Designing conditions for in vitro formation of amyloid protofilaments and fibrils

We have been able to convert a small α/β protein, acylphosphatase, from its soluble and native form into insoluble amyloid fibrils of the type observed in a range of pathological conditions. This was achieved by allowing slow growth in a solution containing moderate concentrations of trifluoroethanol. When analyzed with electron microscopy, the protein aggregate present in the sample after long incubation times consisted of extended, unbranched filaments of 30–50 Å in width that assemble subsequently into higher order structures. This fibrillar material possesses extensive β-sheet structure as revealed by far-UV CD and IR spectroscopy. Furthermore, the fibrils exhibit Congo red birefringence, increased fluorescence with thioflavine T and cause a red-shift of the Congo red absorption spectrum. All of these characteristics are typical of amyloid fibrils. The results indicate that formation of amyloid occurs when the native fold of a protein is destabilized under conditions in which noncovalent interactions, and in particular hydrogen bonding, within the polypeptide chain remain favorable. We suggest that amyloid formation is not restricted to a small number of protein sequences but is a property common to many, if not all, natural polypeptide chains under appropriate conditions.

Heme is an effector molecule for iron-dependent degradation of the bacterial iron response regulator (Irr) protein

The bacterial iron response regulator (Irr) protein mediates iron-dependent regulation of heme biosynthesis. Pulse–chase and immunoprecipitation experiments showed that Irr degraded in response to 6 μM iron with a half-life of ≈30 min and that this regulated stability was the principal determinant of control by iron. Irr contains a heme regulatory motif (HRM) near its amino terminus. A role for heme in regulation was implicated by the retention of Irr in heme synthesis mutants in the presence of iron. Addition of heme to low iron (0.3 μM) cultures was sufficient for the disappearance of Irr in cells of the wild-type and heme mutant strains. Spectral and binding analyses of purified recombinant Irr showed that the protein bound heme with high affinity and caused a blue shift in the absorption spectrum of heme to a shorter wavelength. A Cys29 → Ala substitution within the HRM of Irr (IrrC29A) abrogated both high affinity binding to heme and the spectral blue shift. In vivo turnover experiments showed that, unlike wild-type Irr, IrrC29A was stable in the presence of iron. We conclude that iron-dependent degradation of Irr involves direct binding of heme to the protein at the HRM. The findings implicate a regulatory role for heme in protein degradation and provide direct evidence for a functional HRM in a prokaryote.

Identification, retinoid binding, and x-ray analysis of a human retinol-binding protein

Two cellular retinol-binding proteins (CRBP I and II) with distinct tissue distributions and retinoid-binding properties have been recognized thus far in mammals. Here, we report the identification of a human retinol-binding protein resembling type I (55.6% identity) and type II (49.6% identity) CRBPs, but with a unique H residue in the retinoid-binding site and a distinctively different tissue distribution. Additionally, this binding protein (CRBP III) exhibits a remarkable sequence identity (62.2%) with the recently identified ι-crystallin/CRBP of the diurnal gecko Lygodactylus picturatus [Werten, P. J. L., Röll, B., van Alten, D. M. F. & de Jong, W. W. (2000) Proc. Natl. Acad. Sci. USA 97, 3282–3287 (First Published March 21, 2000; 10.1073/pnas.050500597)]. CRBP III and all-trans-retinol form a complex (Kd ≈ 60 nM), the absorption spectrum of which is characterized by the peculiar fine structure typical of the spectra of holo-CRBP I and II. As revealed by a 2.3-Å x-ray molecular model of apo-CRBP III, the amino acid residues that line the retinol-binding site in CRBP I and II are positioned nearly identically in the structure of CRBP III. At variance with the human CRBP I and II mRNAs, which are most abundant in ovary and intestine, respectively, the CRBP III mRNA is expressed at the highest levels in kidney and liver thus suggesting a prominent role for human CRBP III as an intracellular mediator of retinol metabolism in these tissues.

Close correspondence between the action spectra for the blue light responses of the guard cell and coleoptile chloroplasts, and the spectra for blue light-dependent stomatal opening and coleoptile phototropism.

Fluorescence spectroscopy was used to characterize blue light responses from chloroplasts of adaxial guard cells from Pima cotton (Gossypium barbadense) and coleoptile tips from corn (Zea mays). The chloroplast response to blue light was quantified by measurements of the blue light-induced enhancement of a red light-stimulated quenching of chlorophyll a fluorescence. In adaxial (upper) guard cells, low fluence rates of blue light applied under saturating fluence rates of red light enhanced the red light-stimulated fluorescence quenching by up to 50%. In contrast, added blue light did not alter the red light-stimulated quenching from abaxial (lower) guard cells. This response pattern paralleled the blue light sensitivity of stomatal opening in the two leaf surfaces. An action spectrum for the blue light-induced enhancement of the red light-stimulated quenching showed a major peak at 450 nm and two minor peaks at 420 and 470 nm. This spectrum matched closely an action spectrum for blue light-stimulated stomatal opening. Coleoptile chloroplasts also showed an enhancement by blue light of red light-stimulated quenching. The action spectrum of this response, showing a major peak at 450 nm, a minor peak at 470 nm, and a shoulder at 430 nm, closely matched an action spectrum for blue light-stimulated coleoptile phototropism. Both action spectra match the absorption spectrum of zeaxanthin, a chloroplastic carotenoid recently implicated in blue light photoreception of both guard cells and coleoptiles. The remarkable similarity between the action spectra for the blue light responses of guard cells and coleoptile chloroplasts and the spectra for blue light-stimulated stomatal opening and phototropism, coupled to the recently reported evidence on a role of zeaxanthin in blue light photoreception, indicates that the guard cell and coleoptile chloroplasts specialize in sensory transduction.

Association of a polynuclear iron-sulfur center with a mutant FNR protein enhances DNA binding.

In the facultative anaerobe Escherichia coli, the transcription factor FNR (fumarate nitrate reduction) regulates gene expression in response to oxygen deprivation. To investigate how the activity of FNR is regulated by oxygen availability, two mutant proteins, DA154 and LH28-DA154, which have enhanced in vivo activity in the presence of oxygen, were purified and compared. Unlike other previously examined FNR preparations, the absorption spectrum of LH28-DA154 had two maxima at 324 nm and 419 nm, typical of iron-sulfur (Fe-S)-containing proteins. Consistent with these data, metal analysis showed that only the LH28-DA154 protein contained a significant amount of iron and acid-labile sulfide, and, by low temperature EPR spectroscopy, a signal typical of a [3Fe-4S]+ cluster was detected. The LH28-DA154 protein that contained the Fe-S cluster also contained a higher proportion of dimers and had a 3- to 4-fold higher apparent affinity for the target DNA than the DA154 protein. In agreement with this, we found that when the LH28-DA154 protein was treated with an iron chelator (alpha,alpha'-dipyridyl), it lost its characteristic absorption and the apparent affinity for DNA was reduced 6-fold. However, increased DNA binding and the characteristic absorption spectrum could be restored by in vitro reconstitution of the Fe-S center. DNA binding of the LH28-DA154 protein was also affected by the redox state of the Fe-S center, since protein exposed to oxygen bound 1/10th as much DNA as the protein reduced anaerobically with dithionite. The observation that DNA binding is enhanced when the Fe-S center is reduced indicates that the redox state of the Fe-S center affects the DNA-binding activity of this protein and suggests a possible mechanism for regulation of the wild-type protein.

Basis of guanylate cyclase activation by carbon monoxide.

Kinetics of CO association with guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] and dissociation from carboxy guanylate cyclase have been studied at pH 7.5 by flash photolysis, yielding rate constants at 23 degrees C of 1.2 +/- 0.1 x 10(5) M-1.sec-1 and 28 +/- 2 sec-1, respectively. While the CO combination rate constant is the same as for the T state of hemoglobin, the CO dissociation rate constant is much higher than expected for a six-coordinate carboxyheme protein; yet the absorption spectrum is indicative of a six-coordinate heme. The two observations are reconciled by a reaction mechanism in which CO dissociation proceeds via a five-coordinate intermediate. This intermediate is structurally very similar to the five-coordinate nitrosyl heme derivative of guanylate cyclase and is presumably responsible for the observed 4-fold activation of guanylate cyclase by CO. Thus, we provide a model that explains enzyme activities of the nitrosyl and carboxy forms of the enzyme on the basis of a common mechanism.

Método simples e rápido para seleção de fungos filamentosos produtores de compostos absorvedores de radiação UV para aplicação em protetores solares

Foram estudadas trinta e uma cepas fúngicas não identificadas, as quais foram denominadasX1 a X31. O potencial fotoprotetor foi avaliado pela medida espectrofotométrica da absorçãodos extratos na região do UV (280-400 nm). Os extratos com os melhores perfis de absorção em cultura estacionária foram X1, X2, X6, X12, X13, X18, X19, X22, X24 e X31 e, em cultura agitada X4 e X17. A reprodutibilidade do processo foi avaliada e as cepas fúngicas que apresentaram coeficiente de variação menor que 15% foram selecionadas para o estudo de fotoestabilidade. A fotoestabilidade dos extratos foi avaliada pela medida da viabilidade celular de fibroblastos L929 tratados com extratos previamente irradiados sob radiação UVA (11,2 J/cm2) e UVB (3,43 J/cm2) e extratos não irradiados, bem como, pela comparação das áreas sob as curvas de absorção na região do UV dos extratos irradiados e não irradiados. Os extratos selecionados para o estudo de fotoestabilidade foram X4, X12, X19, X22, X24 e X31. Os extratos não irradiados apresentaram os seguintes valores deIC50 para viabilidade celular (citotoxidade): X4-130µg/ml, X19-20µg/ml, X22-10 µg/ml e X24-60µg/ml. Após a radiação UVA e UVB, os extratos apresentaram redução significativa da viabilidade celular em relação ao IC50 dos extratos não irradiados. Sob luz UVB, os extratos X12 (IC50 35µg/ml) e X31 (IC50 70µg/ml) mantiveram a mesma porcentagem de redução da viabilidade celular quando comparado ao IC50 dos extratos não irradiados. No entanto após exposição à luz UVA, o extrato X12 aumentou a viabilidade celular de 50% (quando não irradiado) para 75% (irradiado). Enquanto que o extrato X31, mesmo após a radiação UVA, manteve a mesma redução de 50% da viabilidade celular. Nessa etapa os extratos selecionados foram os X12 e X31. O espectro de absorção na região do UV obtido para o extrato X12 mostrou uma redução da absorbância de 28,3% sob radiação UVB e de 60% sob radiação UVA em relação ao extrato não irradiado. O extrato X31 apresentou uma redução da absorbância de 17,6% e30% sob radiação UVB e UVA respectivamente, em relação ao extrato não irradiado. Os fungos selecionados foram identificados por PCR, sugerindo que o fungo X12 seja o Aspergillus terreus e o X31 seja o Talaromyces pinophilus. Por fim, foi feita a identificação da substância ativa do extrato X12 empregando a técnica de desreplicação, a qual fez o uso da instrumentação analítica acoplada UHPLC-DAD-(ESI)-HRMS associada ao banco de dados Chapman& Hall\'s Dictionary of Natural Products (DNP). No extrato X12 o composto majoritário foi identificado como sendo a citreoviridina. Assim, os resultados do presente trabalho permitiu estabelecer um procedimento para a seleção de fungos produtores de compostos absorvedores de radiação UV, que poderia ser aplicado na obtenção de novos filtros orgânicos naturais para protetores solares.

Avaliação do efeito de diferentes concentrações de fotoiniciadores em propriedades mecânicas e na cor de um adesivo dental experimental

O presente trabalho teve por foco prover uma revisão de conceitos relacionados às resinas dentais fotopolimeráveis e, também, analisar como diferentes concentrações de fotoiniciadores de uso comum em odontologia podem influenciar no desempenho mecânico e na cor de resinas dentais. Concentrações de 0,01%, 0,10%, 0,25% 0,50%, 0,75%, 1,00% e 10% em massa de fotoiniciador foram adicionadas em uma matriz polimérica composta por TEGDMA/BisGMA 50%/50% em massa de forma que, fixando variáveis externas referentes à fonte de luz e à preparação das amostras, foi possível avaliar o efeito da concentração de fotoiniciador nas propriedades mecânicas de resistência à fratura por compressão, resistência à fratura pela aplicação de tensão diametral e dureza Barcol. Por fim, o efeito da concentração na cor aferida através do método CIELab foi investigada. Os fotoiniciadores utilizados foram o Irgacure® 819, a 1-fenil-1,2-propanodiona (PPD) e a Canforoquinona a qual foi utilizada juntamente com ocoiniciador Etil-4 dimetilaminobenzoato (EDB) na concentração constante de 1% em massa. Os resultados mecânicos e de cor foram comparados entre as diferentes amostras através de análises estatísticas (ANOVA) e, especificamente para cor, o parâmetro de diferença de cor (ΔE) foi interpretado verificar se as alterações observadas são perceptíveis ao olho humano. Para fundamentar os estudos, análises do espectro de absorção das amostras, do espectro de irradiação da fonte de luz e do grau de conversão das amostras foram realizadas. Os resultados revelaram que, independentemente do fotoiniciador, graus de conversão da mesma ordem de grandeza de um adesivo comercial foram obtidos, porém as velocidades de reação foram diferentes (maior velocidade para as amostras de CQ e menor para PPD). Curvas características foram obtidas para os espectros de absorção e de irradiação confirmando, pela sobreposição das curvas, que a fonte de luz é efetiva para iniciação da polimerização das amostras. As análises de resistência à fratura revelaram que a quebra catastrófica do material ocorrerá preferencialmente quando a força é aplicada ao longo do diâmetro para os corpos de prova preparados. Quanto à dureza, verificou-se que que para a CQ houve um máximo, para Irgacure um comportamento assintótico com aumento da concentração e, para a PPD, não foi possível a obtenção de amostras (verificado que a PPD necessita de tempo de irradiação superior ao convencionalmente utilizado na prática odontológica). Os testes de ANOVA permitiram verificar que o Irgacure é capaz de fornecer resultados mecânicos semelhante em relação à CQ ocasionando, em contrapartida, um menor escurecimento e amarelamento. Analisando o parâmetro ΔE, confirmou-se que a alteração na cor se torna evidente à medida que ocorre o aumento da concentração de fotoiniciador independentemente do fotoiniciador utilizado, porém a alteração é maior para a CQ.

The photochemical decomposition of gaseous formic acid /

Includes reprint of "The Ultraviolet Absorption Spectrum of Formic Acid" by H. C. Ramsperger and C. W. Porter.

Multilevel coherence effects in a two-level atom driven by a trichromatic field

We study the absorption and dispersion properties of a weak probe field monitoring a two-level atom driven by a trichromatic field. We calculate the steady-state linear susceptibility and find that the system can produce a number of multilevel coherence effects predicted for atoms composed of three and more energy levels. Although the atom has only one transition channel, the multilevel effects are possible because there are multichannel transitions between dressed states induced by the driving field. In particular, we show that the system can exhibit multiple electromagnetically induced transparency and can also produce a strong amplification at the central frequency which is not attributed to population inversion in both the atomic bare states and in the dressed atomic states. Moreover, we show that the absorption and dispersion of the probe field is sensitive to the initial relative phase of the components of the driving field. In addition, we show that the group velocity of the probe field can be controlled by changing the initial relative phases or frequencies of the driving fields and can also be varied from subluminal to superluminal. (C) 2003 Elsevier Science B.V. All rights reserved.

Chemical and structural disorder in eumelanins: A possible explanation for broadband absorbance

We report the results of an experimental and theoretical study of the electronic and structural properties of a key eumelanin precursor-5,6,-dihydroxyindole-2-carboxylic acid ( DHICA) - and its dimeric forms. We have used optical spectroscopy to follow the oxidative polymerization of DHICA to eumelanin and observe red shifting and broadening of the absorption spectrum as the reaction proceeds. First principles density functional theory calculations indicate that DHICA oligomers ( possible reaction products of oxidative polymerization) have the gap between the highest occupied molecular orbital and the lowest unoccupied molecular orbital red-shifted gaps with respect to the monomer. Furthermore, different bonding configurations ( leading to oligomers with different structures) produce a range of gaps. These experimental and theoretical results lend support to the chemical disorder model where the broadband monotonic absorption characteristic of all melanins is a consequence of the superposition of a large number of nonhomogeneously broadened Gaussian transitions associated with each of the components of a melanin ensemble. These results suggest that the traditional model of eumelanin as an amorphous organic semiconductor is not required to explain its optical properties and should be thoroughly reexamined. These results have significant implications for our understanding of the physics, chemistry, and biological function of these important biological macromolecules. Indeed, one may speculate that the robust functionality of melanins in vitro is a direct consequence of its heterogeneity, i.e., chemical disorder is a "low cost" natural resource in these systems

Quantitative scattering of melanin solutions

The optical scattering coefficient of a dilute, well-solubilized eumelanin solution has been accurately measured as a function of incident wavelength, and found to contribute < 6% of the total optical attenuation between 210 and 325 nm. At longer wavelengths (325-800 nm), the scattering was less than the minimum sensitivity of our instrument. This indicates that ultraviolet and visible optical density spectra can be interpreted as true absorption with a high degree of confidence. The scattering coefficient versus wavelength was found to be consistent with Rayleigh theory for a particle radius of 38 6 1 nm. Our results shed important light on the role of melanins as photoprotectants.

Morphology and material stability in polymer solar cells

Polymer solar cells are promising in that they are inexpensive to produce, and due to their mechanical flexibility have the potential for use in applications not possible for more traditional types of solar cells. The performance of polymer solar cells depends strongly on the distribution of electron donor and acceptor material in the active layer. Understanding the connection between morphology and performance as well as how to control the morphology, is therefore of great importance. Furthermore, improving the lifetime of polymer solar cells has become at least as important as improving the efficiency.   In this thesis, the relation between morphology and solar cell performance is studied, and the material stability for blend films of the thiophene-quinoxaline copolymer TQ1 and the fullerene derivatives PCBM and PC70BM. Atomic force microscopy (AFM) and scanning transmission X-ray microscopy (STXM) are used to investigate the lateral morphology, secondary ion mass spectrometry (SIMS) to measure the vertical morphology and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy to determine the surface composition. Lateral phase-separated domains are observed whose size is correlated to the solar cell performance, while the observed TQ1 surface enrichment does not affect the performance. Changes to the unoccupied molecular orbitals as a result of illumination in ambient air are observed by NEXAFS spectroscopy for PCBM, but not for TQ1. The NEXAFS spectrum of PCBM in a blend with TQ1 changes more than that of pristine PCBM. Solar cells in which the active layer has been illuminated in air prior to the deposition of the top electrode exhibit greatly reduced electrical performance. The valence band and absorption spectrum of TQ1 is affected by illumination in air, but the effects are not large enough to account for losses in solar cell performance, which are mainly attributed to PCBM degradation at the active layer surface.

Processing Bio-Argo nitrate concentration at the DAC Level

The only method used to date to measure dissolved nitrate concentration (NITRATE) with sensors mounted on profiling floats is based on the absorption of light at ultraviolet wavelengths by nitrate ion (Johnson and Coletti, 2002; Johnson et al., 2010; 2013; D’Ortenzio et al., 2012). Nitrate has a modest UV absorption band with a peak near 210 nm, which overlaps with the stronger absorption band of bromide, which has a peak near 200 nm. In addition, there is a much weaker absorption due to dissolved organic matter and light scattering by particles (Ogura and Hanya, 1966). The UV spectrum thus consists of three components, bromide, nitrate and a background due to organics and particles. The background also includes thermal effects on the instrument and slow drift. All of these latter effects (organics, particles, thermal effects and drift) tend to be smooth spectra that combine to form an absorption spectrum that is linear in wavelength over relatively short wavelength spans. If the light absorption spectrum is measured in the wavelength range around 217 to 240 nm (the exact range is a bit of a decision by the operator), then the nitrate concentration can be determined. Two different instruments based on the same optical principles are in use for this purpose. The In Situ Ultraviolet Spectrophotometer (ISUS) built at MBARI or at Satlantic has been mounted inside the pressure hull of a Teledyne/Webb Research APEX and NKE Provor profiling floats and the optics penetrate through the upper end cap into the water. The Satlantic Submersible Ultraviolet Nitrate Analyzer (SUNA) is placed on the outside of APEX, Provor, and Navis profiling floats in its own pressure housing and is connected to the float through an underwater cable that provides power and communications. Power, communications between the float controller and the sensor, and data processing requirements are essentially the same for both ISUS and SUNA. There are several possible algorithms that can be used for the deconvolution of nitrate concentration from the observed UV absorption spectrum (Johnson and Coletti, 2002; Arai et al., 2008; Sakamoto et al., 2009; Zielinski et al., 2011). In addition, the default algorithm that is available in Satlantic sensors is a proprietary approach, but this is not generally used on profiling floats. There are some tradeoffs in every approach. To date almost all nitrate sensors on profiling floats have used the Temperature Compensated Salinity Subtracted (TCSS) algorithm developed by Sakamoto et al. (2009), and this document focuses on that method. It is likely that there will be further algorithm development and it is necessary that the data systems clearly identify the algorithm that is used. It is also desirable that the data system allow for recalculation of prior data sets using new algorithms. To accomplish this, the float must report not just the computed nitrate, but the observed light intensity. Then, the rule to obtain only one NITRATE parameter is, if the spectrum is present then, the NITRATE should be recalculated from the spectrum while the computation of nitrate concentration can also generate useful diagnostics of data quality.

Photocurrents in P3MeT Schottky barrier diodes

The photocurrent action spectrum of a Schottky diode formed from electrodeposited poly(3-methylthiophene) is shown to follow closely the UV-visible absorption spectrum. At low forward bias, the peak photocurrent asymptotes to the expected square-root dependence on total potential, V-t, across the depletion region. At high reverse bias the superlinear dependence of the photocurrent on V-t suggests that internal photoemission from the rectifying aluminium electrode may be the dominant process.