RESUMO
Phenomenological neural models, such as the leaky integrate-and-fire model, normally have a fixed refractory time-course that is independent of the stimulus. The recovery of threshold following an action potential is typically based on physiological experiments that use a two-pulse paradigm in which the first pulse is suprathreshold and causes excitation and the second pulse is used to determine the threshold at various intervals following the first. In such experiments, the nerve is completely unstimulated between the two pulses. This contrasts the receptor stimuli in normal physiological systems and the electrical stimuli used by cochlear implants and other neural prostheses. A numerical study of the Frankenhaeuser-Huxley conductance-based model of nerve fibre was therefore undertaken to investigate the effect of stimulation on refractoriness. We found that the application of a depolarizing stimulus during the later part of what is classically regarded as the absolute refractory period could effectively prolong the absolute refractory period, while leaving the refractory time-constants and other refractory parameters largely unaffected. Indeed, long depolarizing pulses, which would have been suprathreshold if presented to a resting nerve fibre, appeared to block excitation indefinitely. Stimulation during what is classically regarded as the absolute refractory period can therefore greatly affect the temporal response of a nerve. We conclude that the classical definition of absolute refractory period should be refined to include only the initial period following an action potential when an ongoing stimulus would not affect threshold; this period was found to be about half as long as the classical absolute refractory period. We further conclude that the stimulus-dependent nature of the relative refractory period must be considered when developing a phenomenological nerve model for complex stimuli.
Assuntos
Modelos Neurológicos , Período Refratário Eletrofisiológico , Potenciais de Ação , Estimulação Elétrica , Análise Numérica Assistida por Computador , Fatores de TempoRESUMO
To evaluate coding strategies for cochlear implants a model of the human cochlear nerve is required. Nerve models based on voltage-clamp experiments, such as the Frankenhaeuser-Huxley model of myelinated nerve, can have over forty parameters and are not amenable for fitting to physiological data from a different animal or type of nerve. Phenomenological nerve models, such as leaky integrate-and-fire (LIF) models, have fewer parameters but have not been validated with a wide range of stimuli. In the absence of substantial cochlear nerve data, we have used data from a toad sciatic nerve for validation (50 Hz to 2 kHz with levels up to 20 dB above threshold). We show that the standard LIF model with fixed refractory properties and a single set of parameters cannot adequately predict the toad rate-level functions. Given the deficiency of this standard model, we have abstracted the dynamics of the sodium inactivation variable in the Frankenhaeuser-Huxley model to develop a phenomenological LIF model with a dynamic threshold. This nine-parameter model predicts the physiological rate-level functions much more accurately than the standard LIF model. Because of the low number of parameters, we expect to be able to optimize the model parameters so that the model is more appropriate for cochlear implant simulations.
Assuntos
Nervo Coclear/fisiologia , Modelos Neurológicos , Bainha de Mielina/metabolismo , Estimulação Elétrica , HumanosRESUMO
There is much evidence supporting the efficacy of communication skills training; however, very little of this evidence comes from patient feedback. The primary aim of this pilot study was to evaluate whether the advanced communications skills training improves patients' experience of consultations. Healthcare professionals working in oncology and palliative care services from the North East of England were invited to participate in this study. Interactions between healthcare professionals (n = 21) and patients (n = 1103) were evaluated using the Consultation and Relational Empathy (CARE) Measure, which is a brief questionnaire designed to assess the patients' perceptions of relational empathy in the consultation. Additional demographic variables, such as patient age, length of consultation, familiarity with healthcare professional and overall satisfaction with consultation, were also collected. Healthcare professionals were either part of the intervention group who attended a 3-day communication skills training course or part of the control group who were on the waiting list for training. No differences in the patients' ratings on the CARE measure were found between Time 1 (before training) and Time 2 (after training) for the intervention group. Possible explanations for the findings are explored and implications for communication skills training are discussed.
Assuntos
Comunicação , Oncologia/educação , Neoplasias/terapia , Cuidados Paliativos , Satisfação do Paciente , Encaminhamento e Consulta/normas , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Inglaterra , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Relações Médico-Paciente , Projetos Piloto , Competência Profissional , Adulto JovemRESUMO
Profoundly deaf people, who gain no benefit from conventional hearing aids, can receive speech cues by direct electrical stimulation of the cochlear nerve. This is achieved by an electronic device, a cochlear implant, which is surgically inserted into the ear. Here we show physiological results from the isolated sciatic nerve of the toad Xenopus laevis, used to predict the response of the human cochlear nerve to vowels coded by a cochlear implant. These results suggest that standard analogue cochlear implants do not evoke the patterns of neural excitation that are normally associated with acoustic stimulation. Adding noise to the stimulus, however, enhanced distinguishing features of the vowel encoded by the fine time structure of neural discharges. On the basis of these results, and those concerning stochastic resonance, we advocate a cochlear implant coding strategy in which noise is deliberately added to cochlear implant signals.
Assuntos
Implantes Cocleares , Nervo Isquiático/fisiologia , Potenciais de Ação , Animais , Surdez/fisiopatologia , Surdez/terapia , Estimulação Elétrica , Feminino , Humanos , Técnicas In Vitro , Idioma , Ruído , Xenopus laevisRESUMO
Studies in vivo and in vitro have shown that the packaging of DNA into chromatin can affect gene expression. Here, binding of the yeast transcriptional activator GAL4 to DNA in chromatin has been investigated in vivo with a yeast episome. A positioned nucleosome that is present in cells grown in glucose and contains a single GAL4 binding site is disrupted by GAL4 binding in galactose. GAL4 can also bind to DNA in chromatin when the carboxyl-terminal activation domain of GAL4 is either masked by GAL80 or is absent. These results show that a transcription factor can bind to its site in vivo in what would appear to be a repressive chromatin structure.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , Sítios de Ligação , Galactose/metabolismo , Glucose/metabolismo , Dados de Sequência Molecular , Plasmídeos , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Aflatoxin B1, B2, G1, and G2 are produced when a variant of Aspergillus oryzae (NRRL strain 1988) is grown on cowpeas or rice. The present study indicates that a strain of Aspergillus oryzae approved for use in food processing is variable and the resulting variant, unlike the parent strain, has a propensity to produce aflatoxin.
Assuntos
Aflatoxinas/biossíntese , Aspergillus oryzae/metabolismo , Aspergillus/metabolismo , Contaminação de Alimentos , Verduras , Aflatoxinas/toxicidade , Animais , Microbiologia de Alimentos , Conservação de Alimentos , Variação Genética , Masculino , RatosRESUMO
Direct stimulation of the pyramidal tract increases the size of the excitatory receptive fields of neurons in the somatosensory cortex of the cat. This effect reflects greater transmission of cutaneous information through the dorsal column nuclei as a result of the facilitation of cells in these nuclei by pyramidal tract fibers.
Assuntos
Córtex Cerebral/fisiologia , Condução Nervosa/fisiologia , Neurônios/fisiologia , Tratos Piramidais/fisiologia , Animais , GatosRESUMO
The optical properties of the ice at the geographical South Pole have been investigated at depths between 0.8 and 1 kilometer. The absorption and scattering lengths of visible light ( approximately 515 nanometers) have been measured in situ with the use of the laser calibration setup of the Antarctic Muon and Neutrino Detector Array (AMANDA) neutrino detector. The ice is intrinsically extremely transparent. The measured absorption length is 59 +/- 3 meters, comparable with the quality of the ultrapure water used in the Irvine-Michigan-Brookhaven and Kamiokande proton-decay and neutrino experiments and more than twice as long as the best value reported for laboratory ice. Because of a residual density of air bubbles at these depths, the trajectories of photons in the medium are randomized. If the bubbles are assumed to be smooth and spherical, the average distance between collisions at a depth of 1 kilometer is about 25 centimeters. The measured inverse scattering length on bubbles decreases linearly with increasing depth in the volume of ice investigated.
RESUMO
In eukaryotes, DNA that is transcribed is packaged first into nucleosomes and then into chromatin fibres. How does transcription proceed through chromatin? Studies of transcription through nucleosomes in vitro suggest that the intracellular environment may provide factors which alleviate the inhibitory effect that nucleosomes have on transcription, possibly via positive supercoiling induced by the migrating polymerase. Stable changes in nucleosome structure have been correlated with transcriptionally active chromatin, but the precise mechanism by which RNA polymerase transcribes through nucleosomal DNA remains unknown.
Assuntos
Cromatina/fisiologia , Transcrição Gênica , Animais , DNA/genética , RNA Polimerases Dirigidas por DNA/fisiologia , Drosophila , Células Eucarióticas , Histonas/fisiologia , Nucleossomos/fisiologiaRESUMO
OBJECTIVE: The application of moisture to the ear is anecdotally claimed to relieve the pain from otic barotrauma that can arise during aircraft descent. This claim was tested in a randomised double-blind study on an aircraft with eight participants heavily predisposed to barotrauma. METHODS: On the outward flight, half the participants wore 'active' devices that applied moisture to the external ear; the remainder wore placebo devices that contained no moisture, but were otherwise identical. On the return flight, the groups were reversed. Participants wore the devices from just before descent until landing, unless they experienced symptoms of barotrauma, in which case they switched to what they knew was an active device. RESULTS: There were no significant differences between conditions regarding the appearance of the tympanic membrane on landing or the discomfort levels immediately before and after any switch. CONCLUSION: Applying moisture is ineffective for passengers heavily predisposed to otic barotrauma.
Assuntos
Barotrauma/epidemiologia , Orelha Média/lesões , Dor de Orelha/prevenção & controle , Membrana Timpânica/lesões , Testes de Impedância Acústica/métodos , Adulto , Medicina Aeroespacial/métodos , Viagem Aérea/estatística & dados numéricos , Barotrauma/tratamento farmacológico , Barotrauma/prevenção & controle , Método Duplo-Cego , Orelha Média/efeitos dos fármacos , Orelha Média/patologia , Dor de Orelha/etiologia , Feminino , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Placebos/efeitos adversos , Índice de Gravidade de Doença , Resultado do Tratamento , Membrana Timpânica/efeitos dos fármacos , Membrana Timpânica/patologiaRESUMO
RAP1 (repressor/activator protein 1) from budding yeast is well known for its involvement in gene activation and repression, telomere structure and function, and replication. Recent studies have examined additional roles for RAP1 in heterochromatin boundary-element formation, creation of hotspots for meiotic recombination, and chromatin opening. These studies provide new insight into the ability of this abundant DNA-binding protein to participate in a diverse array of functions taking place in a chromatin environment.
Assuntos
Proteínas rap1 de Ligação ao GTP/genética , Proteínas rap1 de Ligação ao GTP/fisiologia , Cromatina/fisiologia , Heterocromatina/fisiologia , Meiose/fisiologia , Modelos Genéticos , Recombinação Genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologiaRESUMO
Chromatin-modifying enzymes such as the histone acetyltransferase GCN5 can contribute to transcriptional activation at steps subsequent to the initial binding of transcriptional activators. However, few studies have directly examined dependence of chromatin remodeling in vivo on GCN5 or other acetyltransferases, and none have examined remodeling via nucleosomal activator binding sites. In this study, we have monitored chromatin perturbation via nucleosomal binding sites in the yeast episome TALS by GAL4 derivatives in GCN5(+) and gcn5Delta yeast cells. The strong activator GAL4 shows no dependence on GCN5 for remodeling TALS chromatin, whereas GAL4-estrogen receptor-VP16 shows substantial, albeit not complete, GCN5 dependence. Mini-GAL4 derivatives having weakened interactions with TATA-binding protein and TFIIB exhibit a strong dependence on GCN5 for both transcriptional activation and TALS remodeling not seen for native GAL4. These results indicate that GCN5 can contribute to chromatin remodeling at activator binding sites and that dependence on coactivator function for a given activator can vary according to the type and strength of contacts that it makes with other factors. We also found a weaker dependence for chromatin remodeling on SPT7 than on GCN5, indicating that GCN5 can function via pathways independent of the SAGA complex. Finally, we examine dependence on GCN5 and SWI-SNF at two model promoters and find that although these two chromatin-remodeling and/or modification activities may sometimes work together, in other instances they act in complementary fashion.
Assuntos
Acetiltransferases/metabolismo , Cromatina/fisiologia , Proteínas de Ligação a DNA , Proteínas Fúngicas/metabolismo , Proteína Vmw65 do Vírus do Herpes Simples/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/metabolismo , Ativação Transcricional , Acetiltransferases/genética , Sítios de Ligação , Proteínas Fúngicas/genética , Proteína Vmw65 do Vírus do Herpes Simples/genética , Histona Acetiltransferases , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Proteínas Quinases/genética , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genéticaRESUMO
Transcriptional activators function in vivo via binding sites that may be packaged into chromatin. Here we show that whereas the transcriptional activator GAL4 is strongly able to perturb chromatin structure via a nucleosomal binding site in yeast, GCN4 does so poorly. Correspondingly, GCN4 requires assistance from an accessory protein, RAP1, for activation of the HIS4 promoter, whereas GAL4 does not. The requirement for RAP1 for GCN4-mediated HIS4 activation is dictated by the DNA-binding domain of GCN4 and not the activation domain, suggesting that RAP1 assists GCN4 in gaining access to its binding site. Consistent with this, overexpression of GCN4 partially alleviates the requirement for RAP1, whereas HIS4 activation via a weak GAL4 binding site requires RAP1. RAP1 is extremely effective at interfering with positioning of a nucleosome containing its binding site, consistent with a role in opening chromatin at the HIS4 promoter. Furthermore, increasing the spacing between binding sites for RAP1 and GCN4 by 5 or 10 bp does not impair HIS4 activation, indicating that cooperative protein-protein interactions are not involved in transcriptional facilitation by RAP1. We conclude that an important role of RAP1 is to assist activator binding by opening chromatin.
Assuntos
Cromatina/metabolismo , DNA Fúngico/genética , Proteínas de Ligação a DNA/fisiologia , Proteínas Fúngicas/fisiologia , Regulação Fúngica da Expressão Gênica , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Ativação Transcricional , Oxirredutases do Álcool , Aminoidrolases , Cromatina/ultraestrutura , DNA Fúngico/metabolismo , Proteínas de Drosophila , Proteínas Fúngicas/genética , Proteínas de Homeodomínio/metabolismo , Ligação Proteica , Proteínas Quinases/fisiologia , Pirofosfatases , Saccharomyces cerevisiae/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologiaRESUMO
The yeast transcriptional activator Gal4p can bind to sites in nucleosomal DNA in vivo which it is unable to access in vitro. One event which could allow proteins to bind to otherwise inaccessible sites in chromatin in living cells is DNA replication. To determine whether replication is required for Gal4p to bind to nucleosomal sites in yeast, we have used previously characterized chromatin reporters in which Gal4p binding sites are incorporated into nucleosomes. We find that Gal4p is able to perturb nucleosome positioning via nucleosomal binding sites in yeast arrested either in G1, with alpha-factor, or in G2/M, with nocodazole. Similar results were obtained whether Gal4p synthesis was induced from the endogenous promoter by growth in galactose medium or by an artificial, hormone-inducible system. We also examined binding of the Drosophila transcriptional activator Bicoid, which belongs to the homeodomain class of transcription factors. We show that Bicoid, like Gal4p, can bind to nucleosomal sites in SWI+ and swi1Delta yeast and in the absence of replication. Our results indicate that some feature of the intracellular environment other than DNA replication or the SWI-SNF complex permits factor access to nucleosomal sites.
Assuntos
Proteínas Fúngicas/metabolismo , Proteínas de Homeodomínio/metabolismo , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Antineoplásicos/farmacologia , Proteínas Cromossômicas não Histona , Replicação do DNA , Proteínas de Ligação a DNA , Proteínas de Drosophila , Proteínas Fúngicas/biossíntese , Fase G1 , Fase G2 , Genes Fúngicos , Fator de Acasalamento , Mitose , Modelos Genéticos , Nocodazol/farmacologia , Peptídeos/farmacologia , Ligação Proteica , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
Incorporation into a positioned nucleosome of a cis-acting element essential for replication in Saccharomyces cerevisiae disrupts the function of the element in vivo [R. T. Simpson, Nature (London) 343:387-389, 1990]. Furthermore, nucleosome positioning has been implicated in repression of transcription by RNA polymerase II in yeast cells. We have now asked whether the function of cis-acting elements essential for transcription of a gene transcribed by RNA polymerase III can be similarly affected. A tRNA gene was fused to either of two nucleosome positioning signals such that the predicted nucleosome would incorporate near its center the tRNA start site and essential A-box element. These constructs were then introduced into yeast cells on stably maintained, multicopy plasmids. Competent tRNA genes were transcribed in vivo and were not incorporated into positioned nucleosomes. Mutated, inactive tRNA genes were incorporated into nucleosomes whose positions were as predicted. This finding demonstrates that the transcriptional competence of the tRNA gene determined its ability to override a nucleosome positioning signal in vivo and establishes that a hierarchy exists between cis-acting elements and nucleosome positioning signals.
Assuntos
Nucleossomos/fisiologia , RNA de Transferência/genética , Saccharomyces cerevisiae/genética , Transcrição Gênica , Sequência de Bases , Northern Blotting , DNA Fúngico , Regulação Fúngica da Expressão Gênica , Dados de Sequência Molecular , Mutação , Regiões Operadoras Genéticas , Sequências Repetitivas de Ácido Nucleico , Replicon , Saccharomyces cerevisiae/metabolismoRESUMO
The SWI-SNF complex in yeast and related complexes in higher eukaryotes have been implicated in assisting gene activation by overcoming the repressive effects of chromatin. We show that the ability of the transcriptional activator GAL4 to bind to a site in a positioned nucleosome is not appreciably impaired in swi mutant yeast cells. However, chromatin remodeling that depends on a transcriptional activation domain shows a considerable, although not complete, SWI-SNF dependence, suggesting that the SWI-SNF complex exerts its major effect at a step subsequent to activator binding. We tested this idea further by comparing the SWI-SNF dependence of a reporter gene based on the GAL10 promoter, which has an accessible upstream activating sequence and a nucleosomal TATA element, with that of a CYC1-lacZ reporter, which has a relatively accessible TATA element. We found that the GAL10-based reporter gene showed a much stronger SWI-SNF dependence than did the CYC1-lacZ reporter with several different activators. Remarkably, transcription of the GAL10-based reporter by a GAL4-GAL11 fusion protein showed a nearly complete requirement for the SWI-SNF complex, strongly suggesting that SWI-SNF is needed to allow access of TFIID or the RNA polymerase II holoenzyme. Taken together, our results demonstrate that chromatin remodeling in vivo can occur by both SWI-SNF-dependent and -independent avenues and suggest that the SWI-SNF complex exerts its major effect in transcriptional activation at a step subsequent to transcriptional activator-promoter recognition.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas Fúngicas/fisiologia , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Transativadores/fisiologia , Fatores de Transcrição , Ativação Transcricional , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Genes Reporter , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , TATA Box , Transativadores/metabolismoRESUMO
Transcriptional activators are believed to work in part by recruiting general transcription factors, such as TATA-binding protein (TBP) and the RNA polymerase II holoenzyme. Activation domains also contribute to remodeling of chromatin in vivo. To determine whether these two activities represent distinct functions of activation domains, we have examined transcriptional activation and chromatin remodeling accompanying artificial recruitment of TBP in yeast (Saccharomyces cerevisiae). We measured transcription of reporter genes with defined chromatin structure by artificial recruitment of TBP and found that a reporter gene whose TATA element was relatively accessible could be activated by artificially recruited TBP, whereas two promoters, GAL10 and CHA1, that have accessible activator binding sites, but nucleosomal TATA elements, could not. A third reporter gene containing the HIS4 promoter could be activated by GAL4-TBP only when a RAP1 binding site was present, although RAP1 alone could not activate the reporter, suggesting that RAP1 was needed to open the chromatin structure to allow activation. Consistent with this interpretation, artificially recruited TBP was unable to perturb nucleosome positioning via a nucleosomal binding site, in contrast to a true activator such as GAL4, or to perturb the TATA-containing nucleosome at the CHA1 promoter. Finally, we show that activation of the GAL10 promoter by GAL4, which requires chromatin remodeling, can occur even in swi gcn5 yeast, implying that remodeling pathways independent of GCN5, the SWI-SNF complex, and TFIID can operate during transcriptional activation in vivo.
Assuntos
Cromatina/genética , Proteínas de Ligação a DNA/genética , Fatores de Transcrição/genética , Cromatina/química , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética , TATA Box , Proteína de Ligação a TATA-Box , Ativação TranscricionalRESUMO
We have investigated information transmission in an array of threshold units that have signal-dependent noise and a common input signal. We demonstrate a phenomenon similar to stochastic resonance and suprathreshold stochastic resonance with additive noise and show that information transmission can be enhanced by a nonzero level of noise. By comparing system performance to one with additive noise we also demonstrate that the information transmission of weak signals is significantly better with signal-dependent noise. Indeed, information rates are not compromised even for arbitrary small input signals. Furthermore, by an appropriate selection of parameters, we observe that the information can be made to be (almost) independent of the level of the noise, thus providing a robust method of transmitting information in the presence of noise. These result could imply that the ability of hair cells to code and transmit sensory information in biological sensory systems is not limited by the level of signal-dependent noise.
RESUMO
BACKGROUND: Genetically modified (GM) crops that express insecticidal protein toxins are an integral part of modern agriculture. Proteins produced by Bacillus thuringiensis (Bt) during sporulation mediate the pathogenicity of Bt toward a spectrum of insect larvae whose breadth depends upon the Bt strain. These transmembrane channel-forming toxins are stored in Bt as crystalline inclusions called Cry proteins. These proteins are the active agents used in the majority of biorational pesticides and insect-resistant transgenic crops. Though Bt toxins are promising as a crop protection alternative and are ecologically friendlier than synthetic organic pesticides, resistance to Bt toxins by insects is recognized as a potential limitation to their application. RESULTS: We have determined the 2.2 A crystal structure of the Cry2Aa protoxin by multiple isomorphous replacement. This is the first crystal structure of a Cry toxin specific to Diptera (mosquitoes and flies) and the first structure of a Cry toxin with high activity against larvae from two insect orders, Lepidoptera (moths and butterflies) and Diptera. Cry2Aa also provides the first structure of the proregion of a Cry toxin that is cleaved to generate the membrane-active toxin in the larval gut. CONCLUSIONS: The crystal structure of Cry2Aa reported here, together with chimeric-scanning and domain-swapping mutagenesis, defines the putative receptor binding epitope on the toxin and so may allow for alteration of specificity to combat resistance or to minimize collateral effects on nontarget species. The putative receptor binding epitope of Cry2Aa identified in this study differs from that inferred from previous structural studies of other Cry toxins.
Assuntos
Bacillus thuringiensis , Proteínas de Bactérias/química , Toxinas Bacterianas/química , Endotoxinas/química , Proteínas de Insetos , Receptores de Superfície Celular/química , Sequência de Aminoácidos , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Sítios de Ligação , Cristalografia por Raios X , Endotoxinas/genética , Proteínas Hemolisinas , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de ProteínaRESUMO
Spin-labeled stearic acid derivatives (N-DS) can be used to determine the rate at which lipid-derived drugs can cross a phospholipid bilayer (flip-flop). The flip-flop rate of N-DS (where N=5, 6, 7, 9, 10, 12, 16), was measured using vectorial photoreduction of nitroxides to their corresponding hydroxylamine by FMN, a charged, membrane-impermeable flavin, by hydrogen atom transfer from EDTA. From the time difference in the photoreduction rates of N-DS located in the outer and inner half of the bilayer, the flip-flop rate of N-DS across the bilayer can be determined. The results show that at pH 8.0 or lower, the photoreduction of 5-DS on one side of the membrane by FMN is slower than the flip-flop rate of 5-DS across phospholipid bilayers. For 5-DS at pH 7.0, this rate is at least 33.8+/-4.24 s or faster. Stearic acids with the spin label at different positions along the acyl chain (N=5, 6, 7, 9, 10, 12) have similar flip-flop rates in the liposomes at pH 7.0 although 16-DS is slower, probably due to the inaccessibility of the nitroxide moiety to FMN. It is most likely that the fast distribution of 5-DS in cells is due to the fast movement of acidic form, but not the salt form, of 5-DS across membrane bilayers. The oxazolidine (nitroxide moiety) does not seem to affect the pKa ( approximately 8.3) of stearic acid at air-water interface. Thus, N-DS are good probes for studying the distribution kinetics of stearic acid derivatives in biological systems.