RESUMO
The Difficult Airway Society recommends that all patients should be pre-oxygenated before the induction of general anaesthesia, but this may not always be easy or comfortable and anaesthesia may often be induced without full pre-oxygenation. We tested the hypothesis that high-flow nasal oxygen cannulae would be easier and more comfortable than facemasks for pre-oxygenation. We randomly allocated 199 patients undergoing elective surgery aged ≥ 10 years to pre-oxygenation using either high-flow nasal oxygen or facemask. Ease and comfort were assessed by anaesthetists and patients on 10-cm visual analogue scale and six-point smiley face scale, respectively. Secondary endpoints included end-tidal oxygen fraction after securing a definitive airway and time to secure an airway. A mean difference (95%CI) between groups in ratings of -0.76 (-1.25 to -0.27) cm for ease of use (p = 0.003) and -0.45 (-0.75 to -0.13) points for comfort (p = 0.006), both favoured high-flow nasal oxygen. A mean difference (95%CI) between groups in end-tidal oxygen fraction of 3.89% (2.41-5.37%) after securing a definitive airway also favoured high-flow nasal oxygen (p < 0.001). There was no significant difference between groups in the number of patients with hypoxaemia (Sp O2 < 90%) or severe hypoxaemia (Sp O2 < 85%) lasting ≥ 1 min or ≥ 2 min; in the proportion of patients with an end-tidal oxygen fraction < 87% in the first 5 min after tracheal intubation (52.2% vs. 58.9% in facemask and high-flow nasal oxygen groups, respectively; p = 0.31); or in time taken to secure an airway (11.6 vs. 12.2 min in facemask and high-flow nasal oxygen groups, respectively; p = 0.65). In conclusion, we found pre-oxygenation with high-flow nasal oxygen to be easier for anaesthetists and more comfortable for patients than pre-oxygenation with a facemask, with no clinically relevant differences in end-tidal oxygen fraction after securing a definitive airway or time to secure an airway. The differences in ease and comfort were modest.
Assuntos
Máscaras , Oxigênio , Humanos , Cânula , Administração Intranasal , Hipóxia , OxigenoterapiaRESUMO
Homophilous behaviour plays a central role in the formation of human friendships. Individuals form social ties with others that show similar phenotypic traits, independently of relatedness. Evidence of such homophily can be found in bottlenose dolphins ( Tursiops aduncus) in Shark Bay, Western Australia, where females that use marine sponges as foraging tools often associate with other females that use sponges. 'Sponging' is a socially learned, time-consuming behaviour, transmitted from mother to calf. Previous research illustrated a strong female bias in adopting this technique. The lower propensity for males to engage in sponging may be due to its incompatibility with adult male-specific behaviours, particularly the formation of multi-level alliances. However, the link between sponging and male behaviour has never been formally tested. Here, we show that male spongers associated significantly more often with other male spongers irrespective of their level of relatedness. Male spongers spent significantly more time foraging, and less time resting and travelling, than did male non-spongers. Interestingly, we found no difference in time spent socializing. Our study provides novel insights into the relationship between tool use and activity budgets of male dolphins, and indicates social homophily in the second-order alliance composition of tool-using bottlenose dolphins.
Assuntos
Comportamento Animal , Golfinho Nariz-de-Garrafa/fisiologia , Comportamento Social , Comportamento de Utilização de Ferramentas , Animais , Feminino , Masculino , Caracteres Sexuais , Aprendizado SocialRESUMO
Monitoring the presence of commensal and pathogenic respiratory microorganisms is of critical global importance. However, community-based surveillance is difficult because nasopharyngeal swabs are uncomfortable and painful for a wide age range of participants. We designed a methodology for minimally invasive self-sampling at home and assessed its use for longitudinal monitoring of the oral, nasal and hand microbiota of adults and children within families. Healthy families with two adults and up to three children, living in and near Liverpool, United Kingdom, self-collected saliva, nasal lining fluid using synthetic absorptive matrices and hand swabs at home every two weeks for six months. Questionnaires were used to collect demographic and epidemiological data and assess feasibility and acceptability. Participants were invited to take part in an exit interview. Thirty-three families completed the study. Sampling using our approach was acceptable to 25/33 (76%) families, as sampling was fast (76%), easy (76%) and painless (60%). Saliva and hand sampling was acceptable to all participants of any age, whereas nasal sampling was accepted mostly by adults and children older than 5 years. Multi-niche self-sampling at home can be used by adults and children for longitudinal surveillance of respiratory microorganisms, providing key data for design of future studies.
Assuntos
Microbiota , Nariz , Adulto , Criança , Humanos , Pré-Escolar , Inquéritos e Questionários , Manejo de Espécimes/métodos , SalivaRESUMO
We measured a change in the current transport of an antenna-coupled, multigate, GaAs/AlGaAs field-effect transistor when terahertz electromagnetic waves irradiated the transistor and attribute the change to bolometric heating of the electrons in the two dimensional electron channel. The observed terahertz absorption spectrum indicates coherence between plasmons excited under adjacent biased device gates. The experimental results agree quantitatively with a theoretical model we developed that is based on a generalized plasmonic transmission line formalism and describes an evolution of the plasmonic spectrum with increasing electron density modulation from homogeneous to the crystal limit. These results demonstrate an electronically induced and dynamically tunable plasmonic band structure.
RESUMO
In many Asian populations, the commonest form of severe thalassemia results from the coinheritance of HbE and beta thalassemia. The management of this disease is particularly difficult because of its extreme clinical diversity; although some genetic and adaptive factors have been identified as phenotypic modifiers, the reasons remain unclear. Because the role of the environment in the course of severe thalassemia has been neglected completely and because malaria due to both Plasmodium falciparum and Plasmodium vivax has been prevalent in Sri Lanka, we carried out a pilot study of patients with HbE beta thalassemia that showed high frequencies of antibodies to both parasite species and that 28.6% of the children had DNA-based evidence of current infection with P. vivax. Malarial antibodies then were assessed in patients with HbE beta thalassemia compared with those in age-matched controls. There was a significant increase in the frequency of antibodies in the thalassemic patients, particularly against P. vivax and in young children. There was also a higher frequency in those who had been splenectomized compared with those with intact spleens, although in the latter it was still higher than that in the controls. The thalassemic patients showed significant correlations between malaria antibody status and phenotype. Patients with HbE beta thalassemia may be more prone to malaria, particularly P. vivax, which is reflected in their clinical severity. Because P. vivax malaria is widespread in Asia, further studies of its interaction with HbE beta thalassemia and related diseases are required urgently as a part of ongoing thalassemia control programs.
Assuntos
Povo Asiático , Malária/complicações , Talassemia beta/complicações , Talassemia beta/patologia , Adolescente , Adulto , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Estudos de Casos e Controles , Criança , Exposição Ambiental , Humanos , Malária/epidemiologia , Malária/imunologia , Fenótipo , Projetos Piloto , Prevalência , Esplenectomia , Sri Lanka/epidemiologia , Talassemia beta/imunologiaRESUMO
We previously have described a model of multiple sclerosis (MS) in which constitutive expression of murine interleukin (IL)-2 by herpes simplex virus type 1 (HSV-1) (HSV-IL-2) causes central nervous system (CNS) demyelination in different strains of mice. In the current study, we investigated whether this HSV-IL-2-induced demyelination can be blocked using recombinant viruses expressing different cytokines or by injection of plasmid DNA. We have found that coinfection of HSV-IL-2-infected mice with recombinant viruses expressing IL-12p35, IL-12p40 or IL-12p35+IL-12p40 did not block the CNS demyelination, and that coinfection with a recombinant virus expressing interferon (IFN)-γ exacerbated it. In contrast, coinfection with a recombinant virus expressing IL-4 reduced demyelination, whereas coinfection of HSV-IL-2-infected mice with a recombinant HSV-1 expressing the IL-12 heterodimer (HSV-IL-12p70) blocked the CNS demyelination in a dose-dependent manner. Similarly, injection of IL-12p70 DNA blocked HSV-IL-2-induced CNS demyelination in a dose-dependent manner and injection of IL-35 DNA significantly reduced CNS demyelination. Injection of mice with IL-12p35 DNA, IL-12p40 DNA, IL-12p35+IL-12p40 DNA or IL-23 DNA did not have any effect on HSV-IL-2-induced demyelination, whereas injection of IL-27 DNA increased the severity of the CNS demyelination in the HSV-IL-2-infected mice. This study demonstrates for the first time that IL-12p70 can block HSV-IL-2-induced CNS demyelination and that IL-35 can also reduce this demyelination, whereas IFN-γ and IL-27 exacerbated the demyelination in the CNS of the HSV-IL-2-infected mice. Our results suggest a potential role for IL-12p70 and IL-35 signaling in the inhibition of HSV-IL-2-induced immunopathology by preventing development of autoaggressive T cells.
Assuntos
Citocinas/genética , Doenças Desmielinizantes/prevenção & controle , Vetores Genéticos , Herpesvirus Humano 1/genética , Animais , Sistema Nervoso Central/patologia , Feminino , Imunoterapia , Interleucina-12/genética , Interleucina-2/genética , Interleucinas/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
The purpose of this study was to quantify the magnitude and frequency content of surface-measured accelerations at each major human body segment from foot to head during impact landings. Twelve males performed two single leg drop landings from each of 0.15 m, 0.30 m, and 0.45 m. Triaxial accelerometers (2000 Hz) were positioned over the: first metatarsophalangeal joint; distal anteromedial tibia; superior to the medial femoral condyle; L5 vertebra; and C6 vertebra. Analysis of acceleration signal power spectral densities revealed two distinct components, 2-14 Hz and 14-58 Hz, which were assumed to correspond to time domain signal joint rotations and elastic wave tissue deformation, respectively. Between each accelerometer position from the metatarsophalangeal joint to the L5 vertebra, signals exhibited decreased peak acceleration, increased time to peak acceleration, and decreased power spectral density integral of both the 2-14 Hz and 14-58 Hz components, with no further attenuation beyond the L5 vertebra. This resulted in peak accelerations close to vital organs of less than 10% of those at the foot. Following landings from greater heights, peak accelerations measured distally were greater, as was attenuation prior to the L5 position. Active and passive mechanisms within the lower limb therefore contribute to progressive attenuation of accelerations, preventing excessive accelerations from reaching the torso and head, even when distal accelerations are large.
Assuntos
Corrida , Aceleração , Fenômenos Biomecânicos , Pé , Humanos , Masculino , TíbiaRESUMO
We report four patients with pandemic H1N1 2009 influenza virus and secondary bacterial infection who were treated with extracorporeal membrane oxygenation (ECMO) for cardiorespiratory failure. Three of the four patients had profound shock, necessitating support with venoarterial ECMO. Two patients died during ECMO support. The two survivors had prolonged hospital stays, which were complicated by renal failure and limb ischaemia.
Assuntos
Infecções Bacterianas/complicações , Oxigenação por Membrana Extracorpórea/métodos , Vírus da Influenza A Subtipo H1N1 , Influenza Humana/terapia , Síndrome do Desconforto Respiratório/terapia , Injúria Renal Aguda/microbiologia , Adolescente , Adulto , Evolução Fatal , Feminino , Humanos , Influenza Humana/complicações , Masculino , Infecções Oportunistas/complicações , Síndrome do Desconforto Respiratório/virologiaRESUMO
This study aimed to measure neuromuscular function for the masticatory muscles under a range of occlusal conditions in healthy, dentate adults. Forty-one subjects conducted maximum voluntary clenches under nine different occlusal loading conditions encompassing bilateral posterior teeth contacts with the mandible in different positions, anterior teeth contacts and unilateral posterior teeth contacts. Surface electromyography was recorded bilaterally from the anterior temporalis, superficial masseter, sternocleidomastoid, anterior digastric and trapezius muscles. Clench condition had a significant effect on muscle function (P = 0.0000) with the maximum function obtained for occlusions with bilateral posterior contacts and the mandible in a stable centric position. The remaining contact points and moving the mandible to a protruded position, whilst keeping posterior contacts, resulted in significantly lower muscle activities. Clench condition also had a significant effect on the per cent overlap, anterior-posterior and torque coefficients (P = 0.0000-0.0024), which describe the degree of symmetry in these muscle activities. Bilateral posterior contact conditions had significantly greater symmetry in muscle activities than anterior contact conditions. Activity in the sternocleidomastoid, anterior digastric and trapezius was consistently low for all clench conditions, i.e. <20% of the maximum voluntary contraction level. In conclusion, during maximum voluntary clenches in a healthy population, maximum masticatory muscle activity requires bilateral posterior contacts and the mandible to be in a stable centric position, whilst with anterior teeth contacts, both the muscle activity and the degree of symmetry in muscle activity are significantly reduced.
Assuntos
Oclusão Dentária , Músculos da Mastigação/fisiologia , Junção Neuromuscular/fisiologia , Adulto , Dente Pré-Molar/anatomia & histologia , Força de Mordida , Oclusão Dentária Central , Eletromiografia , Feminino , Humanos , Incisivo/anatomia & histologia , Masculino , Mandíbula/anatomia & histologia , Músculo Masseter/fisiologia , Dente Molar/anatomia & histologia , Contração Muscular/fisiologia , Músculo Esquelético/fisiologia , Músculos do Pescoço/fisiologia , Músculo Temporal/fisiologia , Fatores de Tempo , Torque , Adulto JovemRESUMO
A plasmonic terahertz detector that integrates a voltage-controlled planar barrier into a grating gated GaAs/AlGaAs high electron mobility transistor has been fabricated and experimentally characterized. The plasmonic response at fixed grating gate voltage has a full width at half-maximum of 40 GHz at â¼405 GHz. Substantially increased responsivity is achieved by introducing an independently biased narrow gate that produces a lateral potential barrier electrically in series with the resonant grating gated region. DC electrical characterization in conjunction with bias-dependent terahertz responsivity and time constant measurements indicate that a hot electron bolometric effect is the dominant response mechanism at 20 K.
RESUMO
Nerve growth factor (NGF) is involved in the development and maintenance of the nervous system. NGF binds with high affinity to the extracellular region of the tyrosine kinase receptor TrkA. This domain comprises leucine and cysteine rich motifs, followed by two immunoglobulin like (Ig-like) domains. We describe the expression and purification of recombinant Ig-like domains. Fluorescence and circular dichroism spectroscopy show that the protein is folded into a compact globular structure and contains mainly beta-sheet secondary structure. Recombinant protein binds to NGF and can inhibit NGF bioactivity both in vitro and in vivo.
Assuntos
Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Fatores de Crescimento Neural/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Ligação Competitiva , Biotecnologia , Dicroísmo Circular , Humanos , Imunoglobulinas/química , Técnicas In Vitro , Masculino , Dados de Sequência Molecular , Fatores de Crescimento Neural/antagonistas & inibidores , Células PC12 , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas Proto-Oncogênicas/química , Ratos , Ratos Wistar , Receptores Proteína Tirosina Quinases/química , Receptor trkA , Receptores de Fator de Crescimento Neural/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de FluorescênciaRESUMO
The method described here cannot fully replace the analysis of large columns by small test columns (microcolumns). The procedure, however, is suitable for speeding up the determination of adsorption parameters of dye onto the adsorbent and for speeding up the initial screening of a large adsorbent collection that can be tedious if a several adsorbents and adsorption conditions must be tested. The performance of methylene blue (MB), a basic dye, Cibacron reactive black (RB) and Cibacron reactive yellow (RY) was predicted in this way and the influence of initial dye concentration and other adsorption conditions on the adsorption behaviour were demonstrated. On the basis of the experimental results, it can be concluded that the adsorption of RY onto manganese oxides modified diatomite (MOMD) exhibited a characteristic "S" shape and can be simulated effectively by the Thomas model. It is shown that the adsorption capacity increased as the initial dye concentration increased. The increase in the dye uptake capacity with the increase of the adsorbent mass in the column was due to the increase in the surface area of adsorbent, which provided more binding sites for the adsorption. It is shown that the use of high flow rates reduced the time that RY in the solution is in contact with the MOMD, thus allowing less time for adsorption to occur, leading to an early breakthrough of RY. A rapid decrease in the column adsorption capacity with an increase in particle size with an average 56% reduction in capacity resulting from an increase in the particle size from 106-250 microm to 250-500 microm. The experimental data correlated well with calculated data using the Thomas equation and the bed depth-service time (BDST) equation. Therefore, it might be concluded that the Thomas equation and the BDST equations can produce accurate predication for variation of dye concentration, mass of the adsorbent, flow rate and particle size. In general, the values of adsorption isotherm capacity obtained in a batch system show the maximum values and are considerably higher than those obtained in a fixed-bed.
Assuntos
Corantes/química , Terra de Diatomáceas/química , Poluentes Químicos da Água/química , Adsorção , Cloretos/química , Compostos de Manganês/química , Azul de Metileno/química , Tamanho da Partícula , Hidróxido de Sódio/química , Fatores de Tempo , Eliminação de Resíduos Líquidos/métodos , Purificação da Água/métodosRESUMO
Sexual displays enriched by object carrying serve to increase individual male fitness, yet are uncommon phenomena in the animal kingdom. While they have been documented in a variety of taxa, primarily birds, they are rare outside non-human mammals. Here, we document marine sponge presenting associated with visual and acoustic posturing found in several, geographically widespread populations of Australian humpback dolphins (Sousa sahulensis) over ten years of observation. Only adult males presented marine sponges, typically doing so in the presence of sexually mature females, although social groups predominantly consisted of mixed age and sex classes. Male humpback dolphins appear to be using sponges for signalling purposes in multi-modal sexual displays. Further, based on limited behavioural and genetic data, we hypothesise that pairs of adult male Sousa form at least temporary coalitions or alliances. The use of objects in sexual displays by non-human mammals is rare and, moreover, cooperation between males in the pursuit of an indivisible resource is an evolutionary hurdle relatively few species have overcome. These findings suggest a hitherto unrecognised level of social complexity in humpback dolphins.
Assuntos
Golfinhos , Comportamento Sexual Animal , Agressão , Animais , Austrália , Comportamento Cooperativo , Golfinhos/psicologia , Feminino , Masculino , Oceanos e Mares , PoríferosRESUMO
During a prospective study of red cell variants and severe malaria in children, a surprising observation was the occurrence of dark urine. Children were grouped according to urine findings: 22 had dark urine that contained a haem protein (Group I), 93 had urine of normal colour that contained a haem protein (Group II) and 236 had normal urine (Group III). To investigate the cause of dark urine, haemolysis and muscle cell injury were assessed. Intravascular haemolysis was greater in Group I than in Groups II and III. However, anaemia was more severe in Group III and is likely to have resulted mainly from extravascular haemolysis. Median plasma myoglobin concentrations were greater in Groups I and II than Group III (P = 0.00060). Plasma myoglobin was greater in children with cerebral malaria, hyperlactataemia and those who died but was not associated with acidosis. Urine myoglobin was greater in Group I than Groups II and III (P = 0.00054). It is likely that both haemoglobin and myoglobin contributed to dark urine. The association between muscle cell injury and coma suggests sequestration of parasitized red cells as a common underlying pathology. In malaria, hyperlactataemia may result directly from breakdown of muscle protein as well as tissue hypoxia.
Assuntos
Febre Hemoglobinúrica/etiologia , Hemólise , Células Musculares/patologia , Anemia Hemolítica/sangue , Anemia Hemolítica/complicações , Anemia Hemolítica/urina , Bilirrubina/análise , Febre Hemoglobinúrica/sangue , Febre Hemoglobinúrica/urina , Criança , Pré-Escolar , Eritrócitos/patologia , Feminino , Hemoglobinas/análise , Hemoglobinúria/sangue , Hemoglobinúria/complicações , Hemoglobinúria/urina , Humanos , Lactente , Fígado/enzimologia , Masculino , Mioglobina/análise , Mioglobinúria/sangue , Mioglobinúria/complicações , Mioglobinúria/urina , Papua Nova Guiné , Estudos ProspectivosRESUMO
BACKGROUND: The binding of neurotrophin ligands to their respective Trk cellular receptors initiates intracellular signals essential for the growth and survival of neurons. The site of neurotrophin binding has been located to the fifth extracellular domain of the Trk receptor, with this region regulating both the affinity and specificity of Trk receptor:neurotrophin interaction. Neurotrophin function has been implicated in a number of neurological disorders, including Alzheimer's disease and Parkinson's disease. RESULTS: We have determined the 2.7 A crystal structure of neurotrophin-4/5 bound to the neurotrophin binding domain of its high-affinity receptor TrkB (TrkB-d5). As previously seen in the interaction of nerve growth factor with TrkA, neurotrophin-4/5 forms a crosslink between two spatially distant receptor molecules. The contacts formed in the TrkB-d5:neurotrophin-4/5 complex can be divided into a conserved area similar to a region observed in the TrkA-d5:NGF complex and a second site-unique in each ligand-receptor pair-formed primarily by the ordering of the neurotrophin N terminus. CONCLUSIONS: Together, the structures of the TrkB-d5:NT-4/5 and TrkA-d5:NGF complexes confirm a consistent pattern of recognition in Trk receptor:neurotrophin complex formation. In both cases, the N terminus of the neurotrophin becomes ordered only on complex formation. This ordering appears to be directed largely by the receptor surface, with the resulting complementary surfaces providing the main determinant of receptor specificity. These features provide an explanation both for the limited crossreactivity observed between the range of neurotrophins and Trk receptors and for the high-affinity binding associated with respective ligand-receptor pairs.
Assuntos
Fatores de Crescimento Neural/química , Receptor trkB/química , Motivos de Aminoácidos , Animais , Sítios de Ligação , Cromatografia em Gel , Sequência Conservada , Cristalografia por Raios X , Dimerização , Escherichia coli/metabolismo , Humanos , Ligantes , Modelos Moleculares , Fatores de Crescimento Neural/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptor trkB/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Bacteriorhodopsin functions as a light-driven proton pump in Halobacterium salinarium. The functional protein consists of an apoprotein, bacterioopsin, with seven transmembrane alpha helices together with a covalently bound all-trans retinal chromophore. In order to study the role of the interhelical loop conformations in the structure and function of bacteriorhodopsin, we have constructed bacterioopsin genes where each loop is replaced, one at a time, by a peptide linker consisting of Gly-Gly-Ser- repeat sequences, which are believed to have flexible conformations. These mutant proteins have been expressed in Escherichia coli, purified and reconstituted with all-trans retinal in l-alpha-1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/3-(3-cholamidopropyl)dimethylammonio-1-propane sulfonate (CHAPS)/SDS and l-alpha-1,2-dihexanoylphosphatidylcholine (DHPC)/DMPC/SDS micelles. Wild-type-like chromophore formation was observed in all the mutants containing single loop replacements. In the BC and FG mutants, an additional chromophore band with an absorption band at about 480 nm was observed, which was in equilibrium with the 550 nm, wild-type band. The position of the equilibrium depended on temperature, SDS and relative DMPC concentration. The proton pumping activity of all of the mutants was comparable to that of wild-type bR except for the BC and FG mutants, which had lower activity. All of the loop mutants were more sensitive to denaturation by SDS than the wild-type protein, except the mutant where the DE loop was replaced. These results suggest that a specific conformation of all the loops of bR, except the DE loop, contributes to bR stability and is required for the correct folding and function of the protein. An increase in the relative proportion of DHPC in DHPC/DMPC micelles, which reduces the micelle rigidity and alters the micelle shape, resulted in lower folding yields of all loop mutants except the BC and DE mutants. This effect of micelle rigidity on the bR folding yield correlated with a loss in stability of a partially folded, seven-transmembrane apoprotein intermediate state in SDS/DMPC/CHAPS micelles. The folding yield and stability of the apoprotein intermediate state both decreased for the loop mutants in the order WT approximately BC approximately DE>FG>AB>EF> or =CD, where the EF and CD loop mutants were the least stable.
Assuntos
Bacteriorodopsinas/química , Bacteriorodopsinas/metabolismo , Halobacterium salinarum/química , Dobramento de Proteína , Sequência de Aminoácidos , Bacteriorodopsinas/genética , Ácidos Cólicos/metabolismo , Dimiristoilfosfatidilcolina/metabolismo , Halobacterium salinarum/genética , Hidroxilamina/metabolismo , Transporte de Íons/efeitos da radiação , Cinética , Luz , Micelas , Modelos Moleculares , Dados de Sequência Molecular , Éteres Fosfolipídicos/metabolismo , Desnaturação Proteica , Engenharia de Proteínas , Estrutura Secundária de Proteína , Prótons , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Retinaldeído/química , Retinaldeído/metabolismo , Dodecilsulfato de Sódio/metabolismo , Análise Espectral , Relação Estrutura-Atividade , TermodinâmicaRESUMO
The loops connecting the seven transmembrane helices of bacteriorhodopsin have each been replaced in turn by structureless linkers of Gly-Gly-Ser repeat sequences, and the effect on the protein folding kinetics has been determined. An SDS-denatured state of each loop mutant bacterio-opsin was folded in l-alpha-1,2-dihexanoylphosphatidylcholine/l-alpha-1,2-dimyristoylphosphatidylcholine micelles, containing retinal, to give functional bacteriorhodopsin. Stopped-flow mixing was used to initiate the folding reaction, giving a time resolution of milliseconds, and changes in protein fluorescence were used to monitor folding. All loop mutant proteins folded according to the same reaction scheme as wild-type protein. The folding kinetics of the AB, BC and DE loop mutants were the same as wild-type protein, despite the blue-shifted chromophore band of the BC loop mutant bR state. A partially folded apoprotein intermediate state of the AB loop mutant did however appear to decay in the absence of retinal. The most significant effects on the folding kinetics were seen for mutant protein with structureless linkers in place of the CD, EF and FG loops. The rate-limiting apoprotein folding step of the CD loop mutant was about ten times slower than wild-type, whilst that of the EF loop mutant was almost four times slower than wild-type. Wild-type behaviour was observed for the other folding and retinal binding events of the CD and EF loop mutant proteins. These effects of the CD and EF loop mutations on apoprotein folding correlate with the fact that these two loop mutants also have the least stable, partially folded apoprotein intermediate of all the loop mutants, and are the most affected by a decrease in lipid lateral pressure. In contrast, the FG loop mutant exhibited wild-type apoprotein folding, but altered covalent binding of retinal and final folding to bacteriorhodopsin. This correlates with the fact that the FG loop mutant bacteriorhodopsin is the most susceptible to denaturation by SDS of all the loop mutants, but its partially folded apoprotein intermediate is more stable than that of the CD and EF mutants. Thus the CD and EF loops may contribute to the transition state for the rate-limiting apoprotein folding step and the FG loop to that for final folding and covalent binding of retinal.
Assuntos
Bacteriorodopsinas/química , Bacteriorodopsinas/metabolismo , Halobacterium salinarum/química , Dobramento de Proteína , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/metabolismo , Bacteriorodopsinas/genética , Ácidos Cólicos/metabolismo , Dimiristoilfosfatidilcolina/metabolismo , Fluorescência , Halobacterium salinarum/genética , Cinética , Micelas , Mutação/genética , Éteres Fosfolipídicos/metabolismo , Renaturação Proteica , Retinaldeído/metabolismo , Dodecilsulfato de Sódio/metabolismo , Análise Espectral , Relação Estrutura-Atividade , TermodinâmicaRESUMO
Free-Electron Lasers (FELs) collectively operate from the terahertz through the ultraviolet range and via intracavity Compton backscattering into the X-ray and gamma-ray regimes. FELs are continuously tunable and can provide optical powers, pulse structures and polarizations that are not matched by conventional lasers. Representative research in the biological and biomedical sciences and condensed matter and material research are described to illustrate the breadth and impact of FEL applications. These include terahertz dynamics in materials far from equilibrium, infrared nonlinear vibrational spectroscopy to investigate dynamical processes in condensed-phase systems, infrared resonant-enhanced multiphoton ionization for gas-phase spectroscopy and spectrometry, infrared matrix-assisted laser-desorption-ionization and infrared matrix-assisted pulsed laser evaporation for analysis and processing of organic materials, human neurosurgery and ophthalmic surgery using a medical infrared FEL and ultraviolet photoemission electron microscopy for nanoscale characterization of materials and nanoscale phenomena. The ongoing development of ultraviolet and X-ray FELs are discussed in terms of future opportunities for applications research.