protein must enter and move down the axon to fulfill its biological task of stabilizing axonal microtubules. Therefore, cellular systems for distributing Tau molecules are needed. This review discusses different mechanisms that have been proposed to contribute to the dispersion of Tau molecules in neurons. They include (1) directed transport along microtubules as cargo of tubulin complexes and/or motor proteins, (2) diffusion, either through the cytosolic space or along microtubules, and (3) mRNA-based mechanisms such as transport of Tau mRNA into axons and local translation. Diffusion along the microtubule lattice or through the cytosol appear to be the major mechanisms for axonal distribution of Tau protein in the short-to-intermediate range over distances of up to a millimetre. The high diffusion coefficients ensure that Tau can distribute evenly throughout the axonal volume as well as along microtubules. Motor protein-dependent transport of Tau dominates over longer distances and time scales. At low near-physiological levels, Tau is co-transported along with short microtubules from cell bodies into axons by cytoplasmic dynein and kinesin family members at rates of slow axonal transport.
Assuntos
Neurônios/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/metabolismo , Animais , Transporte Axonal , Difusão , Dineínas/metabolismo , Humanos , Cinesinas/metabolismo , Microtúbulos/metabolismo , Modelos Moleculares , Transporte Proteico , RNA Mensageiro/metabolismoRESUMO
Coupling of ATP hydrolysis to structural changes in the motor domain is fundamental to the driving of motile functions by myosins. Current understanding of this chemomechanical coupling is primarily based on ensemble average measurements in solution and muscle fibers. Although important, the averaging could potentially mask essential details of the chemomechanical coupling, particularly for mixed populations of molecules. Here, we demonstrate the potential of studying individual myosin molecules, one by one, for unique insights into established systems and to dissect mixed populations of molecules where separation can be particularly challenging. We measured ATP turnover by individual myosin molecules, monitoring appearance and disappearance of fluorescent spots upon binding/dissociation of a fluorescent nucleotide to/from the active site of myosin. Surprisingly, for all myosins tested, we found two populations of fluorescence lifetimes for individual myosin molecules, suggesting that termination of fluorescence occurred by two different paths, unexpected from standard kinetic schemes of myosin ATPase. In addition, molecules of the same myosin isoform showed substantial intermolecular variability in fluorescence lifetimes. From kinetic modeling of our two fluorescence lifetime populations and earlier solution data, we propose two conformers of the active site of myosin, one that allows the complete ATPase cycle and one that dissociates ATP uncleaved. Statistical analysis and Monte Carlo simulations showed that the intermolecular variability in our studies is essentially due to the stochastic behavior of enzyme kinetics and the limited number of ATP binding events detectable from an individual myosin molecule with little room for static variation among individual molecules, previously described for other enzymes.
Assuntos
Trifosfato de Adenosina/metabolismo , Modelos Químicos , Contração Muscular/fisiologia , Miosinas/química , Miosinas/metabolismo , Conformação Proteica , Simulação por Computador , Hidrólise , Cinética , Microscopia de Fluorescência , Método de Monte Carlo , Miosinas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fatores de TempoRESUMO
OBJECTIVE: The objective of this pilot study was to investigate the potential for long-term overall survival (OS) after liver transplantation for colorectal liver metastases (CLMs). BACKGROUND: Patients with nonresectable CLMs have poor prognosis, and few survive beyond 5 years. CLMs are currently considered an absolute contraindication for liver transplantation, although liver transplantation for primary and some secondary liver malignancies shows excellent outcome in selected patients. Before 1995, several liver transplantations for CLMs were performed, but outcome was poor (5-year survival rate: 18%) and liver transplantation for CLMs was abandoned. Since then, the survival rate after liver transplantation in general has improved by almost 30%. On the basis of this, a 5-year survival rate of about 50% after liver transplantation for CLMs could be anticipated. METHODS: In a prospective pilot study, liver transplantation for nonresectable CLMs was performed (n = 21). Main inclusion criteria were liver-only CLMs, excised primary tumors, and at least 6 weeks of chemotherapy. RESULTS: Kaplan-Meier estimates of the OS rate at 1, 3, and 5 years were 95%, 68%, and 60%, respectively. Metastatic recurrence of disease was common (mainly pulmonary). However, a significant proportion of the recurrences were accessible for surgery, and at follow-up (after median of 27 months; range, 8-60), 33% had no evidence of disease. Hepatic tumor load before liver transplantation, time from primary surgery to liver transplantation, and progressive disease on chemotherapy were identified as significant prognostic factors. CONCLUSIONS: OS exceeds by far reported outcome for chemotherapy, which is the only treatment option available for this patient group. Furthermore, OS is comparable with liver resection for resectable CLMs and survival after repeat liver transplantation for nonmalignant diseases. Selection strategies based on prognostic factors may further improve the outcome (ClinicalTrials.gov: NCT01311453).
Assuntos
Neoplasias Colorretais/patologia , Neoplasias Hepáticas/secundário , Neoplasias Hepáticas/cirurgia , Transplante de Fígado , Idoso , Antineoplásicos/uso terapêutico , Quimioterapia Adjuvante , Neoplasias Colorretais/mortalidade , Feminino , Seguimentos , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/mortalidade , Neoplasias Pulmonares/secundário , Masculino , Pessoa de Meia-Idade , Terapia Neoadjuvante , Recidiva Local de Neoplasia , Projetos Piloto , Estudos Prospectivos , Análise de Sobrevida , Resultado do TratamentoRESUMO
Current models for the intracellular transport of Tau protein suggest motor protein-dependent co-transport with microtubule fragments and diffusion of Tau in the cytoplasm, whereas Tau is believed to be stationary while bound to microtubules and in equilibrium with free diffusion in the cytosol. Observations that members of the microtubule-dependent kinesin family show Brownian motion along microtubules led us to hypothesize that diffusion along microtubules could also be relevant in the case of Tau. We used single-molecule total internal reflection fluorescence microscopy to probe for diffusion of individual fluorescently labeled Tau molecules along microtubules. This allowed us to avoid the problem that microtubule-dependent diffusion could be masked by excess of labeled Tau in solution that might occur in in vivo overexpression experiments. We found that approximately half of the individually detected Tau molecules moved bidirectionally along microtubules over distances up to several micrometers. Diffusion parameters such as diffusion coefficient, interaction time, and scanned microtubule length did not change with Tau concentration. Tau binding and diffusion along the microtubule lattice, however, were sensitive to ionic strength and pH and drastically reduced upon enzymatic removal of the negatively charged C termini of tubulin. We propose one-dimensional Tau diffusion guided by the microtubule lattice as one possible additional mechanism for Tau distribution. By such one-dimensional microtubule lattice diffusion, Tau could be guided to both microtubule ends, i.e. the sites where Tau is needed during microtubule polymerization, independently of directed motor-dependent transport. This could be important in conditions where active transport along microtubules might be compromised.
Assuntos
Microtúbulos/metabolismo , Proteínas tau/química , Trifosfato de Adenosina/química , Doença de Alzheimer/metabolismo , Sítios de Ligação , Transporte Biológico , Biofísica/métodos , Citosol/metabolismo , Difusão , Humanos , Concentração de Íons de Hidrogênio , Íons , Microscopia de Fluorescência/métodos , Microtúbulos/química , Doenças Neurodegenerativas/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Tubulina (Proteína)/químicaRESUMO
We aimed to establish reference parameters to identify functional effects of familial hypertrophic cardiomyopathy-related point mutations in the ß-cardiac/slow skeletal muscle myosin heavy chain (ß-cardiac/MyHC-1). We determined mechanical and kinetic parameters of the ß-cardiac/MyHC-1 using human soleus muscle fibers that express the same myosin heavy chain (MyHC-1) as ventricular myocardium (ß-cardiac). The observed parameters are compared to previously reported data for rabbit psoas muscle fibers. We found all of the examined kinetic parameters to be slower in soleus fibers than in rabbit psoas muscle. Somewhat surprisingly, however, we also found that the stiffness of the ß-cardiac/MyHC-1 head domain is more than 3-fold lower than the stiffness of the fast isoform of psoas fibers. Furthermore, and different from rabbit psoas muscle, in human soleus fibers both the occupancy of force-generating cross-bridge states as well as the elastic extension of force-generating heads increase with temperature. Thus, a myosin head in the force generating states makes an increasing contribution to force with temperature. We support some of our fiber data by data from in vitro motility and optical trapping assays. Initial findings with FHC-related point mutations in the converter imply that the differences in stiffness of the head domain between the slow and fast isoform may well be due to particular differences in the amino acid sequence of the converter. We show that the slower kinetics may be linked to a larger flexibility of the ß-cardiac/MyHC-1 isoform compared to fast MyHC isoforms.
Assuntos
Miosinas de Músculo Esquelético/metabolismo , Actinas/metabolismo , Adenosina Trifosfatases/metabolismo , Humanos , Cinética , Músculo Esquelético/metabolismo , TemperaturaRESUMO
The mitotic spindle in eukaryotic cells is composed of a bipolar array of microtubules (MTs) and associated proteins that are required during mitosis for the correct partitioning of the two sets of chromosomes to the daughter cells. In addition to the well-established functions of MT-associated proteins (MAPs) and MT-based motors in cell division, there is increasing evidence that the F-actin-based myosin motors are important mediators of F-actin-MT interactions during mitosis. Here, we report the functional characterization of the long-tailed class-1 myosin myosin-1C from Dictyostelium discoideum during mitosis. Our data reveal that myosin-1C binds to MTs and has a role in maintenance of spindle stability for accurate chromosome separation. Both myosin-1C motor function and tail-domain-mediated MT-F-actin interactions are required for the cell-cycle-dependent relocalization of the protein from the cell periphery to the spindle. We show that the association of myosin-1C with MTs is mediated through the tail domain. The myosin-1C tail can inhibit kinesin motor activity, increase the stability of MTs, and form crosslinks between MTs and F-actin. These data illustrate that myosin-1C is involved in the regulation of MT function during mitosis in D. discoideum.
Assuntos
Dictyostelium/citologia , Dictyostelium/metabolismo , Microtúbulos/metabolismo , Miosinas/metabolismo , Fuso Acromático/metabolismo , Actinas/genética , Actinas/metabolismo , Divisão Celular/genética , Divisão Celular/fisiologia , Dictyostelium/genética , Microscopia de Fluorescência , Miosinas/genética , Ligação Proteica , Fuso Acromático/genéticaRESUMO
BACKGROUND: Formation of lymphocele (accumulation of lymphatic fluid) is a common surgical complication following kidney transplantation. This open randomized trial evaluated the effect of prophylactic fenestration on lymphocele formation. METHODS: Adult recipients of kidney grafts from deceased donors were randomized to undergo peritoneal fenestration during the transplantation or to standard surgical procedure without fenestration. The incidence of symptomatic lymphocele in the two groups was compared at 1 year after transplantation. A protocol-based ultrasound examination was performed in the 1st, 5th, and 10th postoperative week. Any hypoechoic perirenal collection was registered. RESULTS: One hundred thirty recipients were randomized to peritoneal fenestration (n = 69) or standard therapy (n = 61). Six patients were excluded. Nine of 58 (15.5%) patients in the standard group developed symptomatic lymphoceles requiring treatment during the first postoperative year, versus 2 of 66 (3.0%) in the fenestration group (relative risk = 0.20, 95% confidence interval: 0.04-0.82, P = 0.015). Seven major surgical procedures and five percutaneous drainages due to lymphoceles were performed in the standard group, compared with two percutaneous drainages in the fenestration group. The prevalence of fluid collections in the fifth postoperative week was significantly higher in the standard group (66% vs. 37%; relative risk = 0.57, 95% confidence interval: 0.37-0.71, P = 0.005). CONCLUSION: Prophylactic fenestration reduced the risk of lymphoceles and the need for invasive procedures to treat this condition. The results need to be confirmed in a population of transplant recipients on lower steroids and with the use of wound drains.
Assuntos
Transplante de Rim , Linfocele/prevenção & controle , Peritônio/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Drenagem , Feminino , Humanos , Incidência , Linfocele/epidemiologia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Fatores de Risco , Resultado do TratamentoRESUMO
Many viruses depend on host microtubule motors to reach their destined intracellular location. Viral particles of neurotropic alphaherpesviruses such as herpes simplex virus 1 (HSV1) show bidirectional transport towards the cell center as well as the periphery, indicating that they utilize microtubule motors of opposing directionality. To understand the mechanisms of specific motor recruitment, it is necessary to characterize the molecular composition of such motile viral structures. We have generated HSV1 capsids with different surface features without impairing their overall architecture, and show that in a mammalian cell-free system the microtubule motors dynein and kinesin-1 and the dynein cofactor dynactin could interact directly with capsids independent of other host factors. The capsid composition and surface was analyzed with respect to 23 structural proteins that are potentially exposed to the cytosol during virus assembly or cell entry. Many of these proteins belong to the tegument, the hallmark of all herpesviruses located between the capsid and the viral envelope. Using immunoblots, quantitative mass spectrometry and quantitative immunoelectron microscopy, we show that capsids exposing inner tegument proteins such as pUS3, pUL36, pUL37, ICP0, pUL14, pUL16, and pUL21 recruited dynein, dynactin, kinesin-1 and kinesin-2. In contrast, neither untegumented capsids exposing VP5, VP26, pUL17 and pUL25 nor capsids covered by outer tegument proteins such as vhs, pUL11, ICP4, ICP34.5, VP11/12, VP13/14, VP16, VP22 or pUS11 bound microtubule motors. Our data suggest that HSV1 uses different structural features of the inner tegument to recruit dynein or kinesin-1. Individual capsids simultaneously accommodated motors of opposing directionality as well as several copies of the same motor. Thus, these associated motors either engage in a tug-of-war or their activities are coordinately regulated to achieve net transport either to the nucleus during cell entry or to cytoplasmic membranes for envelopment during assembly.
Assuntos
Capsídeo/metabolismo , Microtúbulos/metabolismo , Proteínas Motores Moleculares/metabolismo , Simplexvirus/ultraestrutura , Animais , Sítios de Ligação , Proteínas do Capsídeo/metabolismo , Sistema Livre de Células , Complexo Dinactina , Dineínas/metabolismo , Humanos , Cinesinas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Transporte ProteicoRESUMO
BACKGROUND: The potential anti-cancer effects of mammalian target of rapamycin (mTOR) inhibitors are being intensively studied. To date, however, few randomised clinical trials (RCT) have been performed to demonstrate anti-neoplastic effects in the pure oncology setting, and at present, no oncology endpoint-directed RCT has been reported in the high-malignancy risk population of immunosuppressed transplant recipients. Interestingly, since mTOR inhibitors have both immunosuppressive and anti-cancer effects, they have the potential to simultaneously protect against immunologic graft loss and tumour development. Therefore, we designed a prospective RCT to determine if the mTOR inhibitor sirolimus can improve hepatocellular carcinoma (HCC)-free patient survival in liver transplant (LT) recipients with a pre-transplant diagnosis of HCC. METHODS/DESIGN: The study is an open-labelled, randomised, RCT comparing sirolimus-containing versus mTOR-inhibitor-free immunosuppression in patients undergoing LT for HCC. Patients with a histologically confirmed HCC diagnosis are randomised into 2 groups within 4-6 weeks after LT; one arm is maintained on a centre-specific mTOR-inhibitor-free immunosuppressive protocol and the second arm is maintained on a centre-specific mTOR-inhibitor-free immunosuppressive protocol for the first 4-6 weeks, at which time sirolimus is initiated. A 21/2 -year recruitment phase is planned with a 5-year follow-up, testing HCC-free survival as the primary endpoint. Our hypothesis is that sirolimus use in the second arm of the study will improve HCC-free survival. The study is a non-commercial investigator-initiated trial (IIT) sponsored by the University Hospital Regensburg and is endorsed by the European Liver and Intestine Transplant Association; 13 countries within Europe, Canada and Australia are participating. DISCUSSION: If our hypothesis is correct that mTOR inhibition can reduce HCC tumour growth while simultaneously providing immunosuppression to protect the liver allograft from rejection, patients should experience less post-transplant problems with HCC recurrence, and therefore could expect a longer and better quality of life. A positive outcome will likely change the standard of posttransplant immunosuppressive care for LT patients with HCC. TRIAL REGISTER: Trial registered at http://www.clinicaltrials.gov: NCT00355862(EudraCT Number: 2005-005362-36).
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/cirurgia , Imunossupressores/uso terapêutico , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/cirurgia , Transplante de Fígado , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Sirolimo/uso terapêutico , Austrália , Canadá , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/mortalidade , Intervalo Livre de Doença , Europa (Continente) , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/mortalidade , Transplante de Fígado/efeitos adversos , Transplante de Fígado/mortalidade , Estudos Prospectivos , Proteínas Serina-Treonina Quinases/metabolismo , Recidiva , Fatores de Risco , Serina-Treonina Quinases TOR , Fatores de Tempo , Transplante Homólogo , Resultado do TratamentoRESUMO
BACKGROUND: In Norway, liver transplantation has been the treatment of choice for irreversible acute and chronic liver failure for 25 years. The aim of this article is to present a summary of the results obtained. MATERIAL AND METHODS: All liver transplants performed in Norway in the period 25.02.84-31.12.08 have been reviewed retrospectively with respect to patient and donor epidemiology, survival and recurrence. RESULTS: 651 transplants have been performed in this period. The annual number of transplants increased gradually up to the year 2000 (31), and more steeply afterwards - to 79 in 2008. Also the number of organ donations has increased and reached 98 (20 pr. million inh.) in 2008. 5-year patient survival was 53 % in the period 1984-1994. In the period 2001-2008, 1-year survival was 90 % and 5-year survival was 83 %. INTERPRETATION: The gradual improvement of results should be interpreted in light of improvements within transplant surgery, medicine and anaesthesiology and the increased local experience due to the increasing number of transplants performed. The transplant centre at Rikshospitalet has developed into being among the largest of its kind within the Nordic Countries and the results compare well with the best international data.
Assuntos
Transplante de Fígado , Adolescente , Adulto , Criança , Pré-Escolar , História do Século XX , História do Século XXI , Humanos , Lactente , Falência Hepática/diagnóstico , Falência Hepática/cirurgia , Transplante de Fígado/história , Transplante de Fígado/mortalidade , Transplante de Fígado/estatística & dados numéricos , Pessoa de Meia-Idade , Noruega/epidemiologia , Sistema de Registros , Estudos Retrospectivos , Taxa de Sobrevida , Doadores de Tecidos , Listas de Espera , Adulto JovemRESUMO
BACKGROUND: Periodontitis is the primary clinical indication for enamel matrix derivative (EMD). Recent investigations, showing that EMD inhibits the production of tumor necrosis factor-alpha (TNF-alpha) when added to human whole blood, indicate a novel role for EMD as a modulator of systemic inflammation. In the present study, we investigated the systemic effects of EMD in lipopolysaccharide (LPS)-challenged pigs. METHODS: In a preparatory study, seven pigs received a prophylactic EMD bolus injection (5 mg/kg), followed by a continuous infusion (50 mg/kg/min). Thirty minutes later, a continuous infusion of LPS (1.7 mcg/kg/h) was started. An additional 12 pigs were randomized into two groups. Six of these animals were given the same treatment, except that EMD was administered 30 min after LPS. The remainder served as controls. The groups were compared according to organ injury and function, hemodynamics, and systemic markers of inflammation. RESULTS: Prophylactic administration of EMD triggered transient hemodynamic instability in two of seven pigs. In the randomized pigs, no or only nonspecific changes were observed in biopsies from vital organs, independent of treatment. Enamel matrix derivative did not modify systemic TNF-alpha, interleukin (IL)-1 beta, or IL-6 concentrations. CONCLUSIONS: In the formulation and dosages used, EMD did not modulate the inflammatory response. No true allergic or immunotoxic reactions were seen. To be usable for systemic application, a new formulation should be developed, or the active part of the protein(s) should be identified and produced in a soluble form designed for infusion. The potential of EMD as a systemic immune modulator is still unsettled.
Assuntos
Anti-Inflamatórios/farmacologia , Proteínas do Esmalte Dentário/farmacologia , Endotoxemia/prevenção & controle , Animais , Modelos Animais de Doenças , Interleucina-1beta/efeitos dos fármacos , Interleucina-6/imunologia , Lipopolissacarídeos/efeitos adversos , Lipopolissacarídeos/imunologia , Sus scrofa , Fator de Necrose Tumoral alfa/efeitos dos fármacosRESUMO
CONTEXT: Pheochromocytomas are rare tumors of predominantly adrenal origin that often produce and secrete catecholamines. Malignancy occurs in a variable percentage of cases depending on genetic background and tumor location. Definitive diagnosis relies on the detection of distant metastases. Treatments for malignant pheochromocytoma include surgical debulking, pharmacological control of hormone-mediated symptoms, targeted methods such as external irradiation, and systemic antineoplastic therapy. Different agents and protocols for this purpose are reviewed, and their therapeutic potential is discussed. EVIDENCE ACQUISITION: Literature on antineoplastic therapies for malignant pheochromocytoma was identified by searching the PubMed database with restriction to articles published in English during the past 30 yr. EVIDENCE SYNTHESIS: Because of the rarity of the condition, no randomized clinical trials concerning the treatment of malignant pheochromocytoma have been performed. The strategy established best is [131I]meta-iodobenzylguanidine (MIBG) therapy, which is well tolerated. Similar to cytotoxic chemotherapy with cyclophosphamide, vincristine, and dacarbazine, MIBG can induce remission for a limited period in a significant proportion of patients. Octreotide as a single agent seems to be largely ineffective. CONCLUSIONS: MIBG radiotherapy and cyclophosphamide, vincristine, and dacarbazine chemotherapy are comparable with respect to response rate and toxicity. It is unclear whether combining both can improve the outcome. Future developments may include new multimodal concepts with focus on inhibition of angiogenetic factors and heat shock protein 90. Any present or new therapeutic approach must take into account the highly variable natural course of the disease.
Assuntos
Neoplasias das Glândulas Suprarrenais/tratamento farmacológico , Neoplasias das Glândulas Suprarrenais/radioterapia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Feocromocitoma/tratamento farmacológico , Feocromocitoma/radioterapia , 3-Iodobenzilguanidina/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Terapia Combinada , Ciclofosfamida/administração & dosagem , Dacarbazina/administração & dosagem , Humanos , Radioisótopos do Iodo/uso terapêutico , Vincristina/administração & dosagemRESUMO
BACKGROUND: Very few randomized studies on laparoscopic (L) versus open (O) living-donor nephrectomy (LDN) have been presented. The largest randomized series reported so far included 80 donors. In 2000, an Australian safety group concluded that the evidence base for L-LDN is inadequate to make recommendations regarding safety and efficacy. METHODS: With this background, at our single national center, 122 donors were randomized to left-sided L-LDN (n=63) or O-LDN (n=59), from February 2001 to May 2004. This article summarizes our experiences, in particular regarding complications and safety. RESULTS: There were significant differences in favor of O-LDN regarding operative time, warm ischemia time, and vessel lengths, whereas the analgesic requirements and pain data were significantly in favor of the laparoscopic procedure. In the L-LDN group, there were five major postoperative complications resulting in reoperations (8%), including two intestinal perforations. No major complications occurred in the O-LDN group. CONCLUSIONS: These results from our randomized study do suggest that conventional O-LDN is a very secure procedure, superior to L-LDN regarding donor safety. There has been an unacceptably high rate of reoperations in our L-LDN series but without mortality or significant sequelae. A careful look at some other L-LDN series also suggests increased morbidity/mortality. Our data do, however, support the view that a perfect, uncomplicated L-LDN appears to be the superior procedure, and the laparoscopic procedure is still evolving. Donor safety may be improved by avoiding obese donors, stapling of the renal artery (not clipping), and perhaps by hand assistance. Furthermore, we will consider the retroperitoneal approach.
Assuntos
Laparoscopia/métodos , Laparoscopia/normas , Doadores Vivos , Nefrectomia/métodos , Complicações Pós-Operatórias/epidemiologia , Analgésicos/uso terapêutico , Perda Sanguínea Cirúrgica , Humanos , Nefrectomia/normas , Complicações Pós-Operatórias/prevenção & controle , Período Pós-Operatório , SegurançaRESUMO
To characterize elastic properties and geometrical parameters of individual, whole myosin molecules during their interaction with actin we sparsely adsorbed myosin molecules to nanometer-sized microspheres. Thermally driven position fluctuations of these microspheres were recorded with the three-dimensional detection scheme of the photonic force microscope. Upon binding of single myosin molecules to immobilized actin filaments in the absence of ATP, these thermally driven position fluctuations of the microspheres change significantly. From three-dimensional position fluctuations stiffness and geometrical information of the tethering molecule can be derived. Axial stiffness was found to be asymmetric, approximately 0.04 pN/nm for extension, approximately 0.004 pN/nm for compression. Observed stiffness of whole myosin molecules is much less than estimated for individual myosin heads in muscle fibers or for single-molecule studies on myosin fragments. The stiffness reported here, however, is identical to stiffness found in other single-molecule studies with full-length myosin suggesting that the source of this low stiffness is located outside the myosin head domain. Analysis of geometrical properties of tethering myosin molecules by Brownian dynamics computer simulations suggests a linker length of approximately 130 nm that is divided by a free hinge located approximately 90 nm above the substrate. This pivot location coincides with myosin's hinge region. We demonstrate the general applicability of thermal fluctuation analysis to determine elastic properties and geometrical factors of individual molecules.