RESUMO
We show that quantum detector tomography can be applied to the human visual system to explore human perception of photon number states. In detector tomography, instead of using very hard-to-produce photon number states, the response of a detector to light pulses with known photon statistics of varying intensity is recorded, and a model is fitted to the experimental outcomes, thereby inferring the detector's photon number state response. Generally, light pulses containing a Poisson-distributed number of photons are utilized, which are very easy to produce in the lab. This technique has not been explored to study the human visual system before because it usually requires a very large number of repetitions not suitable for experiments on humans. Yet, in the present study we show that detector tomography is feasible for human experiments. Assuming a simple model for this accuracy, the results of our simulations show that detector tomography is able to reconstruct the model using Bayesian inference with as few as 5000 trials. We then optimize the experimental parameters in order to maximize the probability of showing that the single-photon accuracy is above chance. As such, our study opens the road to study human perception on the quantum level.
Assuntos
Fótons , Tomografia , Humanos , Estudos de Viabilidade , Teorema de BayesRESUMO
The analysis of amino acids has become a central task in many aspects. While amino acid analysis has traditionally mainly been carried out using either gas chromatography (GC) in combination with flame ionization detection or liquid chromatography (LC) with either post-column derivatization using ninhydrin or pre-column derivatization using o-phthalaldehyde, many of today's analysis platforms are based on chromatography in combination with mass spectrometry (MS). While derivatization is mandatory for the GC-based analysis of amino acids, several LC platforms have emerged, particularly in the dawn of targeted metabolite profiling using hydrophilic interaction liquid chromatography (HILIC) coupled to MS, allowing the analysis of underivatized amino acids. Among the numerous analytical platforms available for amino acid analysis today, we here compare three prominent approaches, being GC-MS and LC-MS after amino acid derivatization using chloroformate and HILIC-MS of underivatized amino acids. We compare and discuss practical issues as well as performance characteristics, e.g., the use of (13)C-labeled internal standards, of the different platforms and present data on their practical implementation in our laboratory. Finally, we compare the real-life applicability of all three platforms for a complex biological sample. While all three platforms are very-well suited for the analysis of complex biological samples they all show advantages and disadvantages for some analytes as discussed in detail in this manuscript.
Assuntos
Aminoácidos/análise , Cromatografia Líquida/métodos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Aminoácidos/química , Calibragem , Isótopos de Carbono/química , Técnicas de Química Analítica , Ionização de Chama/métodos , Formiatos/análise , Interações Hidrofóbicas e Hidrofílicas , Íons , Limite de Detecção , Ninidrina/química , Hidrolisados de Proteína , Reprodutibilidade dos Testes , o-Ftalaldeído/químicaRESUMO
BACKGROUND: The success of the Diabetes Prevention Program (DPP) lifestyle intervention has led to community-based translation efforts in a variety of settings. One community setting which holds promise for the delivery of prevention intervention is the worksite; however, information regarding recruitment in this setting is limited. The current effort describes the initial processes surrounding provision of an adapted DPP lifestyle intervention at a corporate worksite. METHODS: Investigators and key management at the worksite collaborated to develop and implement a recruitment plan for the intervention focusing on 1) in-person onsite activities and 2) implementation of a variety of media recruitment tools and methods. RESULTS: Adult, non-diabetic overweight/obese employees and family members with pre-diabetes and/or the metabolic syndrome were eligible for the study. Telephone pre-screening was completed for 176 individuals resulting in 171 eligible for onsite screening. Of that number, 160 completed onsite screening, 107 met eligibility criteria, and 89 enrolled in the study. Support from worksite leadership, an invested worksite planning team and a solid recruitment plan consisting of multiple strategies were identified as crucial elements of this effective workplace recruitment effort. CONCLUSION: A worksite team successfully developed and implemented a recruitment plan using existing mechanisms appropriate to that worksite in order to identify and enroll eligible individuals. The results of this effort indicate that employee recruitment in a worksite setting is feasible as the first step in offering onsite behavioral lifestyle intervention programs as part of a widespread dissemination plan to prevent diabetes and lower risk for cardiovascular disease.
Assuntos
Diabetes Mellitus Tipo 2/prevenção & controle , Terapia por Exercício/métodos , Síndrome Metabólica/terapia , Obesidade/terapia , Serviços de Saúde do Trabalhador , Seleção de Pacientes , Estado Pré-Diabético/terapia , Programas de Redução de Peso/métodos , Adulto , Comportamento Alimentar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Atividade Motora , Sobrepeso/terapia , Comportamento de Redução do Risco , Local de TrabalhoRESUMO
CONTEXT: Diisocyanates have been associated with respiratory and dermal sensitization. Limited number of case reports, and a few case studies, media, and other references suggest potential neurotoxic effects from exposures to toluene diisocyanate (TDI), 1,6 hexamethylene diisocyanate (HDI), and methylene diisocyanate (MDI). However, a systematic review of the literature evaluating the causal association on humans does not exist to support this alleged association. OBJECTIVE: To perform systematic review examining the body of epidemiologic evidence and provide assessment of causal association based on principles of the Sir Austin Bradford Hill criteria or considerations for causal analysis. METHODS: A comprehensive search of public databases for published abstracts, case reports, cross-sectional surveys, and cohort studies using key search terms was conducted. Additional searches included regulatory reviews, EU IUCLID and EU Risk Assessment databases, and unpublished reports in the International Isocyanate Institute database. An expert panel consisting of physicians, toxicologists, and an epidemiologist critically reviewed accepted papers, providing examination of epidemiologic evidence of each report. Finally, the Hill criteria for causation were applied to the summative analysis of identified reports to estimate probability of causal association. RESULTS: Twelve papers reporting exposed populations with a variety of neurological symptoms or findings suitable for analysis were identified, including eleven case or case series reports, and one cross-sectional study. Three papers reported on the same population. Each of the papers was limited by paucity of diisocyanate exposure estimates, the presence of confounding exposures to known or suspected neurotoxicants, a lack of objective biological measures of exposure or neurotoxic effects, and lack of relative strength of association measures. Additionally, reported health symptoms and syndromes lacked consistency or specificity. No plausible mechanism of toxicity was found. Application of a predictive mathematical model for determining probability of causal association for neurotoxicity was calculated to be 21%. CONCLUSION: There is insufficient evidence for a causal association of neurotoxic effects and diisocyanate exposure based on lack of evidence in all categories of the Hill criteria for causality except for temporal association of reported symptoms and alleged exposure. Future reports should attempt to address more rigorous exposure assessment and control for confounding exposures.
Assuntos
Fármacos do Sistema Nervoso Central/toxicidade , Poluentes Ambientais/toxicidade , Prática Clínica Baseada em Evidências , Isocianatos/toxicidade , Síndromes Neurotóxicas/epidemiologia , Fármacos do Sistema Nervoso Periférico/toxicidade , Animais , Causalidade , Ecotoxicologia/métodos , Exposição Ambiental/efeitos adversos , Guias como Assunto , Humanos , Exposição Ocupacional/efeitos adversos , Tolueno 2,4-Di-Isocianato/toxicidadeRESUMO
Cheese making is a process in which enzymatic coagulation of milk is followed by protein separation, carbohydrate removal, and an extended bacterial fermentation. The number of variables in this complex process that influence cheese quality is so large that the developments of new manufacturing protocols are cumbersome. To reduce screening costs, several models have been developed to miniaturize the cheese manufacturing process. However, these models are not able to accommodate the throughputs required for systematic screening programs. Here, we describe a protocol that allows the parallel manufacturing of approximately 600 cheeses in individual cheese vats each with individual process specifications. Protocols for the production of miniaturized Gouda- and Cheddar-type cheeses have been developed. Starting with as little as 1.7 mL of milk, miniature cheeses of about 170 mg can be produced and they closely resemble conventionally produced cheese in terms of acidification profiles, moisture and salt contents, proteolysis, flavor profiles, and microstructure. Flavor profiling of miniature cheeses manufactured with and without mixed-strain adjunct starter cultures allowed the distinguishing of the different cheeses. Moreover, single-strain adjunct starter cultures engineered to overexpress important flavor-related enzymes revealed effects similar to those described in industrial cheese. Benchmarking against industrial cheese produced from the same raw materials established a good correlation between their proteolytic degradation products and their flavor profiles. These miniature cheeses, referred to as microcheeses, open new possibilities to study many aspects of cheese production, which will not only accelerate product development but also allow a more systematic approach to investigate the complex biochemistry and microbiology of cheese making.
Assuntos
Queijo/microbiologia , Queijo/normas , Manipulação de Alimentos/métodos , Bactérias/crescimento & desenvolvimento , Fenômenos Fisiológicos Bacterianos , Queijo/análise , Contagem de Colônia Microbiana , Concentração de Íons de Hidrogênio , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
Characteristic bursting behavior is observed in a driven, two-dimensional viscous flow, confined to a square domain and subject to no-slip boundaries. Passing a critical parameter value, an existing chaotic attractor undergoes a crisis, after which the flow initially enters a transient bursting regime. Bursting is caused by ejections from and return to a limited subdomain of the phase space, whereas the precrisis chaotic set forms the asymptotic attractor of the flow. For increasing values of the control parameter the length of the bursting regime increases progressively. Passing another critical parameter value, a second crisis leads to the appearance of a secondary type of bursting, of very large dynamical range. Within the bursting regime the flow then switches in irregular intervals from the primary to the secondary type of bursting. Peak enstrophy levels for both types of bursting are associated to the collapse of a primary vortex into a quadrupolar state.
RESUMO
We demonstrate the emergence of complexity from remarkably simple and ubiquitous systems: draining thin-film suspensions exhibiting a striking transition between two classes of self-organizing patterns. Vertical channels form when attractive forces lead to transient gelation, while horizontal bands result from granular mixtures. We propose an explanation whereby the generic physical mechanisms require only the existence of viscous and excluded-volume couplings among the particles, solvent, and substrate. System-specific, small inhomogeneities trigger large-scale pattern formation, through collective dynamics, where jamming plays a crucial role. Our results shed light on emergent complexity in bio- and geophysical processes and have implications for coatings and food industries.
RESUMO
AIMS: The purpose of this study was to determine the global transcriptional response in a food-associated lactic acid bacterium during bile stress. METHODS AND RESULTS: Clone-based DNA micro-arrays were employed to describe the global transcriptional response of Lactobacillus plantarum WCFS1 towards 0.1% porcine bile. Comparison of differential transcript profiles obtained during growth of Lact. plantarum on plates with and without bile revealed 28 and 62 putative genes, of which the expression was at least 2.5-fold up- or down-regulated by bile, respectively. Approximately, 50% of these genes appeared genetically linked, and 12 bile-responsive gene clusters were identified. Seven of the identified bile-responsive genes and gene clusters encode typical stress-related functions, including glutathione reductase and glutamate decarboxylase, involved in oxidative and acid stress, respectively. Moreover, 14 bile-responsive genes and gene clusters were identified that encode proteins that are located in the cell envelope, including the dlt operon and the F1F0 ATPase. CONCLUSIONS: The identification of a relatively high number of genes encoding cell envelope functions indicates a major impact of bile acids on the integrity and/or functionality of the cytoplasmic membrane and cell wall. SIGNIFICANCE AND IMPACT OF THE STUDY: The data presented here provide valuable clues towards the defence mechanisms that play a role during bile stress in Lact. plantarum.
Assuntos
Bile/fisiologia , Microbiologia de Alimentos , Lactobacillus plantarum/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Animais , Proteínas de Bactérias/genética , Clonagem Molecular/métodos , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica/genética , Genes Bacterianos/genética , Glutamato Descarboxilase/genética , Glutationa Redutase/genética , Lactobacillus plantarum/enzimologia , Família Multigênica/genética , Estresse Oxidativo/genética , Suínos , Transcrição GênicaRESUMO
For a two-dimensional fluid in a square domain with no-slip walls, new direct numerical simulations reveal that the transition from steady to chaotic flow occurs through a sequence of various periodic and quasiperiodic flows, similar to the well-known Ruelle-Takens-Newhouse scenario. For all solutions beyond the ground state, the phenomenology is dominated by a domain-filling circulation cell, whereas the associated symmetry is reduced from the full symmetry group of the square to rotational symmetry over an angle pi. The results complement both laboratory experiments in containers with rigid walls and numerical simulations on double-periodic domains.
Assuntos
Antivirais/efeitos adversos , Hepatite C Crônica/tratamento farmacológico , Interferon-alfa/efeitos adversos , Miastenia Gravis/etiologia , Ribavirina/efeitos adversos , Adulto , Antivirais/uso terapêutico , Humanos , Interferon-alfa/uso terapêutico , Masculino , Miastenia Gravis/diagnóstico , Ribavirina/uso terapêuticoRESUMO
Like many other bacteria, Corynebacterium glutamicum possesses two types of L-malate dehydrogenase, a membrane-associated malate:quinone oxidoreductase (MQO; EC 1.1.99.16) and a cytoplasmic malate dehydrogenase (MDH; EC 1.1.1.37) The regulation of MDH and of the three membrane-associated dehydrogenases MQO, succinate dehydrogenase (SDH), and NADH dehydrogenase was investigated. MQO, MDH, and SDH activities are regulated coordinately in response to the carbon and energy source for growth. Compared to growth on glucose, these activities are increased during growth on lactate, pyruvate, or acetate, substrates which require high citric acid cycle activity to sustain growth. The simultaneous presence of high activities of both malate dehydrogenases is puzzling. MQO is the most important malate dehydrogenase in the physiology of C. glutamicum. A mutant with a site-directed deletion in the mqo gene does not grow on minimal medium. Growth can be partially restored in this mutant by addition of the vitamin nicotinamide. In contrast, a double mutant lacking MQO and MDH does not grow even in the presence of nicotinamide. Apparently, MDH is able to take over the function of MQO in an mqo mutant, but this requires the presence of nicotinamide in the growth medium. It is shown that addition of nicotinamide leads to a higher intracellular pyridine nucleotide concentration, which probably enables MDH to catalyze malate oxidation. Purified MDH from C. glutamicum catalyzes oxaloacetate reduction much more readily than malate oxidation at physiological pH. In a reconstituted system with isolated membranes and purified MDH, MQO and MDH catalyze the cyclic conversion of malate and oxaloacetate, leading to a net oxidation of NADH. Evidence is presented that this cyclic reaction also takes place in vivo. As yet, no phenotype of an mdh deletion alone was observed, which leaves a physiological function for MDH in C. glutamicum obscure.
Assuntos
Membrana Celular/enzimologia , Ciclo do Ácido Cítrico , Corynebacterium/enzimologia , Citoplasma/enzimologia , Malato Desidrogenase/metabolismo , Corynebacterium/genética , Meios de Cultura , Malato Desidrogenase/genética , Malatos/metabolismo , Dados de Sequência Molecular , Mutação , NADH Desidrogenase/genética , NADH Desidrogenase/metabolismo , Ácido Oxaloacético/farmacologia , Quinona Redutases/genética , Quinona Redutases/metabolismo , Succinato Desidrogenase/antagonistas & inibidores , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismoRESUMO
Oxidation of malate to oxaloacetate in Escherichia coli can be catalyzed by two enzymes: the well-known NAD-dependent malate dehydrogenase (MDH; EC 1.1.1.37) and the membrane-associated malate:quinone-oxidoreductase (MQO; EC 1.1.99.16), encoded by the gene mqo (previously called yojH). Expression of the mqo gene and, consequently, MQO activity are regulated by carbon and energy source for growth. In batch cultures, MQO activity was highest during exponential growth and decreased sharply after onset of the stationary phase. Experiments with the beta-galactosidase reporter fused to the promoter of the mqo gene indicate that its transcription is regulated by the ArcA-ArcB two-component system. In contrast to earlier reports, MDH did not repress mqo expression. On the contrary, MQO and MDH are active at the same time in E. coli. For Corynebacterium glutamicum, it was found that MQO is the principal enzyme catalyzing the oxidation of malate to oxaloacetate. These observations justified a reinvestigation of the roles of MDH and MQO in the citric acid cycle of E. coli. In this organism, a defined deletion of the mdh gene led to severely decreased rates of growth on several substrates. Deletion of the mqo gene did not produce a distinguishable effect on the growth rate, nor did it affect the fitness of the organism in competition with the wild type. To investigate whether in an mqo mutant the conversion of malate to oxaloacetate could have been taken over by a bypass route via malic enzyme, phosphoenolpyruvate synthase, and phosphenolpyruvate carboxylase, deletion mutants of the malic enzyme genes sfcA and b2463 (coding for EC 1.1.1.38 and EC 1.1.1.40, respectively) and of the phosphoenolpyruvate synthase (EC 2.7.9.2) gene pps were created. They were introduced separately or together with the deletion of mqo. These studies did not reveal a significant role for MQO in malate oxidation in wild-type E. coli. However, comparing growth of the mdh single mutant to that of the double mutant containing mdh and mqo deletions did indicate that MQO partly takes over the function of MDH in an mdh mutant.
Assuntos
Membrana Celular/enzimologia , Ciclo do Ácido Cítrico , Citoplasma/metabolismo , Escherichia coli/enzimologia , Malato Desidrogenase/metabolismo , Proteínas Repressoras , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Carbono/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Óperon Lac , Malato Desidrogenase/genética , Malatos/metabolismo , Fosfotransferases (Aceptores Pareados)/metabolismo , Quinona Redutases/genética , Quinona Redutases/metabolismo , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The only enzyme of the citric acid cycle for which no open reading frame (ORF) was found in the Helicobacter pylori genome is the NAD-dependent malate dehydrogenase. Here, it is shown that in this organism the oxidation of malate to oxaloacetate is catalyzed by a malate:quinone oxidoreductase (MQO). This flavin adenine dinucleotide-dependent membrane-associated enzyme donates electrons to quinones of the electron transfer chain. Similar to succinate dehydrogenase, it is part of both the electron transfer chain and the citric acid cycle. MQO activity was demonstrated in isolated membranes of H. pylori. The enzyme is encoded by the ORF HP0086, which is shown by the fact that expression of the HP0086 sequence from a plasmid induces high MQO activity in mqo deletion mutants of Escherichia coli or Corynebacterium glutamicum. Furthermore, this plasmid was able to complement the phenotype of the C. glutamicum mqo deletion mutant. Interestingly, the protein predicted to be encoded by this ORF is only distantly related to known or postulated MQO sequences from other bacteria. The presence of an MQO shown here and the previously demonstrated presence of a 2-ketoglutarate:ferredoxin oxidoreductase and a succinyl-coenzyme A (CoA):acetoacetyl-CoA transferase indicate that H. pylori possesses a complete citric acid cycle, but one which deviates from the standard textbook example in three steps.
Assuntos
Ciclo do Ácido Cítrico/genética , Helicobacter pylori/genética , Malatos/metabolismo , Proteínas de Membrana/genética , Ácido Oxaloacético/metabolismo , Quinona Redutases/genética , Clonagem Molecular , Teste de Complementação Genética , Helicobacter pylori/enzimologia , Modelos Biológicos , Dados de Sequência Molecular , Oxirredução , Quinona Redutases/metabolismo , Frações Subcelulares/enzimologiaRESUMO
A new method to record transient-evoked otoacoustic emissions (TEOAEs) is introduced. Click stimuli were presented both with and without a simultaneously presented wide-band noise burst. Subtraction of the recorded signal evoked by the noise burst plus click from the signal evoked by the click alone, cancelled the eardrum reflection components of the response and resulted in a measure of the emission. This was used to obtain the TEOAEs from 21 subjects for peak click stimulus levels of 48-66 dB SPL. The root-mean-square (RMS) level of the noise burst was set 10 dB higher than the peak click level, and resulted in suppression of the TEOAE by up to 20 dB. The TEOAE waveforms obtained by the new method were compared to those obtained with Kemp's non-linear method, and were indistinguishable in 20 of the 21 subjects. On basis of the emission spectra, they were indistinguishable in 18 out of 21 subjects. The latencies of narrow-band filtered components from the TEOAEs obtained with the two methods were also similar. This suggests that this noise-suppression method produces similar results as Kemp's non-linear method with the advantage that emission components with very short latencies can be obtained.
Assuntos
Ruído , Emissões Otoacústicas Espontâneas , Estimulação Acústica/métodos , Adulto , Meato Acústico Externo/fisiologia , Humanos , Dinâmica não Linear , Tempo de Reação/fisiologia , Fatores de TempoRESUMO
Transient-evoked otoacoustic emissions (TEOAEs) were decomposed into cochlear place specific components using high-pass noise suppression. This was performed using high-pass filtered noise with cut-off frequencies between 0.7 and 5.6 kHz in 0.5-octave steps. Subtraction of the TEOAEs obtained in the presence of two high-pass noise suppressors with 0.5-octave difference in their cut-off frequencies, f(A) and f(B), should theoretically result in TEOAE components with frequencies between f(A) and f(B). The reconstructed wide-band emission power spectrum obtained by summing the narrow-band emission power spectra, was nearly identical to the power spectrum of the original wide-band emission. This suggests that no phase-cancellation occurs and that the individual narrow-band TEOAEs are uncorrelated, and thus that their generators are potentially independent. About 66% of the derived narrow-band emissions had spectral components that extended below the cut-off frequency of the lower high-pass noise filter. These tail components were interpreted as resulting from high-frequency side suppression of the high-pass noise on the click emission and potentially distortion product components from the TEOAE.
Assuntos
Ruído , Emissões Otoacústicas Espontâneas/fisiologia , Estimulação Acústica/métodos , Adulto , Humanos , Tempo de Reação/fisiologia , Fatores de TempoRESUMO
An improved method for the electrotransformation of wild-type Corynebacterium glutamicum (ATCC 13032) is described. The two crucial alterations to previously developed methods are: cultivation of cells used for electrotransformation at 18 degrees C instead of 30 degrees C, and application of a heat shock immediately following electrotransformation. Cells cultivated at sub optimal temperature have a 100-fold improved transformation efficiency (10(8) cfu micrograms-1) for syngeneic DNA (DNA isolated from the same species). A heat shock applied to these cells following electroporation improved the transformation efficiency for xenogeneic DNA (DNA isolated from a different species). In combination, low cultivation temperature and heat shock act synergistically and increased the transformation efficiency by four orders of magnitude to 2.5 x 10(6) cfu micrograms-1 xenogeneic DNA. The method was used to generate gene disruptions in C. glutamicum.
Assuntos
Corynebacterium/genética , Eletroporação , Temperatura Alta , Transformação Bacteriana , Corynebacterium/crescimento & desenvolvimento , DNA Bacteriano/genética , Plasmídeos/genética , Temperatura , Fatores de TempoRESUMO
OBJECTIVE: To determine whether the Neurologic Disability Score (NDS), the Neuropathic Symptom Score (NSS), and the Medical Research Council (MRC) "sumscore" are reliable, and to determine whether they provide information regarding the functional status of patients with peripheral neuropathies. METHODS: The authors analyzed homogeneity of the frequently used outcome measures in 97 patients using Cronbach's alpha coefficient and corrected item-total correlations. Their association with functional status (sickness impact profile and modified Rankin score) was analyzed univariately with Pearson's and Spearman's correlation coefficients, and multivariately with linear regression analysis. RESULTS: The NDS and MRC scales were homogeneous (range of Cronbach's alpha, 0.81 to 0.97) compared with the NSS scales (range, 0.20 to 0.63). The correlation patterns between neurologic signs and symptoms and functional status ranged from 0.13 to 0.65. Multivariate linear regression analyses showed that 40% or less of patients' functional status could be explained by the three tested outcome measures. CONCLUSION: The NDS and MRC are reliable measures, but these measures do not correlate with measures of functional status.
Assuntos
Avaliação da Deficiência , Doenças do Sistema Nervoso Periférico/diagnóstico , Doenças do Sistema Nervoso Periférico/reabilitação , Atividades Cotidianas , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Atividade Motora , Análise Multivariada , Debilidade Muscular/diagnóstico , Debilidade Muscular/reabilitaçãoRESUMO
In addition to a cytoplasmic, NAD-dependent malate dehydrogenase (EC 1.1.1.37), Corynebacterium glutamicum possesses a highly active membrane-associated malate dehydrogenase (acceptor) (EC 1.1.99.16). This enzyme also takes part in the citric acid cycle. It oxidizes L-malate to oxaloacetate and donates electrons to ubiquinone-1 and other artificial acceptors or, via the electron transfer chain, to oxygen. NAD is not an acceptor and the natural direct acceptor for the enzyme is most likely a quinone. The enzyme is therefore called malate:quinone oxidoreductase, abbreviated to Mqo. Mqo is a peripheral membrane protein and can be released from the membrane by addition of chelators. The solubilized form was partially purified and characterized biochemically. FAD is probably a tightly but non-covalently bound prosthetic group, and the enzyme is activated by lipids. A C. glutamicum mutant completely lacking Mqo activity was isolated. It grows poorly on several substrates tested. The mutant possesses normal levels of cytoplasmic NAD-dependent malate dehydrogenase. A plasmid containing the gene from C. glutamicum coding for Mqo was isolated by complementation of the Mqo-negative phenotype. It leads to overexpression of Mqo activity in the mutant. The nucleotide sequence of the mqo gene was determined and is the first sequence known for this enzyme. The derived protein sequence is similar to hypothetical proteins from Escherichia coli, Klebsiella pneumoniae, and Mycobacterium tuberculosis.
Assuntos
Corynebacterium/enzimologia , Corynebacterium/genética , Malato Desidrogenase/química , Malato Desidrogenase/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Ativação Enzimática/efeitos dos fármacos , Flavina-Adenina Dinucleotídeo/farmacologia , Genes Bacterianos , Lipídeos/farmacologia , Malato Desidrogenase/metabolismo , Malatos/metabolismo , Membranas/enzimologia , Dados de Sequência Molecular , Mutação , Oligodesoxirribonucleotídeos/genética , Oxaloacetatos/metabolismo , Oxirredução , Homologia de Sequência de Aminoácidos , Solubilidade , Especificidade por Substrato , Ubiquinona/metabolismo , Ubiquinona/farmacologiaRESUMO
OBJECTIVE: To investigate the additional diagnostic value of sural nerve biopsy of 64 patients in whom chronic inflammatory demyelinating polyneuropathy (CIDP) was considered, as sural nerve biopsy is recommended in the research criteria of an ad hoc subcommittee to diagnose CIDP. METHODS: Firstly, the additional diagnostic value of sural nerve biopsy was analysed with multivariate logistic regression. Six clinical features (remitting course, symmetric sensorimotor neuropathy in arms and legs, areflexia, raised CSF protein concentration, nerve conduction studies consistent with demyelination, and absence of comorbidity or relevant laboratory abnormalities) were entered into a logistic model. Afterwards, all significant features identified from this model, as well as the results of sural nerve biopsy were forced into a second logistic model. Secondly, the diagnostic performance of a neurologist experienced in diagnosis of peripheral nerve disorders was studied by receiver operating characteristics (ROC) curve analysis. RESULTS: The results of the first logistic analysis showed that CSF protein concentration >1 g/l (odds ratio (OR)=38.5) and neurophysiological studies consistent with demyelination (OR=51.7) were strong predictors of CIDP. When forcing the significant features and the sural nerve biopsy data into the model, an independent predictive value of sural nerve biopsy could not be found. The neurologist was able to discriminate patients with and without CIDP (area under the curve (AUC)=0.95). His diagnostic performance did not improve significantly by offering him the results of sural nerve biopsy. CONCLUSION: Any additional diagnostic value of sural nerve biopsy in the diagnosis of CIDP could not be shown.