RESUMO
BACKGROUND: A preponderance of behavioural symptoms is assumed to be the main difference in the manifestation of symptoms of post-traumatic stress disorder (PTSD) in people with intellectual disability (ID). However, no study so far has assessed the relationship between challenging behaviour (CB) and PTSD. The present study aims to explore this relationship by exploring whether CB is directly related to trauma exposure or whether this relationship is mediated through core symptoms of PTSD. METHODS: Trauma exposure and current symptoms of PTSD were assessed in 43 adults with mild to moderate ID. Parallel versions were administered to 43 caregivers, including the Aberrant Behaviour Checklist to measure CB. Bayesian mediation analyses were conducted using self-rated and informant-rated data. RESULTS: The self-report data showed no associations of CB with trauma exposure or PTSD symptoms. The association between informant-rated trauma exposure and irritability was mediated by severity and frequency of PTSD symptoms. The associations between informant-reported trauma exposure and the Aberrant Behaviour Checklist subscales hyperactivity and inappropriate speech were mediated by PTSD symptom severity. CONCLUSIONS: The relationship between trauma exposure and CB was mediated by PTSD symptoms. PTSD core symptoms should be considered as underlying causes of CB, highlighting the necessity to explore trauma biography and symptoms of PTSD. The improvement of self-report assessment in people with ID is an important task for future studies.
Assuntos
Deficiência Intelectual/fisiopatologia , Humor Irritável/fisiologia , Comportamento Problema , Trauma Psicológico/fisiopatologia , Transtornos de Estresse Pós-Traumáticos/fisiopatologia , Adulto , Idoso , Teorema de Bayes , Comorbidade , Feminino , Humanos , Deficiência Intelectual/epidemiologia , Masculino , Análise de Mediação , Pessoa de Meia-Idade , Trauma Psicológico/epidemiologia , Índice de Gravidade de Doença , Transtornos de Estresse Pós-Traumáticos/epidemiologia , Adulto JovemRESUMO
OBJECTIVES: Tobacco use and alcohol use have their origin in adolescence, and risky use of these substances is amongst the leading preventable causes of morbidity and mortality. Health literacy (HL) encompasses the skills that are decisive to make appropriate health decisions in this context. Given the paucity of evidence on the link between HL and adolescents' health behaviors, the present study examined overall HL and different components of HL and their associations with smoking and alcohol use among 13 to 17-year-old Austrian students. STUDY DESIGN: Data were obtained from a national survey carried out in Austria (N = 4219; 56% females) as part of the Health Behaviour in School-aged Children: World Health Organization (WHO) collaborative cross-national study. METHODS: We tested two structural equation models, one including the overall HL scale as the predictor and one with the three subscales of HL 'finding,' 'understanding and appraising,' and 'applying' health-related information as predictors of smoking and alcohol use. RESULTS: Although overall HL was related to all indicators of adolescents' smoking and drinking, the three HL components had differential effects on these behaviors. The easier it was for the participants to 'understand and appraise' and 'apply' health-related information, the less frequently they had smoked and drunk alcohol and the less was the amount of alcohol they had consumed. Contrarily, the easier it was for the participants to 'find' information, the more they had smoked and drunk alcohol. CONCLUSIONS: Our results indicate that availability of health-related information may be insufficient on its own to prevent or reduce risky substance use. This is of particular importance for the development and improvement of primary prevention programs targeting adolescent populations.
Assuntos
Comportamento do Adolescente/psicologia , Consumo de Bebidas Alcoólicas/epidemiologia , Letramento em Saúde/estatística & dados numéricos , Assunção de Riscos , Fumar/epidemiologia , Adolescente , Áustria/epidemiologia , Feminino , Humanos , Masculino , Estudantes/psicologia , Estudantes/estatística & dados numéricos , Inquéritos e QuestionáriosRESUMO
As part of the international joint projects working towards the control of taeniosis/cysticercosis in Asia Pacific, epidemiological studies on Taenia solium cysticercosis have been carried out in high-incidence populations, such as minority groups in Thailand. To assess the epidemiology of cysticercotic infections in pigs in the hill-tribe minority villages (Karen) in Tak province, Thailand, we conducted serological screening and necropsies. The patterns of antibody response to T. solium antigens were then investigated using immunoblot assays. Of the 188 pig serum samples tested for antibody responses to partially purified low-molecular-weight antigens of T. solium cyst fluid, positive responses were detected in 37 samples (19.7%). Based on these results, 16 pigs (10 seropositive and 6 seronegative) were necropsied for investigation of cysticerci and intestinal parasites. All seropositive pigs were coinfected with both T. solium and Taenia hydatigena cysticerci, except one, which was infected with T. hydatigena alone. Three of the six seronegative pigs were confirmed to be infected with T. hydatigena. Pigs infected with T. solium showed much stronger antibody responses than those infected with T. hydatigena. Our results demonstrate the co-occurrence of two swine cysticercoses due to T. solium and T. hydatigena in the studied areas. This study also reveals the importance of direct confirmation of the presence of cysticerci by necropsy after serological screening. In addition to the prevalence of swine cysticercosis in these endemic areas, our findings also reveal potential implications for the development of serological diagnostic assays for swine cysticercosis.
Assuntos
Coinfecção/veterinária , Cisticercose/veterinária , Doenças dos Suínos/parasitologia , Taenia/isolamento & purificação , Teníase/veterinária , Animais , Coinfecção/epidemiologia , Coinfecção/parasitologia , Cisticercose/parasitologia , Feminino , Humanos , Masculino , Mianmar/epidemiologia , População Rural , Suínos , Doenças dos Suínos/epidemiologia , Taenia/classificação , Taenia/genética , Taenia solium/genética , Taenia solium/isolamento & purificação , Taenia solium/fisiologia , Teníase/parasitologia , Tailândia/epidemiologiaRESUMO
Bovine cysticercosis causing damage to the beef industry is closely linked to human taeniasis due to Taenia saginata. In African countries, Taenia spp. from wildlife are also involved as possible sources of infections in livestock. To identify the aetiological agents of bovine cysticercosis in Ethiopia, cysticerci were collected from 41 cattle slaughtered in the eastern and central areas during 2010-2012. A single cysticercus per animal was subjected to the polymerase chain reaction (PCR)-based DNA sequencing of mitochondrial cytochrome c oxidase subunit 1 gene, and the resultant sequence was compared with those of members of the genus Taenia. Although 38 out of 41 cysticerci (92.7%) were identified as T. saginata, three samples (7.3%) showed the hitherto unknown sequences of Taenia sp., which is distantly related to Taenia solium, Taenia arctos and Taenia ovis. Old literatures suggest it to be Taenia hyaenae, but morphological identification of species could not be completed by observing only the larval samples.
Assuntos
Doenças dos Bovinos/parasitologia , Cisticercose/veterinária , Variação Genética , Taenia/classificação , Taenia/genética , Animais , Bovinos , Análise por Conglomerados , Cisticercose/parasitologia , DNA de Helmintos/genética , DNA Mitocondrial/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Etiópia , Genótipo , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Homologia de Sequência , Taenia/isolamento & purificaçãoRESUMO
Kudoa ogawai n. sp. (Myxozoa; Multivalvulida) is described from the trunk muscle of Pacific barrelfish Hyperoglyphe japonica caught in Japan. Oval to ellipsoidal cysts, white to yellowish in colour, 1-2 mm in size, were filled with numerous spores having four polar capsules. Spores were stellate with rounded peripheral edges in apical view, and garlic shaped with four prominent apical projections in side view. Average (range) spore size was 13.3 (12.0-14.2) µm in width and 9.0 (8.3-9.7) µm in length. The spore dimensions and the apical projections of K. ogawai n. sp. were clearly distinguishable from the other muscle-infecting species. Molecular analyses of 18S rDNA and 28S rDNA sequences showed that K. ogawai n. sp. is closely related to Kudo iwatai and Kudoa hemiscylli (95% and 92% genetic similarity, respectively), but the present species is morphologically distinct from the two species. Phylogenetic analysis of 18S and 28S rDNA placed K. ogawai n. sp. sister to all Kudoa spp. available in GenBank.
Assuntos
Doenças dos Peixes/parasitologia , Músculos/parasitologia , Myxozoa/classificação , Myxozoa/isolamento & purificação , Doenças Parasitárias em Animais/parasitologia , Perciformes/parasitologia , Animais , Análise por Conglomerados , DNA Ribossômico/química , DNA Ribossômico/genética , Japão , Microscopia , Dados de Sequência Molecular , Myxozoa/anatomia & histologia , Myxozoa/genética , Filogenia , RNA Ribossômico 18S/genética , RNA Ribossômico 28S/genética , Análise de Sequência de DNA , Esporos/citologiaRESUMO
A case of cystic echinococcosis (CE) in a domestic cat is described from Saint Petersburg, Russia. Ultrasonography showed numerous cysts with hyperechoic walls and anechoic contents within the cat's abdominal cavity. Molecular identification based on mitochondrial DNA genes indicated that the causative agent was Echinococcus granulosus sensu stricto (G1 strain). This is the first report of CE in a cat caused by E. granulosus sensu stricto with molecular confirmation.
Assuntos
Doenças do Gato/diagnóstico , Doenças do Gato/patologia , Equinococose/veterinária , Echinococcus granulosus/isolamento & purificação , Abdome/diagnóstico por imagem , Abdome/patologia , Animais , Doenças do Gato/parasitologia , Gatos , DNA Mitocondrial/química , DNA Mitocondrial/genética , Equinococose/diagnóstico , Equinococose/parasitologia , Equinococose/patologia , Feminino , Dados de Sequência Molecular , Federação Russa , Análise de Sequência de DNA , UltrassonografiaRESUMO
Progress in imaging techniques and nano-manipulation of single molecules has been remarkable. These techniques have allowed the accurate determination of myosin-head-induced displacements and of how the mechanical cycles of the actomyosin motor are coupled to ATP hydrolysis. This has been achieved by measuring mechanical and chemical events of actomyosin directly at the single molecule level. Recent studies have made detailed measurements of myosin step size and mechanochemical coupling. The results of these studies suggest a new model for the mechanism of motion underlying actomyosin motors, which differs from the currently accepted lever-arm swinging model.
Assuntos
Actomiosina/química , Actomiosina/metabolismo , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/metabolismo , Animais , Modelos Químicos , Miosinas/química , Miosinas/metabolismo , Conformação ProteicaRESUMO
The early events in signal transduction from the epidermal growth factor (EGF) receptor (EGFR) are dimerization and autophosphorylation of the receptor, induced by binding of EGF. Here we observe these events in living cells by visualizing single molecules of fluorescent-dye-labelled EGF in the plasma membrane of A431 carcinoma cells. Single-molecule tracking reveals that the predominant mechanism of dimerization involves the formation of a cell-surface complex of one EGF molecule and an EGFR dimer, followed by the direct arrest of a second EGF molecule, indicating that the EGFR dimers were probably preformed before the binding of the second EGF molecule. Single-molecule fluorescence-resonance energy transfer shows that EGF-EGFR complexes indeed form dimers at the molecular level. Use of a monoclonal antibody specific to the phosphorylated (activated) EGFR reveals that the EGFR becomes phosphorylated after dimerization.
Assuntos
Membrana Celular/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Microscopia de Fluorescência/métodos , Transdução de Sinais/fisiologia , Anticorpos Monoclonais/farmacologia , Cálcio/metabolismo , Carbocianinas , Carcinoma/metabolismo , Dimerização , Transferência de Energia/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Corantes Fluorescentes , Humanos , Líquido Intracelular/metabolismo , Fosforilação/efeitos dos fármacos , Rodaminas , Transdução de Sinais/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Kinesin is a molecular motor that moves processively by regular 8-nm steps along microtubules. The processivity of this movement is explained by a hand-over-hand model in which the two heads of kinesin work in a coordinated manner. One head remains bound to the microtubule while the other steps from the alphabeta-tubulin dimer behind the attached head to the dimer in front. The overall movement is 8 nm per ATPase cycle. To investigate elementary processes within the 8-nm step, we have developed a new assay that resolves nanometre displacements of single kinesin molecules with microsecond accuracy. Our data show that the 8-nm step can be resolved into fast and slow substeps, each corresponding to a displacement of approximately 4 nm. The substeps are most probably generated by structural changes in one head of kinesin, leading to rectified forward thermal motions of the partner head. It is also possible that the kinesin steps along the 4-nm repeat of tubulin monomers.
Assuntos
Cinesinas/metabolismo , Animais , Bovinos , Microtúbulos/metabolismoRESUMO
Cytomegalovirus (CMV) reinfection of seropositive individuals has been associated with adverse outcomes in organ transplantation and is a frequent cause of congenital infection. Previously we demonstrated that mismatching of CMV glycoprotein H (gH) serotypes was associated with CMV disease after renal transplantation. Because the antigen domain 2 (AD2) epitope of glycoprotein B (gB) is conserved among CMV isolates and is one of the known targets of neutralizing antibodies, in this study we investigated whether antibodies against the epitope contribute to protection from CMV reinfection in renal transplantation, irrespective of gH serological matching. For this purpose, the gB and gH serology and clinical outcomes were analyzed retrospectively for 77 transplant recipients in the donor positive/recipient positive setting, who were managed by preemptive strategy. We found that there was a good negative correlation between the numbers of antigenemia-positive cells and the levels of antibodies against gB AD2 in the CMV-gH antibody matched group, but not in the CMV-gH antibody mismatched group. None of the recipients with antibodies against both gB AD2 and strain-specific epitopes of gH have experienced CMV disease during 6 month after transplantation, while 28% of those who lacked either/both antibody response needed preemptive therapy. Because the outcome was statistically significant, antibodies against gB AD2 can be a useful indicator to predict emergence of CMV disease for preemptive therapy, in addition to antibodies against the mismatched gH types.
Assuntos
Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Infecções por Citomegalovirus/imunologia , Epitopos/imunologia , Transplante de Rim/efeitos adversos , Proteínas do Envelope Viral/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/química , Citomegalovirus/classificação , Citomegalovirus/imunologia , Infecções por Citomegalovirus/virologia , Epitopos/genética , Humanos , Transplante de Rim/imunologia , Sorotipagem , Especificidade da Espécie , Doadores de Tecidos , Proteínas do Envelope Viral/químicaRESUMO
Mass mortality of cultured yellowtail, Seriola quinqueradiata, has recently been reported from fish farms in western Japan. Previous studies revealed that diseased fish were characterized by encephalomyelitis and presporogonic stages of a myxosporean-like parasite in the spinal cord. However, the parasite has remained unidentified because of the lack of mature stages being present. Thus, in the present study, analysis of the small subunit ribosomal DNA (18S rDNA) of the parasite as well as in situ hybridization (ISH) studies using histological sections of the infected tissue was conducted. The 18S rDNA of the myxosporean had higher sequence similarities with those of bile-duct-infecting myxosporeans rather than those infecting nervous tissues and was identified as Myxobolus spirosulcatus. The ISH using specific probes demonstrated that the DNA amplified was derived from the multinuclear organisms found in histological sections. A highly sensitive and specific PCR-based assay for M. spirosulcatus was developed, which revealed a high prevalence of infection in cultured yellowtail that exhibited the clinical signs of encephalomyelitis.
Assuntos
Encefalomielite/veterinária , Doenças dos Peixes/diagnóstico , Pesqueiros/métodos , Myxobolus/fisiologia , Doenças Parasitárias em Animais/diagnóstico , Reação em Cadeia da Polimerase/veterinária , Animais , Encefalomielite/parasitologia , Doenças dos Peixes/parasitologia , Hibridização In Situ/veterinária , Dados de Sequência Molecular , Myxobolus/classificação , Myxobolus/genética , Doenças Parasitárias em Animais/parasitologia , Perciformes , Filogenia , Reação em Cadeia da Polimerase/métodos , RNA Ribossômico 18S/genética , Sensibilidade e Especificidade , Homologia de Sequência do Ácido NucleicoRESUMO
F-actin in a glycerinated muscle fiber was specifically labeled with fluorescent phalloidin-(fluorescein isothiocyanate) FITC complex at 1:1 molar ratio. Binding of phalloidin-FITC to F-actin affected neither contraction of the fiber nor its regulation by Ca2+. Comparison of polarized fluorescence from phalloidin-FITC bound to F-actin in the relaxed state, rigor, and during isometric contraction of the fiber revealed that the changes in polarization accompanying activation are quantitatively as well as qualitatively different from those accompanying transition of the fiber from the relaxed state to rigor. The extent of the changes of polarized fluorescence during isometric contraction increased with decreasing ionic strength, in parallel with increase in isometric tension. On the other hand, polarized fluorescence was not affected by addition of ADP or by stretching of the fiber in rigor solution. It is concluded from these observations that conformational changes in F-actin are involved in the process of active tension development.
Assuntos
Actinas/fisiologia , Contração Muscular , Músculos/fisiologia , Animais , Fluoresceína-5-Isotiocianato , Fluoresceínas , Corantes Fluorescentes , Contração Isométrica , Microscopia de Fluorescência , Relaxamento Muscular , Faloidina/análogos & derivados , Conformação Proteica , Coelhos , TiocianatosRESUMO
Single-molecule imaging techniques were used to reveal the binding of individual cyclic adenosine 3',5'-monophosphate molecules to heterotrimeric guanine nucleotide-binding protein coupled receptors on the surface of living Dictyostelium discoideum cells. The binding sites were uniformly distributed and diffused rapidly in the plane of the membrane. The probabilities of individual association and dissociation events were greater for receptors at the anterior end of the cell. Agonist-induced receptor phosphorylation had little effect on any of the monitored properties, whereas G protein coupling influenced the binding kinetics. These observations illustrate the dynamic properties of receptors involved in gradient sensing and suggest that these may be polarized in chemotactic cells.
Assuntos
Quimiotaxia , AMP Cíclico/metabolismo , Dictyostelium/fisiologia , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Receptores de AMP Cíclico/metabolismo , Transdução de Sinais , Animais , Carbocianinas/metabolismo , Membrana Celular/metabolismo , Dictyostelium/citologia , Dictyostelium/genética , Dictyostelium/metabolismo , Difusão , Guanosina Difosfato/farmacologia , Guanosina Trifosfato/farmacologia , Proteínas Heterotriméricas de Ligação ao GTP/genética , Cinética , Microscopia de Fluorescência , Mutação , Fosforilação , Pseudópodes/metabolismoRESUMO
F0F1, found in mitochondria or bacterial membranes, synthesizes adenosine 5'-triphosphate (ATP) coupling with an electrochemical proton gradient and also reversibly hydrolyzes ATP to form the gradient. An actin filament connected to a c subunit oligomer of F0 was able to rotate by using the energy of ATP hydrolysis. The rotary torque produced by the c subunit oligomer reached about 40 piconewton-nanometers, which is similar to that generated by the gamma subunit in the F1 motor. These results suggest that the gamma and c subunits rotate together during ATP hydrolysis and synthesis. Thus, coupled rotation may be essential for energy coupling between proton transport through F0 and ATP hydrolysis or synthesis in F1.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/metabolismo , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/metabolismo , Actinas/química , Actinas/metabolismo , Sítios de Ligação , Biotinilação , Transferência de Energia , Enzimas Imobilizadas , Escherichia coli/enzimologia , Hidrólise , Força Próton-Motriz , Desacopladores/metabolismo , Desacopladores/farmacologia , Venturicidinas/farmacologia , Gravação em VídeoRESUMO
In recent years, the rapid development and progress of single-molecule detection techniques have opened up a new era of biological research. The advantage of single-molecule studies is that data are not obscured by the ensemble-averaged measurements inherent in classical biochemical experiments. These techniques are shedding light on the dynamic and mechanistic properties of molecular machines, both in vivo and in vitro. This review summarizes the single-molecule experiments that have been designed to investigate molecular motors, enzyme reactions, protein dynamics, DNA transcription and cell signaling.
Assuntos
Biotecnologia/instrumentação , Biotecnologia/métodos , DNA/ultraestrutura , Cinesinas/química , Transcrição Gênica , Actinas/química , Adenosina Trifosfatases/metabolismo , Animais , Humanos , Canais Iônicos/química , Microscopia de Fluorescência , Modelos Biológicos , Miosinas/química , Transdução de SinaisRESUMO
Techniques have been recently developed that allow the direct observation of single actin filaments and their manipulation, using glass microneedles, in the nanometer range. Further development of these techniques has made possible the detection of subpiconewton-level forces of individual myosin heads. This in vitro motility model is sensitive in the submillisecond range and has allowed us to determine the force generation of an actomyosin motor directly at the molecular level. The results have led to a new conceptual framework for chemo-mechanical energy transduction in the molecular motor.
Assuntos
Actomiosina/fisiologia , Actinas/fisiologia , Trifosfato de Adenosina/fisiologia , Animais , Miosinas/fisiologia , Transdução de SinaisRESUMO
The fungal metabolite brefeldin A (BFA) induces the disassembly of the Golgi complex in mammalian cells. The drug seems to accentuate tubule formation and causes the subsequent fusion with the endoplasmic reticulum (ER). To investigate the biochemical requirements and kinetics of BFA-induced Golgi disassembly, we have reconstituted the process of green fluorescent protein-tagged Golgi complex disassembly in streptolysin O-permeabilized semi-intact Chinese hamster ovary cells. For quantitative analysis of the morphological changes to the Golgi complex in semi-intact cells, we developed a novel morphometric analysis. Based on this analysis, we have dissected the BFA-induced Golgi disassembly process biochemically into two processes, Golgi tubule formation and fusion with the ER, and found that the formation is induced by only ATP and the residual factors in the cells and that the subsequent fusion is mediated in an N-ethylmaleimide-sensitive factor-dependent manner via Golgi tubules. Tubulation occurs by two pathways that depend on either microtubule integrity or exogenously added cytosol. In the presence of GTPgammaS, coat protein I inhibited the Golgi tubule fusion with the ER but showed no apparent effect on tubulation. Additionally, we analyzed the kinetics of tubulation and fusion independently in nocodazole-treated and -untreated semi-intact cells and found that tubulation is a rate-limiting step of the Golgi disassembly.
Assuntos
Brefeldina A/farmacologia , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/ultraestrutura , Fusão de Membrana/efeitos dos fármacos , Microtúbulos/ultraestrutura , Animais , Células CHO , Complexo I de Proteína do Envoltório/metabolismo , Cricetinae , Citosol/fisiologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/fisiologia , Genes Reporter , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/fisiologia , Proteínas de Fluorescência Verde , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Cinética , Proteínas Luminescentes/análise , Microtúbulos/efeitos dos fármacos , Microtúbulos/fisiologia , Proteínas Recombinantes de Fusão/análise , TransfecçãoRESUMO
A serological survey of pig cysticercosis was conducted in a hill-tribe village at Thai-Myanmar border, Tak province of Thailand in 2012. Sixteen backyard pigs were examined for pig cysticercosis and gastrointestinal helminth infection. In addition to cysticerci of Taenia solium and Taenia hydatigena found outside the gut, nine other helminth species were found in guts: Echinostoma malayanum, Pseudanoplocephala crawfordi, Ascarops dentata, Physocephalus sexalatus, Gnathostoma doloresi, Ascaris suum, Globocephalus sp., Oesophagostomum dentatum and Bourgelatia diducta. The study presents a report for the first time of adult tapeworm, P. crawfordi infection in pigs from Thailand. For medical importance, E. malayanum, P. crawfordi, G. doloresi and A. suum have been confirmed as potentially zoonotic helminths and pigs may act as one of the reservoir hosts for human helminthiases. Pigs of both gender and all ages appeared to be exposed to the parasites equally and did not show any significant difference to these helminth species in richness and total intensity.
RESUMO
Mechanosensitive biological nanomachines such as motor proteins and ion channels regulate diverse cellular behaviour. Combined optical trapping with single-molecule fluorescence imaging provides a powerful methodology to clearly characterize the mechanoresponse, structural dynamics and stability of such nanomachines. However, this system requires complicated experimental geometry, preparation and optics, and is limited by low data-acquisition efficiency. Here we develop a programmable DNA origami nanospring that overcomes these issues. We apply our nanospring to human myosin VI, a mechanosensory motor protein, and demonstrate nanometre-precision single-molecule fluorescence imaging of the individual motor domains (heads) under force. We observe force-induced transitions of myosin VI heads from non-adjacent to adjacent binding, which correspond to adapted roles for low-load and high-load transport, respectively. Our technique extends single-molecule studies under force and clarifies the effect of force on biological processes.
Assuntos
Cadeias Pesadas de Miosina/química , Nanotecnologia , Transporte Biológico , Humanos , Mecanotransdução Celular , Cadeias Pesadas de Miosina/ultraestrutura , Imagem ÓpticaRESUMO
By using glycerinated single fibers of crab muscle (Sesarma haematocheir) which has long sarcomeres, the birefringence of the I band, H band and the overlapping region between thin and thick filaments was measured separately, under various environmental conditions. At the resting length, the birefringence of the fiber was decreased by the addition of Ca2+ in the absence of ATP, by about 0.35%. This birefringence decrease was found to take place in the overlapping region. The decrease corresponded to about 2% of the birefringence of thin filaments in this region. The birefringence of the fiber was increased by the addition of ATP in the absence of Ca2+, by about 6%. This birefringence increase also took place mostly in the overlapping region. The increase of birefringence by pyrophosphate was about half of that by ATP. The birefringence of the fiber was decreased by the increase of the ionic strength from 0.12 to 0.20. The origin of the observed changes of birefringence is discussed.