RESUMO
Active feedback control in magnetic confinement fusion devices is desirable to mitigate plasma instabilities and enable robust operation. Optical high-speed cameras provide a powerful, non-invasive diagnostic and can be suitable for these applications. In this study, we process high-speed camera data, at rates exceeding 100 kfps, on in situ field-programmable gate array (FPGA) hardware to track magnetohydrodynamic (MHD) mode evolution and generate control signals in real time. Our system utilizes a convolutional neural network (CNN) model, which predicts the n = 1 MHD mode amplitude and phase using camera images with better accuracy than other tested non-deep-learning-based methods. By implementing this model directly within the standard FPGA readout hardware of the high-speed camera diagnostic, our mode tracking system achieves a total trigger-to-output latency of 17.6 µs and a throughput of up to 120 kfps. This study at the High Beta Tokamak-Extended Pulse (HBT-EP) experiment demonstrates an FPGA-based high-speed camera data acquisition and processing system, enabling application in real-time machine-learning-based tokamak diagnostic and control as well as potential applications in other scientific domains.
RESUMO
Conformational change and modification of proteins are involved in many cellular functions. However, they can also have adverse effects that are implicated in numerous diseases. How structural change promotes disease is generally not well-understood. This perspective illustrates how mass spectrometry (MS), followed by toxicological and epidemiological validation, can discover disease-relevant structural changes and therapeutic strategies. We (with our collaborators) set out to characterize the structural and toxic consequences of disease-associated mutations and post-translational modifications (PTMs) of the cytosolic antioxidant protein Cu/Zn-superoxide dismutase (SOD1). Previous genetic studies discovered >180 different mutations in the SOD1 gene that caused familial (inherited) amyotrophic lateral sclerosis (fALS). Using hydrogen-deuterium exchange with mass spectrometry, we determined that diverse disease-associated SOD1 mutations cause a common structural defect - perturbation of the SOD1 electrostatic loop. X-ray crystallographic studies had demonstrated that this leads to protein aggregation through a specific interaction between the electrostatic loop and an exposed beta-barrel edge strand. Using epidemiology methods, we then determined that decreased SOD1 stability and increased protein aggregation are powerful risk factors for fALS progression, with a combined hazard ratio > 300 (for comparison, a lifetime of smoking is associated with a hazard ratio of ~15 for lung cancer). The resulting structural model of fALS etiology supported the hypothesis that some sporadic ALS (sALS, ~80% of ALS is not associated with a gene defect) could be caused by post-translational protein modification of wild-type SOD1. We developed immunocapture antibodies and high sensitivity top-down MS methods and characterized PTMs of wild-type SOD1 using human tissue samples. Using global hydrogen-deuterium exchange, X-ray crystallography and neurotoxicology, we then characterized toxic and protective subsets of SOD1 PTMs. To cap this perspective, we present proof-of-concept that post-translational modification can cause disease. We show that numerous mutations (NâD; QâE), which result in the same chemical structure as the PTM deamidation, cause multiple diseases. Copyright © 2017 John Wiley & Sons, Ltd.
Assuntos
Esclerose Lateral Amiotrófica/genética , Superóxido Dismutase-1/metabolismo , Esclerose Lateral Amiotrófica/epidemiologia , Sítios de Ligação , Simulação por Computador , Cristalografia por Raios X/métodos , Métodos Epidemiológicos , Humanos , Espectrometria de Massas/métodos , Modelos Moleculares , Mutação , Estudo de Prova de Conceito , Ligação Proteica , Conformação Proteica , Processamento de Proteína Pós-Traducional , Proteólise , Eletricidade Estática , Superóxido Dismutase-1/genéticaRESUMO
Domains and domain walls are critical in determining the response of ferroelectrics, and the ability to controllably create, annihilate, or move domains is essential to enable a range of next-generation devices. Whereas electric-field control has been demonstrated for ferroelectric 180° domain walls, similar control of ferroelastic domains has not been achieved. Here, using controlled composition and strain gradients, we demonstrate deterministic control of ferroelastic domains that are rendered highly mobile in a controlled and reversible manner. Through a combination of thin-film growth, transmission-electron-microscopy-based nanobeam diffraction and nanoscale band-excitation switching spectroscopy, we show that strain gradients in compositionally graded PbZr1-xTixO3 heterostructures stabilize needle-like ferroelastic domains that terminate inside the film. These needle-like domains are highly labile in the out-of-plane direction under applied electric fields, producing a locally enhanced piezoresponse. This work demonstrates the efficacy of novel modes of epitaxy in providing new modalities of domain engineering and potential for as-yet-unrealized nanoscale functional devices.
RESUMO
Pyroelectric materials have been widely used for a range of thermal-related applications including thermal imaging/sensing, waste heat energy conversion, and electron emission. In general, the figures of merit for applications of pyroelectric materials are proportional to the pyroelectric coefficient and inversely proportional to the dielectric permittivity. In this context, we explore single-layer and compositionally graded PbZr1-xTixO3 thin-film heterostructures as a way to independently engineer the pyroelectric coefficient and dielectric permittivity of materials and increase overall performance. Compositional gradients in thin films are found to produce large strain gradients which generate large built-in potentials in the films that can reduce the permittivity while maintaining large pyroelectric response. Routes to enhance the figures of merit of pyroelectric materials by 3-12 times are reported, and comparisons to standard materials are made.
RESUMO
We have investigated the contribution of 90° domain walls and thermal expansion mismatch to pyroelectricity in PbZr(0.2)Ti(0.8)O(3) thin films. The first phenomenological models to include extrinsic and secondary contributions to pyroelectricity in polydomain films predict significant extrinsic contributions (arising from the temperature-dependent motion of domain walls) and large secondary contributions (arising from thermal expansion mismatch between the film and the substrate). Phase-sensitive pyroelectric current measurements are applied to model thin films for the first time and reveal a dramatic increase in the pyroelectric coefficient with increasing fraction of in-plane oriented domains and thermal expansion mismatch.
RESUMO
The nephrology workforce is in trouble. Like other consultative-heavy specialties dealing with the chronically ill, it is increasingly difficult to attract and retain trainees. The reasons are multifactorial but include perceptions of a high workload, poor remuneration and, for regional or remote services, isolation. The rate at which new nephrologists are being added to the workforce is insufficient to replace the projected retirements by 2010 and do not allow for any increase in numbers of patients with chronic kidney disease (CKD). However, both CKD incidence and prevalence are inexorably rising, and the patients within this category bear increasingly complex comorbidities. Who will replace the retirees? Sadly, perhaps the answer is no one - an answer which places this challenging specialty under real and imminent threat.
Assuntos
Nefrologia/tendências , Médicos/provisão & distribuição , Médicos/tendências , Austrália , Humanos , Recursos HumanosRESUMO
OBJECTIVE: To investigate the difficulties doctors face in discussing treatment options with patients with acute, life threatening illness and major comorbidities. DESIGN: Observational study of doctor-patient interviews based on a standardised clinical scenario involving high risk surgery in a hypothetical patient (played by an actor) with serious comorbidities. PARTICIPANTS: 30 trainee doctors 3-5 years after graduation. MAIN OUTCOME MEASURES: Adequacy of coverage of various aspects was scored from 3 (good) to 0 (not discussed). RESULTS: The medical situation was considered to be well described (median score 2.7 (interquartile range 2.1-3.0)), whereas the patient's functional status, values, and fears were poorly or minimally addressed (scores 0.5 (0.0-1.0), 0.5 (0.0-1.0), and 0.0 (0.0-1.5), respectively; all P < 0.001 v score for describing the medical situation). Twenty nine of the doctors indicated that they wished to include the patient's family in the discussion, but none identified a preferred surrogate decision maker. Six doctors suggested that the patient alone should speak with his family to reach a decision without the doctor being present. The doctors were reluctant to give advice, despite it being directly requested: two doctors stated that a doctor could not give advice, while 17 simply restated the medical risks, without advocating any particular course. Of the 11 who did offer advice, eight advocated intervention. CONCLUSIONS: Doctors focused on technical medical issues and placed much less emphasis on patient issues such as functional status, values, wishes, and fears. This limits doctors' ability to offer suitable advice about treatment options. Doctors need to improve their communication skills in this difficult but common clinical situation.
Assuntos
Competência Clínica/normas , Corpo Clínico Hospitalar/normas , Relações Médico-Paciente , Revelação da Verdade , Estado Terminal , Tomada de Decisões , Humanos , Variações Dependentes do Observador , Procedimentos Cirúrgicos OperatóriosRESUMO
Previous series that described phenotypes in carriers of Alport's syndrome did not distinguish genetically between carriers of X-linked and autosomal recessive disease. In this study, modes of inheritance in unselected families with Alport's syndrome associated with two city and two provincial hospitals were determined using microsatellite markers, and carriers of disease haplotypes were identified within these families. All 47 carriers (100%) from 18 families with X-linked Alport's syndrome had dysmorphic hematuria on phase-contrast microscopy, but few developed renal failure (3 of 40 carriers; 8%), clinical hearing loss (2 of 45 carriers; 4%), retinopathy (1 of 30 carriers; 3%), or lenticonus (0 of 30 carriers; 0%). Eleven of the 14 carriers (79%) from 2 families with autosomal recessive disease had dysmorphic hematuria, but none had renal failure, clinical hearing loss, retinopathy, or lenticonus. Urinary red blood cell counts in carriers of X-linked Alport's syndrome were greater than those in carriers of autosomal recessive disease (P < 0.0001), but the frequency of proteinuria and hypertension and levels of proteinuria were not different. There was more tubulointerstitial damage in carriers of X-linked disease (P = 0.012); however, carriers of autosomal recessive disease had more widespread and more uniform thinning of the glomerular basement membrane (P < 0.0001) and less lamellation (P < 0.04).
Assuntos
Triagem de Portadores Genéticos , Nefrite Hereditária/diagnóstico , Nefrite Hereditária/genética , Adolescente , Adulto , Distribuição por Idade , Idoso , Idoso de 80 Anos ou mais , Biópsia , Criança , Comorbidade , Anormalidades do Olho/epidemiologia , Feminino , Ligação Genética , Perda Auditiva/epidemiologia , Humanos , Rim/patologia , Glomérulos Renais/ultraestrutura , Túbulos Renais/ultraestrutura , Masculino , Pessoa de Meia-Idade , Nefrite Hereditária/epidemiologia , Fenótipo , Distribuição por SexoAssuntos
Proteínas de Bactérias/metabolismo , Liases de Carbono-Enxofre/metabolismo , Proteínas de Escherichia coli , Proteínas Ferro-Enxofre/biossíntese , Proteínas Ferro-Enxofre/metabolismo , Enxofre/metabolismo , Azotobacter vinelandii/química , Azotobacter vinelandii/metabolismo , Cromatografia Líquida de Alta Pressão , Ditiotreitol/química , Ditiotreitol/farmacologia , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
An IscA homologue within the nif regulon of Azotobacter vinelandii, designated (Nif)IscA, was expressed in Escherichia coli and purified to homogeneity. Purified (Nif)IscA was found to be a homodimer of 11-kDa subunits that contained no metal centers or other prosthetic groups in its as-isolated form. Possible roles for (Nif)IscA in Fe-S cluster biosynthesis were assessed by investigating the ability to bind iron and to assemble Fe-S clusters in a NifS-directed process, as monitored by the combination of UV-vis absorption, Mössbauer, resonance Raman, variable-temperature magnetic circular dichroism, and EPR spectroscopies. Although (Nif)IscA was found to bind ferrous ion in a tetrahedral, predominantly cysteinyl-ligated coordination environment, the low-binding affinity argues against a specific role as a metallochaperone for the delivery of ferrous ion to other Fe-S cluster assembly proteins. Rather, a role for (Nif)IscA as an alternate scaffold protein for Fe-S cluster biosynthesis is proposed, based on the NifS-directed assembly of approximately one labile [4Fe-4S](2+) cluster per (Nif)IscA homodimer, via a transient [2Fe-2S](2+) cluster intermediate. The cluster assembly process was monitored temporally using UV-vis absorption and Mössbauer spectroscopy, and the intermediate [2Fe-2S](2+)-containing species was additionally characterized by resonance Raman spectroscopy. The Mössbauer and resonance Raman properties of the [2Fe-2S](2+) center are consistent with complete cysteinyl ligation. The presence of three conserved cysteine residues in all IscA proteins and the observed cluster stoichiometry of approximately one [2Fe-2S](2+) or one [4Fe-4S](2+) per homodimer suggest that both cluster types are subunit bridging. In addition, (Nif)IscA was shown to couple delivery of iron and sulfur by using ferrous ion to reduce sulfane sulfur. The ability of Fe-S scaffold proteins to couple the delivery of these two toxic and reactive Fe-S cluster precursors is likely to be important for minimizing the cellular concentrations of free ferrous and sulfide ions. On the basis of the spectroscopic and analytical results, mechanistic schemes for NifS-directed cluster assembly on (Nif)IscA are proposed. It is proposed that the IscA family of proteins provide alternative scaffolds to the NifU and IscU proteins for mediating nif-specific and general Fe-S cluster assembly.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Ferro-Enxofre/biossíntese , Sequência de Aminoácidos , Azotobacter vinelandii/genética , Azotobacter vinelandii/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/fisiologia , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Catálise , Genes Bacterianos , Proteínas de Ligação ao Ferro , Proteínas Ferro-Enxofre/metabolismo , Cinética , Dados de Sequência Molecular , Fixação de Nitrogênio/genética , Proteínas de Ligação a TransferrinaRESUMO
Traditionally, it has been advocated that implants planned for use as overdenture abutments be placed parallel to each other to obtain predictable attachment retention and complete seating of the restoration and to prevent premature wear of components. However, it is often difficult or impossible to place implants parallel to each other, and patients with implants that have already been placed in a variety of positions frequently are referred to restorative dentists. This article describes a technique for the fabrication of a matrix-paralleling device as well as 2 of its uses. The device allows proper orientation of the retentive matrices to establish a common path of withdrawal for the prosthesis and all attachments. Provided that the matrices are parallel to each other, spherical overdenture attachments can be used even when the implants are not parallel.
Assuntos
Dente Suporte , Implantes Dentários , Prótese Dentária Fixada por Implante , Planejamento de Dentadura/instrumentação , Retenção de Dentadura/instrumentação , Resinas Acrílicas , Hidróxido de Cálcio , Materiais para Moldagem Odontológica , Desgaste de Restauração Dentária , Encaixe de Precisão de Dentadura , Revestimento de Dentadura , Desenho de Equipamento , Ligas de Ouro , Humanos , Minerais , Cloreto de Polivinila , Polivinil , Rotação , Siloxanas , Propriedades de SuperfícieAssuntos
Anticorpos Anticitoplasma de Neutrófilos/imunologia , Glomerulonefrite Membranosa/complicações , Glomerulonefrite Membranosa/diagnóstico , Idoso , Biomarcadores/sangue , Biópsia , Diagnóstico Diferencial , Glomerulonefrite Membranosa/imunologia , Humanos , Masculino , Vasculite/complicações , Vasculite/diagnóstico , Vasculite/imunologiaRESUMO
There is limited dental literature evaluating the retentive capabilities of luting agents when used between metal components, such as cast metal restorations cemented onto machined metal implant abutments. This study compared the retentive strengths of 5 different classes of luting agents used to cement cast noble metal alloy crowns to 8-degree machined titanium cementable implant abutments from the Straumann ITI Implant System. Sixty prefabricated 5.5-mm solid titanium implant abutments and implants were used; 30 received the standard surface preparation and the other 30 received an anodized surface preparation. Anodized implant components were used to reflect current implant marketing. Sixty castings were fabricated and randomly paired with an abutment and implant. A total of 12 castings were cemented onto the implant-abutment assemblies for each of the 5 different luting agents (zinc phosphate, resin composite, glass ionomer, resin-reinforced glass ionomer, and zinc oxide-non-eugenol). After cementation, the assemblies were stored in a humidor at room temperature prior to thermocycling for 24 hours. Each casting was pulled from its respective abutment, and the force at which bond failure occurred was recorded as retentive strength. A statistically significant difference was found between the 5 cements at P < or = .001. Of the cements used, resin composite demonstrated the highest mean retentive strength. Zinc phosphate and resin-reinforced glass-ionomer cements were the next most retentive, while glass ionomer and zinc oxide-non-eugenol cements demonstrated minimal retention. In addition, retention was not altered by the use of an anodized abutment surface.
Assuntos
Dente Suporte , Ligas Dentárias , Colagem Dentária , Cimentos Dentários , Implantes Dentários , Retenção em Prótese Dentária/métodos , Adesividade , Análise de Variância , Materiais Revestidos Biocompatíveis , Coroas , Técnica de Fundição Odontológica , Cimentos de Ionômeros de Vidro , Teste de Materiais , Fosfatos , Distribuição Aleatória , Cimentos de Resina , Estatísticas não Paramétricas , Propriedades de Superfície , Óxido de Zinco , Cimento de Fosfato de ZincoRESUMO
The Fe-S cluster formation proteins NifU and NifS are essential for viability in the ulcer causing human pathogen Helicobacter pylori. Obtaining viable H. pylori mutants upon mutagenesis of the genes encoding NifU and NifS was unsuccessful even by growing the potential transformants under many different conditions including low O(2) atmosphere and supplementation with both ferric and ferrous iron. When a second copy of nifU was introduced into the chromosome at a unrelated site, creating a mero-diploid strain for nifU, this second copy of the gene could be disrupted at high frequency. This indicates that the procedures used for transformation were capable of nifU mutagenesis, so that the failure to recover mutants is solely due to the requirement of nifU for H. pylori viability. H. pylori NifU and NifS were expressed in Escherichia coli and purified to near homogeneity, and the proteins were characterized. Purified NifU is a red protein that contains approximately 1.5 atoms of iron per monomer. This iron was determined to be in the form of a redox-active [2Fe-2S](2+,+) cluster by characteristic UV-visible, EPR, and MCD spectra. The primary structure of NifU also contains the three conserved cysteine residues which are involved in providing the scaffold for the assembly of a transient Fe-S cluster for insertion into apoprotein. Purified NifS has a yellow color and UV-visible spectra characteristic of a pyridoxal phosphate containing enzyme. NifS is a cysteine desulfurase, releasing sulfur or sulfide (depending on the reducing environment) from L-cysteine, in agreement with its proposed role as a sulfur donor to Fe-S clusters. The results here indicate that the NifU type of Fe-S cluster formation proteins is not specific for maturation of the nitrogenase proteins and, as H. pylori lacks other Fe-S cluster assembly proteins, that the H. pylori NifS and NifU are responsible for the assembly of many (non-nitrogenase) Fe-S clusters.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Helicobacter pylori/fisiologia , Sequência de Aminoácidos , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/fisiologia , Clonagem Molecular , Genes Bacterianos , Genes Letais , Vetores Genéticos/química , Helicobacter pylori/genética , Helicobacter pylori/crescimento & desenvolvimento , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/isolamento & purificação , Proteínas Ferro-Enxofre/fisiologia , Dados de Sequência Molecular , Mutagênese Insercional , Fixação de Nitrogênio/genética , Espectrofotometria UltravioletaRESUMO
Iron-sulfur cluster biosynthesis in both prokaryotic and eukaryotic cells is known to be mediated by two highly conserved proteins, termed IscS and IscU in prokaryotes. The homodimeric IscS protein has been shown to be a cysteine desulfurase that catalyzes the reductive conversion of cysteine to alanine and sulfide. In this work, the time course of IscS-mediated Fe-S cluster assembly in IscU was monitored via anaerobic anion exchange chromatography. The nature and properties of the clusters assembled in discrete fractions were assessed via analytical studies together with absorption, resonance Raman, and Mössbauer investigations. The results show sequential cluster assembly with the initial IscU product containing one [2Fe-2S](2+) cluster per dimer converting first to a form containing two [2Fe-2S](2+) clusters per dimer and finally to a form that contains one [4Fe-4S](2+) cluster per dimer. Both the [2Fe-2S](2+) and [4Fe-4S](2+) clusters in IscU are reductively labile and are degraded within minutes upon being exposed to air. On the basis of sequence considerations and spectroscopic studies, the [2Fe-2S](2+) clusters in IscU are shown to have incomplete cysteinyl ligation. In addition, the resonance Raman spectrum of the [4Fe-4S](2+) cluster in IscU is best interpreted in terms of noncysteinyl ligation at a unique Fe site. The ability to assemble both [2Fe-2S](2+) and [4Fe-4S](2+) clusters in IscU supports the proposal that this ubiquitous protein provides a scaffold for IscS-mediated assembly of clusters that are subsequently used for maturation of apo Fe-S proteins.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Ferro-Enxofre/biossíntese , Azotobacter vinelandii/metabolismo , Cromatografia Líquida , Análise EspectralRESUMO
The NifS and NifU nitrogen fixation-specific gene products are required for the full activation of both the Fe-protein and MoFe-protein of nitrogenase from Azotobacter vinelandii. Because the two nitrogenase component proteins both require the assembly of [Fe-S]-containing clusters for their activation, it has been suggested that NifS and NifU could have complementary functions in the mobilization of sulfur and iron necessary for nitrogenase-specific [Fe-S] cluster assembly. The NifS protein has been shown to have cysteine desulfurase activity and can be used to supply sulfide for the in vitro catalytic formation of [Fe-S] clusters. The NifU protein was previously purified and shown to be a homodimer with a [2Fe-2S] cluster in each subunit. In the present work, primary sequence comparisons, amino acid substitution experiments, and optical and resonance Raman spectroscopic characterization of recombinantly produced NifU and NifU fragments are used to show that NifU has a modular structure. One module is contained in approximately the N-terminal third of NifU and is shown to provide a labile rubredoxin-like ferric-binding site. Cysteine residues Cys35, Cys62, and Cys106 are necessary for binding iron in the rubredoxin-like mode and visible extinction coefficients indicate that up to one ferric ion can be bound per NifU monomer. The second module is contained in approximately the C-terminal half of NifU and provides the [2Fe-2S] cluster-binding site. Cysteine residues Cys137, Cys139, Cys172, and Cys175 provide ligands to the [2Fe-2S] cluster. The cysteines involved in ligating the mononuclear Fe in the rubredoxin-like site and those that provide the [2Fe-2S] cluster ligands are all required for the full physiological function of NifU. The only two other cysteines contained within NifU, Cys272 and Cys275, are not necessary for iron binding at either site, nor are they required for the full physiological function of NifU. The results provide the basis for a model where iron bound in labile rubredoxin-like sites within NifU is used for [Fe-S] cluster formation. The [2Fe-2S] clusters contained within NifU are proposed to have a redox function involving the release of Fe from bacterioferritin and/or the release of Fe or an [Fe-S] cluster precursor from the rubredoxin-like binding site.
Assuntos
Proteínas de Bactérias/química , Genes Bacterianos , Ferro/metabolismo , Sequência de Aminoácidos , Azotobacter vinelandii/genética , Azotobacter vinelandii/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Cisteína/química , DNA/química , DNA/genética , Eletroforese em Gel de Poliacrilamida , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fixação de Nitrogênio/genética , Plasmídeos/química , Plasmídeos/genética , Ligação Proteica/genética , Espectrofotometria Ultravioleta , Análise Espectral RamanRESUMO
The NifS and NifU proteins from Azotobacter vinelandii are required for the full activation of nitrogenase. NifS is a homodimeric cysteine desulfurase that supplies the inorganic sulfide necessary for formation of the Fe-S clusters contained within the nitrogenase component proteins. NifU has been suggested to complement NifS either by mobilizing the Fe necessary for nitrogenase Fe-S cluster formation or by providing an intermediate Fe-S cluster assembly site. As isolated, the homodimeric NifU protein contains one [2Fe-2S](2+, +) cluster per subunit, which is referred to as the permanent cluster. In this report, we show that NifU is able to interact with NifS and that a second, transient [2Fe-2S] cluster can be assembled within NifU in vitro when incubated in the presence of ferric ion, L-cysteine, and catalytic amounts of NifS. Approximately one transient [2Fe-2S] cluster is assembled per homodimer. The transient [2Fe-2S] cluster species is labile and rapidly released on reduction. We propose that transient [2Fe-2S] cluster units are formed on NifU and then released to supply the inorganic iron and sulfur necessary for maturation of the nitrogenase component proteins. The role of the permanent [2Fe-2S] clusters contained within NifU is not yet known, but they could have a redox function involving either the formation or release of transient [2Fe-2S] cluster units assembled on NifU. Because homologs to both NifU and NifS, respectively designated IscU and IscS, are found in non-nitrogen fixing organisms, it is possible that the function of NifU proposed here could represent a general mechanism for the maturation of Fe-S cluster-containing proteins.
Assuntos
Proteínas de Bactérias/química , Proteínas Ferro-Enxofre/química , Substituição de Aminoácidos , Azotobacter vinelandii/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Ferro/química , Substâncias Macromoleculares , Oxirredução , Espectrofotometria Ultravioleta , Análise Espectral RamanRESUMO
The nifE and nifN gene products from Azotobacter vinelandii form an alpha2beta2 tetramer (NifEN complex) that is required for the biosynthesis of the nitrogenase FeMo cofactor. In the current model for NifEN complex organization and function, the complex is structurally analogous to the nitrogenase MoFe protein and provides an assembly site for a portion of FeMo cofactor biosynthesis. In this work, gene fusion and immobilized metal-affinity chromatography strategies were used to elevate the in vivo production of the NifEN complex and to facilitate its rapid and efficient purification. The NifEN complex produced and purified in this way exhibits an FeMo cofactor biosynthetic activity similar to that previously described for the NifEN complex purified by traditional chromatography methods. UV-visible, EPR, variable-temperature magnetic circular dichroism, and resonance Raman spectroscopies were used to show that the NifEN complex contains two identical [4Fe-4S]2+ clusters. These clusters have a predominantly S = 1/2 ground state in the reduced form, exhibit a reduction potential of -350 mV, and are likely to be coordinated entirely by cysteinyl residues on the basis of spectroscopic properties and sequence comparisons. A model is proposed where each NifEN complex [4Fe-4S] cluster is bridged between a NifE-NifN subunit interface at a position analogous to that occupied by the P clusters in the nitrogenase MoFe protein. In contrast to the MoFe protein P clusters, the NifEN complex [4Fe-4S] clusters are proposed to be asymmetrically coordinated to the NifEN complex where NifE cysteines-37, -62, and -124 and NifN cysteine-44 are the coordinating ligands. On the basis of a homology model of the three-dimensional structure of the NifEN complex, the [4Fe-4S] cluster sites are likely to be remote from the proposed FeMo cofactor assembly site and are unlikely to become incorporated into the FeMo cofactor during its assembly.
Assuntos
Azotobacter vinelandii/enzimologia , Dinitrogenase Redutase/química , Hidrogenase/química , Proteínas Ferro-Enxofre/química , Molibdoferredoxina/química , Fusão Gênica Artificial , Azotobacter vinelandii/genética , Azotobacter vinelandii/crescimento & desenvolvimento , Cromatografia de Afinidade , Dicroísmo Circular , Dinitrogenase Redutase/genética , Espectroscopia de Ressonância de Spin Eletrônica , Hidrogenase/genética , Proteínas Ferro-Enxofre/genética , Molibdoferredoxina/genética , Família Multigênica , Mutagênese Sítio-Dirigida , Oxirredução , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Análise Espectral RamanRESUMO
Ocular abnormalities are common in X-linked Alport syndrome, but they have not been studied in patients with the rarer autosomal recessive disease. We have examined the eyes of a family with autosomal recessive Alport syndrome. Four of the eight offspring of a consanguineous marriage had renal failure and deafness by the age of 20 years. The diagnosis of Alport syndrome was confirmed on the ultrastructural demonstration of a lamellated glomerular basement membrane (GBM) in one affected family member. Autosomal recessive inheritance was suggested by the lack of linkage to the COL4A5/COL4A6 locus, and by linkage to the COL4A3/COL4A4 locus. All four affected family members had anterior lenticonus (or had had a lens replacement for this) and the three who were examined had a dot-and-fleck retinopathy. Neither of the two unaffected offspring who were examined nor the father had these abnormalities. The ocular manifestations of autosomal recessive Alport syndrome are probably identical to those for the X-linked form. Although the mutations in these diseases affect genes for different type IV collagen chains, these chains occur together in the basement membranes of the kidney, eye and ear, and abnormalities in any one may result in the same clinical phenotype.