Mastitis in dairy heifers: Prevalence and risk factors

Poor udder health represents a serious problem in dairy production and has been investigated intensively, but heifers generally have not been the main focus of mastitis control. The aim of this study was to evaluate the prevalence, risk factors and consequences of heifer mastitis in Switzerland. The study included 166,518 heifers of different breeds (Swiss Red Pied, Swiss Brown Cattle and Holstein). Monthly somatic cell counts (SCCs) provided by the main dairy breeding organisations in Switzerland were monitored for 3 years; the prevalence of subclinical mastitis (SCM) was determined on the basis of SCCs ≥100,000 cells/mL at the first test date. The probability of having SCM at the first test date during lactation was modelled using logistic regression. Analysed factors included data for the genetic background, morphological traits, geographical region, season of parturition and milk composition. The overall prevalence of SCM in heifers during the period from 2006 to 2010 was 20.6%. Higher frequencies of SCM were present in heifers of the Holstein breed (odds ratio, OR, 1.62), heifers with high fat:protein ratios (OR 1.97) and heifers with low milk urea concentrations combined with high milk protein concentrations (OR 3.97). Traits associated with a low risk of SCM were high set udders, high overall breeding values and low milk breeding values. Heifers with SCM on the first test day had a higher risk of either developing chronic mastitis or leaving the herd prematurely.

Focal Epilepsy: MR Imaging of Nonhemodynamic Field Effects by Using a Phase-cycled Stimulus-induced Rotary Saturation Approach with Spin-Lock Preparation.

Purpose To investigate whether nonhemodynamic resonant saturation effects can be detected in patients with focal epilepsy by using a phase-cycled stimulus-induced rotary saturation (PC-SIRS) approach with spin-lock (SL) preparation and whether they colocalize with the seizure onset zone and surface interictal epileptiform discharges (IED). Materials and Methods The study was approved by the local ethics committee, and all subjects gave written informed consent. Eight patients with focal epilepsy undergoing presurgical surface and intracranial electroencephalography (EEG) underwent magnetic resonance (MR) imaging at 3 T with a whole-brain PC-SIRS imaging sequence with alternating SL-on and SL-off and two-dimensional echo-planar readout. The power of the SL radiofrequency pulse was set to 120 Hz to sensitize the sequence to high gamma oscillations present in epileptogenic tissue. Phase cycling was applied to capture distributed current orientations. Voxel-wise subtraction of SL-off from SL-on images enabled the separation of T2* effects from rotary saturation effects. The topography of PC-SIRS effects was compared with the seizure onset zone at intracranial EEG and with surface IED-related potentials. Bayesian statistics were used to test whether prior PC-SIRS information could improve IED source reconstruction. Results Nonhemodynamic resonant saturation effects ipsilateral to the seizure onset zone were detected in six of eight patients (concordance rate, 0.75; 95% confidence interval: 0.40, 0.94) by means of the PC-SIRS technique. They were concordant with IED surface negativity in seven of eight patients (0.88; 95% confidence interval: 0.51, 1.00). Including PC-SIRS as prior information improved the evidence of the standard EEG source models compared with the use of uninformed reconstructions (exceedance probability, 0.77 vs 0.12; Wilcoxon test of model evidence, P < .05). Nonhemodynamic resonant saturation effects resolved in patients with favorable postsurgical outcomes, but persisted in patients with postsurgical seizure recurrence. Conclusion Nonhemodynamic resonant saturation effects are detectable during interictal periods with the PC-SIRS approach in patients with epilepsy. The method may be useful for MR imaging-based detection of neuronal currents in a clinical environment. (©) RSNA, 2016 Online supplemental material is available for this article.

Corporate Tax Avoidance and High Powered Incentives

This paper analyzes the links between corporate tax avoidance, the growth of highpowered incentives for managers, and the structure of corporate governance. We develop and test a simple model that highlights the role of complementarities between tax sheltering and managerial diversion in determining how high-powered incentives influence tax sheltering decisions. The model generates the testable hypothesis that firm governance characteristics determine how incentive compensation changes sheltering decisions. In order to test the model, we construct an empirical measure of corporate tax avoidance - the component of the book-tax gap not attributable to accounting accruals - and investigate the link between this measure of tax avoidance and incentive compensation. We find that, for the full sample of firms, increases in incentive compensation tend to reduce the level of tax sheltering, suggesting a complementary relationship between diversion and sheltering. As predicted by the model, the relationship between incentive compensation and tax sheltering is a function of a firm.s corporate governance. Our results may help explain the growing cross-sectional variation among firms in their levels of tax avoidance, the .undersheltering puzzle,. and why large book-tax gaps are associated with subsequent negative abnormal returns.

Flow separation and roughness lengths over large bedforms in a tidal environment

This study characterises the shape of the flow separation zone (FSZ) and wake region over large asymmetric bedforms under tidal flow conditions. High resolution bathymetry, flow velocity and turbulence data were measured along two parallel transects in a tidal channel covered with bedforms. The field data are used to verify the applicability of a numerical model for a systematic study using the Delft3D modelling system and test the model sensitivity to roughness length. Three experiments are then conducted to investigate how the FSZ size and wake extent vary depending on tidally-varying flow conditions, water levels and bathymetry. During the ebb, a large FSZ occurs over the steep lee side of each bedform. During the flood, no flow separation develops over the bedforms having a flat crest; however, a small FSZ is observed over the steepest part of the crest of some bedforms, where the slope is locally up to 15°. Over a given bedform morphology and constant water levels, no FSZ occurs for velocity magnitudes smaller than 0.1 m s**-1; as the flow accelerates, the FSZ reaches a stable size for velocity magnitudes greater than 0.4 m s**-1. The shape of the FSZ is not influenced by changes in water levels. On the other hand, variations in bed morphology, as recorded from the high-resolution bathymetry collected during the tidal cycle, influence the size and position of the FSZ: a FSZ develops only when the maximum lee side slope over a horizontal distance of 5 m is greater than 10°. The height and length of the wake region are related to the length of the FSZ. The total roughness along the transect lines is an order of magnitude larger during the ebb than during the flood due to flow direction in relation to bedform asymmetry: during the ebb, roughness is created by the large bedforms because a FSZ and wake develops over the steep lee side. The results add to the understanding of hydrodynamics of natural bedforms in a tidal environment and may be used to better parameterise small-scale processes in large-scale studies.

Ice thickness distribution and glacier bed of Hallstätter Gletscher

Hallstätter Glacier is the northernmost glacier of Austria. Appendant to the northern Limestone Alps, the glacier is located at 47°28'50'' N, 13°36'50'' E in the Dachstein-region. At the same time with its advance linked to the Little Ice Age (LIA), research on changes in size and mass of Hallstätter glacier was started in 1842 by Friedrich Simony. He observed and documented the glacier retreat related to its last maximum extension in 1856. In addition, Hallstätter Glacier is a subject to scientific research to date. In this thesis methods and results of ongoing mass balance measurements are presented and compared to long term volume changes and meteorological observations. The current mass balance monitoring programm using the direct glaciological method was started 2006. In this context, 2009 the ice thickness was measured with ground penetrating radar. The result are used with digital elevation models reconstucted from historical maps and recent digital elevation models to calculate changes in shape and volume of Hallstätter Glacier. Based on current meteorological measurements near the glacier and longtime homogenized climate data provided by HISTALP, time series of precipitation and temperature beginning at the LIA are produced. These monthly precipitation and monthly mean temperature data are used to compare results of a simple degree day model with the volume change calculated from the difference of the digital elevation models. The two years of direct mass balance measurements are used to calibrate the degree day model. A number of possible future scenarios are produced to indicate prospective changes. Within the 150-year-period between 1856 and 2007 the Hallstätter Glacier lost 1940 meters of its length and 2.23 km**2 in area. 37% of the initial volume of 1856 remained. This retreat came along with a change in climate. The application of a running avarage of 30 years shows an increase in precipitation of 18.5% and a warming of 1.3°C near the glacier between 1866 and 1993. The mass loss was continued in the hydrological years 2006/2007 and 2007/2008 showing mean specific mass balance of -376 mm and -700 mm, respectively. Applying a temperature correction for the different minimum elevations of the glacier, the degree day approach based on the two measured mass balances can reproduce sign and order of magnitude of the volume change of Hallstätter Glacier since 1856. Nevertheless, the relative deviation is significant. Future scenarios show, that 30% of the entire glacier volume remains after subtracting the elevation changes between the digital elevation models of 2002 and 2007 ten times from the surface of 2007. The past and present mass changes of Hallstätter Glacier are showing a retreating glacier as a consequence of rising temperatures. Due to high precepitation, increased with previous warming, the Hallstätter Glacier can and will exist in lower elevation compared to inner alpine glaciers.

Criterios de optimización de los incentivos en contratos de concesión ligados a indicadores de calidad de servicio para firmes bituminosos

Actualmente se está empezando a consolidar una nueva forma de gestionar la conservación y mantenimiento de la red viaria de las Administraciones Públicas, basándose en contratos de colaboración público-privadas (PPP). Las motivaciones que están provocando este movimiento son de diversa índole. Por un lado, en el seno de la Unión Europea, existen serias restricciones presupuestarias debido al alto endeudamiento del sector público, lo que está llevando a buscar la forma óptima de disminuir el endeudamiento público, sin dejar de prestar servicios a la sociedad como la conservación y mantenimiento de las redes viarias. Por esta vertiente, se trata de convertir contratos convencionales de conservación viaria a esquemas de colaboración público-privada, donde se transferiría al sector privado el riesgo de disponibilidad de la vía mediante el uso de indicadores de calidad y servicio. Con esta transferencia de riesgo, junto con la transferencia del riesgo de demanda/construcción, no consolidaría la deuda de la sociedad de propósito específico constituida para la gestión del contrato de colaboración público-privada dentro de las cuentas públicas, con lo que se conseguiría no aumentar el déficit público, permitiendo continuar ofreciendo el servicio demandado por la sociedad. Por otro lado, la segunda motivación del desarrollo de este tipo de contratos, no tan economicista como la anterior y más enfocada a la gestión, se trata de utilizar los contratos de gestión basados en el uso de indicadores de calidad de servicio para mejorar las prestaciones de la red viaria competencia de una Administración. Con el uso de estos indicadores, el gestor tiene una herramienta muy útil para controlar la actividad del sector privado y asegurar que se ofrece un buen servicio. En la presente tesis, la investigación se ha centrado más en la vertiente de los indicadores de calidad relacionados con la gestión eficiente de las vías objeto de conservación y mantenimiento mediante el empleo de contratos de gestión privada que utilicen este tipo de herramientas de control, monitorización y gestión. En una primera parte, la presente tesis estudia el estado de la red de carreteras, referido principalmente a España, comparando su estado con el resto de redes de carreteras de Europa, detectando las principales carencias de la misma, sobre todo en cuanto a la gestión y conservación de firmes. En un segundo bloque, la tesis analiza el estado del arte de los nuevos procedimientos de gestión de la conservación y mantenimiento basados en indicadores de calidad del servicio en el mundo, destacándose que se trata de un tema relativamente reciente, con gran interés para el sector de la gestión y financiación de infraestructuras viarias. Al ser tan novedoso, por la falta de experiencias previas, las distintas Administración, tanto propias como foráneas, han pecado de un exceso de celo a la hora de establecer los umbrales sobre los que giran los distintos indicadores de calidad de servicio que permiten controlar la gestión de la conservación y mantenimiento de la vía. Partiendo de la labor de análisis descrita, la tesis realiza una investigación más detallada de los indicadores de calidad de servicio correspondientes a firmes bituminosos, debido a que estos indicadores son los más delicados y decisivos a la hora de realizar una correcta gestión de la vía a largo plazo. Dentro de los indicadores de firmes bituminosos, se ha realizado un modelo específico de evolución de comportamiento a lo largo del tiempo de la regularidad superficial, parámetro básico para numerosas Administraciones y organismos investigadores para poder conocer la evolución de un firme a lo largo del tiempo. A esta metodología se le ha dado el nombre de Modelo JRB para evaluar la racionalidad económica de indicadores de calidad asociados a parámetros de firmes. El modelo propuesto básicamente evalúa el valor óptimo desde la perspectiva económica que ha de tener el parámetro técnico que defina alguna propiedad del firme, aplicado a la definición de los indicadores de calidad de servicio. Esta visión del valor umbral del indicador deja a un lado consideraciones de equidad o de cualquier otra índole, basándose más en una visión económica. La metodología del Modelo JRB se puede aplicar a cualquier indicador de calidad relacionado con firmes, ya que lo que se obtiene es el valor óptimo económico que debería tener el umbral del indicador de calidad. El Modelo JRB consta de varias fases. En las primeras etapas el Modelo realiza el cálculo de los costes totales de transporte utilizando como herramienta el software HDM-IV desarrollado por el Banco Mundial. En etapas posteriores, el Modelo realiza análisis de sensibilidad para distintas propuestas de sección de firme, intensidades de tráfico y restricciones al parámetro técnico que define el indicador de calidad de servicio. Como ejercicio práctico de cara a contrastar la metodología del Modelo JRB se ha realizado un Caso de Estudio. Se ha tomado un tramo teórico, con características similares a la red de carreteras española, y con una flota vehicular similar a la española, donde se ha elegido como indicador de calidad la regularidad superficial (IRI). Con las sensibilidades realizadas con el Modelo JRB, se ha determinado el rango de valores que debería tener un indicador de calidad basado en el IRI para que dichos valores fueran óptimos desde la perspectiva económica Nowadays is becoming a new way to manage O&M (operation and maintenance) in public road networks, based on PPP contracts (public-private partnership). There are several issues which are driving this trend. On the one hand, EU (European Union) has serious budgetary constraints due to the high public sector borrowing. EU politicians are looking for the best way to reduce public debt, keeping services to society such as O&M of road networks. For this aspect, conventional O&M contracts are switching to PPP scenarios, where availability risk would be transfer to private sector using PI (performance indicators), along with demand risk transfer With this risk transference, along with the transfer of demand/construction risk, SPV (specific purpose vehicle) debt doesn’t consolidate in public accounts, so deficit wouldn’t increase, allowing the continuation of services demanded by society. On the other hand, the second motivation for developing this kind of contracts, not so economist as above and more focused to management, it is about using O&M contracts based on the use of PI to improve road network maintenance. Using these indicators, manager has a very useful tool to monitor private sector activity and ensure that it is provided a good service. In this thesis, the research has been focused on PI quality aspect, related with efficient management of PPP contracts for roads, which use these tools for control, monitoring and management. In the first part, this thesis examines the state of road network, based mainly in Spain, comparing with other road networks in Europe, identifying the main gaps in it, especially with regard to the management and maintenance of pavements. In a second block, the thesis analyzes the state of art of new O&M contracts based on PI in the world, emphasizing that they are relatively recent. These kinds of contracts have a great interest in road management and financing sector. Administrations all around the world have launch tenders with very exigent PI thresholds due to several factors: this knowledge is a new area, the lack of previous experiences and the variety of Administrations which have bid these contracts. Building on the described analysis, thesis develops a more detailed research about PI for bituminous pavements, because these PI are the most delicate and decisive in making a proper long term road management. Among bituminous pavements PI, IRI (International Roughness Index) has been analyzed with more detail and has been developed a specific model of behaviour evolution over time for evenness (IRI), basic parameter for many administrations and research departments in order to know the evolution of a pavement over time. This methodology has been given the name of JRB Model to evaluate the economic rationality of performance indicators associated with pavements parameters. The proposed model basically evaluates the optimal value from an economic perspective it must have the technical parameter which defines some pavement characteristic applied to the definition of performance indicators. This point of view of indicator value threshold sets aside justice considerations or otherwise, based more on an economic perspective. JRB Model methodology can be applied to any performance indicator associated to pavements, because what you get is the economic optimum threshold should have the performance indicator. JRB Model consists of several phases. In the early stages, the Model calculates transport total cost using HDM-IV software, developed by the World Bank, as a tool. In later stages, the Model performs sensitivity analyzes for different pavement section, AADT and restrictions to the technical parameter which defines the performance indicator. As a practical exercise to test JRB Model methodology, it has done a Case Study. It has taken a theoretical section, with similar characteristics to Spanish road network, and a vehicles fleet similar to Spanish. Evenness (IRI) was chosen as a performance indicator. JRB Model calculated some sensitivities, which were useful to determined thresholds range for pavement performance indicators based on IRI to be optimal from an economic perspective.

Spatial effects on rural commerce

Commerce in rural territories should not be considered as a needed service, but as a basic infrastructure, that impact not only existent population, but also tourism, and rural industrialization. So, the rural areas need not only agriculture but industry and services, to have a global and balanced development, including for the countryside and the population. In the work presented in this paper, we are considering the formulation of the direct relation between population and the endowment of commerce sites within a geographical territory, the ?area of commercial interactions?. These are the closer set of towns that can gravitate to each other to cover the required needs for the populations within the area. The products retailed, range from basic products for the daily lives, to all other products for industry, agriculture, and services. The econometric spatial model developed to evaluate the interactions and estimate the parameters, is based on the Spatial Error Model, which allows for other spatial hidden effects to be considered without direct interference to the commercial disposition. The data and territory used to test the model correspond to a rural area in the Spanish Palencia territory (NUTS-3 level). The parameters have dependence from population levels, local rent per head, local and regional government budgets, and particular spatial restrictions. Interesting results are emerging form the model. The more significant is that the spatial effects can replace some number of commerce sites in towns, given the right spatial distribution of the sites and the towns. This is equivalent to consider the area of commercial interactions as the unit of measurement for the basic infrastructure and not only the towns.

A Comunicação Mediada por Interfaces Digitais: a interação humana com os jogos digitais em celulares.

O trabalho é um estudo exploratório sobre o processamento de mensagens de entretenimento. O objetivo do trabalho foi propor e testar um modelo de processamento de mensagens dedicado à compreensão de jogos digitais. Para realizar tal tarefa realizou-se um extenso levantamento de técnicas de observação de usuários diante de softwares e mídias, para conhecer as qualidades e limitações de cada uma dessas técnicas, bem como de sua abordagem do problema. Também foi realizado um levantamento dos modelos de processamento de mensagens nas mídias tradicionais e nas novas mídias. Com isso foi possível propor um novo modelo de análise de processamento de mensagens de entretenimento. Uma vez criado o modelo teórico, fez-se preciso testar se os elementos propostos como participantes desse processo estavam corretos e se seriam capazes de capturar adequadamente as semelhanças e diferenças entre a interação entre jogadores e as diferentes mídias. Por essa razão, estruturou-se uma ferramenta de coleta de dados, que foi validada junto a designers de jogos digitais, uma vez que esses profissionais conhecem o processo de criação de um jogo, seus elementos e objetivos. Posteriormente, foi feito um primeiro teste, junto a praticantes de jogos digitais de diversas idades em computadores pessoais e TV digital interativa, a fim e verificar como os elementos do modelo relacionavam-se entre si. O teste seguinte fez a coleta de dados de praticantes de jogos digitais em aparelhos celulares, tendo como objetivo capturar como se dá a formação de uma experiência através do processamento da mensagem de entretenimento num meio cujas limitações são inúmeras: tamanho de tela e teclas, para citar algumas delas. Como resultado, verificou-se, por meio de testes estatísticos, que jogos praticados em meios como computadores pessoais atraem mais por seus aspectos estéticos, enquanto a apreciação de um jogo em aparelhos celulares depende muito mais de sua habilidade de manter a interação que um jogo praticado em PC. Com isso conclui-se que o processamento das mensagens de entretenimento depende da capacidade dos seus criadores em entender os limites de cada meio e usar adequadamente os elementos que compõe o ambiente de um jogo, para conseguir levar à apreciação do mesmo.(AU)

A Comunicação Mediada por Interfaces Digitais: a interação humana com os jogos digitais em celulares.

O trabalho é um estudo exploratório sobre o processamento de mensagens de entretenimento. O objetivo do trabalho foi propor e testar um modelo de processamento de mensagens dedicado à compreensão de jogos digitais. Para realizar tal tarefa realizou-se um extenso levantamento de técnicas de observação de usuários diante de softwares e mídias, para conhecer as qualidades e limitações de cada uma dessas técnicas, bem como de sua abordagem do problema. Também foi realizado um levantamento dos modelos de processamento de mensagens nas mídias tradicionais e nas novas mídias. Com isso foi possível propor um novo modelo de análise de processamento de mensagens de entretenimento. Uma vez criado o modelo teórico, fez-se preciso testar se os elementos propostos como participantes desse processo estavam corretos e se seriam capazes de capturar adequadamente as semelhanças e diferenças entre a interação entre jogadores e as diferentes mídias. Por essa razão, estruturou-se uma ferramenta de coleta de dados, que foi validada junto a designers de jogos digitais, uma vez que esses profissionais conhecem o processo de criação de um jogo, seus elementos e objetivos. Posteriormente, foi feito um primeiro teste, junto a praticantes de jogos digitais de diversas idades em computadores pessoais e TV digital interativa, a fim e verificar como os elementos do modelo relacionavam-se entre si. O teste seguinte fez a coleta de dados de praticantes de jogos digitais em aparelhos celulares, tendo como objetivo capturar como se dá a formação de uma experiência através do processamento da mensagem de entretenimento num meio cujas limitações são inúmeras: tamanho de tela e teclas, para citar algumas delas. Como resultado, verificou-se, por meio de testes estatísticos, que jogos praticados em meios como computadores pessoais atraem mais por seus aspectos estéticos, enquanto a apreciação de um jogo em aparelhos celulares depende muito mais de sua habilidade de manter a interação que um jogo praticado em PC. Com isso conclui-se que o processamento das mensagens de entretenimento depende da capacidade dos seus criadores em entender os limites de cada meio e usar adequadamente os elementos que compõe o ambiente de um jogo, para conseguir levar à apreciação do mesmo.(AU)

Interacting helical faces of subunits a and c in the F1Fo ATP synthase of Escherichia coli defined by disulfide cross-linking

Subunits a and c of Fo are thought to cooperatively catalyze proton translocation during ATP synthesis by the Escherichia coli F1Fo ATP synthase. Optimizing mutations in subunit a at residues A217, I221, and L224 improves the partial function of the cA24D/cD61G double mutant and, on this basis, these three residues were proposed to lie on one face of a transmembrane helix of subunit a, which then interacted with the transmembrane helix of subunit c anchoring the essential aspartyl group. To test this model, in the present work Cys residues were introduced into the second transmembrane helix of subunit c and the predicted fourth transmembrane helix of subunit a. After treating the membrane vesicles of these mutants with Cu(1,10-phenanthroline)2SO4 at 0°, 10°, or 20°C, strong a–c dimer formation was observed at all three temperatures in membranes of 7 of the 65 double mutants constructed, i.e., in the aS207C/cI55C, aN214C/cA62C, aN214C/cM65C, aI221C/cG69C, aI223C/cL72C, aL224C/cY73C, and aI225C/cY73C double mutant proteins. The pattern of cross-linking aligns the helices in a parallel fashion over a span of 19 residues with the aN214C residue lying close to the cA62C and cM65C residues in the middle of the membrane. Lesser a–c dimer formation was observed in nine other double mutants after treatment at 20°C in a pattern generally supporting that indicated by the seven landmark residues cited above. Cross-link formation was not observed between helix-1 of subunit c and helix-4 of subunit a in 19 additional combinations of doubly Cys-substituted proteins. These results provide direct chemical evidence that helix-2 of subunit c and helix-4 of subunit a pack close enough to each other in the membrane to interact during function. The proximity of helices supports the possibility of an interaction between Arg210 in helix-4 of subunit a and Asp61 in helix-2 of subunit c during proton translocation, as has been suggested previously.

Conformational changes couple Na+ and glucose transport

The mechanism by which cotransport proteins couple their substrates across cell membranes is not known. A commonly proposed model is that cotransport results from ligand-induced conformational transitions that change the accessibility of ligand-binding sites from one side of the membrane to the other. To test this model, we have measured the accessibility of covalent probes to a cysteine residue (Q457C) placed in the putative sugar-translocation domain of the Na+/glucose cotransporter (SGLT1). The mutant protein Q457C was able to transport sugar, but transport was abolished after alkylation by methanethiosulfonate reagents. Alkylation blocked sugar translocation but not sugar binding. Accessibility of Q457C to alkylating reagents required external Na+ and was blocked by external sugar and phlorizin. The voltage dependence of accessibility was directly correlated with the presteady–state charge movement of SGLT1. Voltage-jump experiments with rhodamine-6-maleimide-labeled Q457C showed that the time course and level of changes in fluorescence closely followed the presteady–state charge movement. We conclude that conformational changes are responsible for the coupling of Na+ and sugar transport and that Q457 plays a critical role in sugar translocation by SGLT1.

Interactions among enzymes of the Arabidopsis flavonoid biosynthetic pathway

Flavonoids are secondary metabolites derived from phenylalanine and acetate metabolism that perform a variety of essential functions in higher plants. Studies over the past 30 years have supported a model in which flavonoid metabolism is catalyzed by an enzyme complex localized to the endoplasmic reticulum [Hrazdina, G. & Wagner, G. J. (1985) Arch. Biochem. Biophys. 237, 88–100]. To test this model further we assayed for direct interactions between several key flavonoid biosynthetic enzymes in developing Arabidopsis seedlings. Two-hybrid assays indicated that chalcone synthase, chalcone isomerase (CHI), and dihydroflavonol 4-reductase interact in an orientation-dependent manner. Affinity chromatography and immunoprecipitation assays further demonstrated interactions between chalcone synthase, CHI, and flavonol 3-hydroxylase in lysates from Arabidopsis seedlings. These results support the hypothesis that the flavonoid enzymes assemble as a macromolecular complex with contacts between multiple proteins. Evidence was also found for posttranslational modification of CHI. The importance of understanding the subcellular organization of elaborate enzyme systems is discussed in the context of metabolic engineering.

Identification of the vitamin K-dependent carboxylase active site: Cys-99 and Cys-450 are required for both epoxidation and carboxylation

The vitamin K-dependent carboxylase modifies and renders active vitamin K-dependent proteins involved in hemostasis, cell growth control, and calcium homeostasis. Using a novel mechanism, the carboxylase transduces the free energy of vitamin K hydroquinone (KH2) oxygenation to convert glutamate into a carbanion intermediate, which subsequently attacks CO2, generating the γ-carboxylated glutamate product. How the carboxylase effects this conversion is poorly understood because the active site has not been identified. Dowd and colleagues [Dowd, P., Hershline, R., Ham, S. W. & Naganathan, S. (1995) Science 269, 1684–1691] have proposed that a weak base (cysteine) produces a strong base (oxygenated KH2) capable of generating the carbanion. To define the active site and test this model, we identified the amino acids that participate in these reactions. N-ethyl maleimide inhibited epoxidation and carboxylation, and both activities were equally protected by KH2 preincubation. Amino acid analysis of 14C- N-ethyl maleimide-modified human carboxylase revealed 1.8–2.3 reactive residues and a specific activity of 7 × 108 cpm/hr per mg. Tryptic digestion and liquid chromatography electrospray mass spectrometry identified Cys-99 and Cys-450 as active site residues. Mutation to serine reduced both epoxidation and carboxylation, to 0.2% (Cys-99) or 1% (Cys-450), and increased the Kms for a glutamyl substrate 6- to 8-fold. Retention of some activity indicates a mechanism for enhancing cysteine/serine nucleophilicity, a property shared by many active site thiol enzymes. These studies, which represent a breakthrough in defining the carboxylase active site, suggest a revised model in which the glutamyl substrate indirectly coordinates at least one thiol, forming a catalytic complex that ionizes a thiol to initiate KH2 oxygenation.

Genetic drift and within-host metapopulation dynamics of HIV-1 infection

Most HIV replication occurs in solid lymphoid tissue, which has prominent architecture at the histological level, which separates groups of productively infected CD4+ cells. Nevertheless, current population models of HIV assume panmixis within lymphoid tissue. We present a simple “metapopulation” model of HIV replication, where the population of infected cells is comprised of a large number of small populations, each of which is established by a few founder viruses and undergoes turnover. To test this model, we analyzed viral genetic variation of infected cell subpopulations within the spleen and demonstrated the action of founder effects as well as significant variation in the extent of genetic differentiation between subpopulations among patients. The combination of founder effects and subpopulation turnover can result in an effective population size much lower than the actual population size and may contribute to the importance of genetic drift in HIV evolution despite a large number of infected cells.

Copy-up mutants of the plasmid RK2 replication initiation protein are defective in coupling RK2 replication origins.

The broad host range plasmid RK2 replicates and regulates its copy number in a wide range of Gram-negative bacteria. The plasmid-encoded trans-acting replication protein TrfA and the origin of replication oriV are sufficient for controlled replication of the plasmid in all Gram-negative bacteria tested. The TrfA protein binds specifically to direct repeat sequences (iterons) at the origin of replication. A replication control model, designated handcuffing or coupling, has been proposed whereby the formation of coupled TrfA-oriV complexes between plasmid molecules results in hindrance of origin activity and, consequently, a shut-down of plasmid replication under conditions of higher than normal copy number. Therefore, according to this model, the coupling activity of an initiation protein is essential for copy number control and a copy-up initiation protein mutant should have reduced ability to form coupled complexes. To test this model for plasmid RK2, two previously characterized copy-up TrfA mutations, trfA-254D and trfA-267L, were combined and the resulting copy-up double mutant TFrfA protein TrfA-254D/267L was characterized. Despite initiating runaway (uncontrolled) replication in vivo, the copy-up double-mutant TrfA protein exhibited replication kinetics similar to the wild-type protein in vitro. Purified TrfA-254D, TrfA-267L, and TrfA-254D/267L proteins were then examined for binding to the iterons and for coupling activity using an in vitro ligase-catalyzed multimerization assay. It was found that both single and double TrfA mutant proteins exhibited substantially reduced (single mutants) or barely detectable (double mutant) levels of coupling activity while not being diminished in their capacity to bind to the origin of replication. These observations provide direct evidence in support of the coupling model of replication control.