Estimate of the total ice volume of Svalbard glaciers and their potential contribution to sea-level rise = Estimación del volumen de hielo de Svalbard y su contribución potencial a la subida del nivel del mar

El objetivo final de las investigaciones recogidas en esta tesis doctoral es la estimación del volumen de hielo total de los ms de 1600 glaciares de Svalbard, en el Ártico, y, con ello, su contribución potencial a la subida del nivel medio del mar en un escenario de calentamiento global. Los cálculos más exactos del volumen de un glaciar se efectúan a partir de medidas del espesor de hielo obtenidas con georradar. Sin embargo, estas medidas no son viables para conjuntos grandes de glaciares, debido al coste, dificultades logísticas y tiempo requerido por ellas, especialmente en las regiones polares o de montaña. Frente a ello, la determinación de áreas de glaciares a partir de imágenes de satélite sí es viable a escalas global y regional, por lo que las relaciones de escala volumen-área constituyen el mecanismo más adecuado para las estimaciones de volúmenes globales y regionales, como las realizadas para Svalbard en esta tesis. Como parte del trabajo de tesis, hemos elaborado un inventario de los glaciares de Svalbard en los que se han efectuado radioecosondeos, y hemos realizado los cálculos del volumen de hielo de más de 80 cuencas glaciares de Svalbard a partir de datos de georradar. Estos volúmenes han sido utilizados para calibrar las relaciones volumen-área desarrolladas en la tesis. Los datos de georradar han sido obtenidos en diversas campañas llevadas a cabo por grupos de investigación internacionales, gran parte de ellas lideradas por el Grupo de Simulación Numérica en Ciencias e Ingeniería de la Universidad Politécnica de Madrid, del que forman parte la doctoranda y los directores de tesis. Además, se ha desarrollado una metodología para la estimación del error en el cálculo de volumen, que aporta una novedosa técnica de cálculo del error de interpolación para conjuntos de datos del tipo de los obtenidos con perfiles de georradar, que presentan distribuciones espaciales con unos patrones muy característicos pero con una densidad de datos muy irregular. Hemos obtenido en este trabajo de tesis relaciones de escala específicas para los glaciares de Svalbard, explorando la sensibilidad de los parámetros a diferentes morfologías glaciares, e incorporando nuevas variables. En particular, hemos efectuado experimentos orientados a verificar si las relaciones de escala obtenidas caracterizando los glaciares individuales por su tamaño, pendiente o forma implican diferencias significativas en el volumen total estimado para los glaciares de Svalbard, y si esta partición implica algún patrón significativo en los parámetros de las relaciones de escala. Nuestros resultados indican que, para un valor constante del factor multiplicativo de la relacin de escala, el exponente que afecta al área en la relación volumen-área decrece según aumentan la pendiente y el factor de forma, mientras que las clasificaciones basadas en tamaño no muestran un patrón significativo. Esto significa que los glaciares con mayores pendientes y de tipo circo son menos sensibles a los cambios de área. Además, los volúmenes de la población total de los glaciares de Svalbard calculados con fraccionamiento en grupos por tamaño y pendiente son un 1-4% menores que los obtenidas usando la totalidad de glaciares sin fraccionamiento en grupos, mientras que los volúmenes calculados fraccionando por forma son un 3-5% mayores. También realizamos experimentos multivariable para obtener estimaciones óptimas del volumen total mediante una combinación de distintos predictores. Nuestros resultados muestran que un modelo potencial simple volumen-área explica el 98.6% de la varianza. Sólo el predictor longitud del glaciar proporciona significación estadística cuando se usa además del área del glaciar, aunque el coeficiente de determinación disminuye en comparación con el modelo más simple V-A. El predictor intervalo de altitud no proporciona información adicional cuando se usa además del área del glaciar. Nuestras estimaciones del volumen de la totalidad de glaciares de Svalbard usando las diferentes relaciones de escala obtenidas en esta tesis oscilan entre 6890 y 8106 km3, con errores relativos del orden de 6.6-8.1%. El valor medio de nuestras estimaciones, que puede ser considerado como nuestra mejor estimación del volumen, es de 7.504 km3. En términos de equivalente en nivel del mar (SLE), nuestras estimaciones corresponden a una subida potencial del nivel del mar de 17-20 mm SLE, promediando 19_2 mm SLE, donde el error corresponde al error en volumen antes indicado. En comparación, las estimaciones usando las relaciones V-A de otros autores son de 13-26 mm SLE, promediando 20 _ 2 mm SLE, donde el error representa la desviación estándar de las distintas estimaciones. ABSTRACT The final aim of the research involved in this doctoral thesis is the estimation of the total ice volume of the more than 1600 glaciers of Svalbard, in the Arctic region, and thus their potential contribution to sea-level rise under a global warming scenario. The most accurate calculations of glacier volumes are those based on ice-thicknesses measured by groundpenetrating radar (GPR). However, such measurements are not viable for very large sets of glaciers, due to their cost, logistic difficulties and time requirements, especially in polar or mountain regions. On the contrary, the calculation of glacier areas from satellite images is perfectly viable at global and regional scales, so the volume-area scaling relationships are the most useful tool to determine glacier volumes at global and regional scales, as done for Svalbard in this PhD thesis. As part of the PhD work, we have compiled an inventory of the radio-echo sounded glaciers in Svalbard, and we have performed the volume calculations for more than 80 glacier basins in Svalbard from GPR data. These volumes have been used to calibrate the volume-area relationships derived in this dissertation. Such GPR data have been obtained during fieldwork campaigns carried out by international teams, often lead by the Group of Numerical Simulation in Science and Engineering of the Technical University of Madrid, to which the PhD candidate and her supervisors belong. Furthermore, we have developed a methodology to estimate the error in the volume calculation, which includes a novel technique to calculate the interpolation error for data sets of the type produced by GPR profiling, which show very characteristic data distribution patterns but with very irregular data density. We have derived in this dissertation scaling relationships specific for Svalbard glaciers, exploring the sensitivity of the scaling parameters to different glacier morphologies and adding new variables. In particular, we did experiments aimed to verify whether scaling relationships obtained through characterization of individual glacier shape, slope and size imply significant differences in the estimated volume of the total population of Svalbard glaciers, and whether this partitioning implies any noticeable pattern in the scaling relationship parameters. Our results indicate that, for a fixed value of the factor in the scaling relationship, the exponent of the area in the volume-area relationship decreases as slope and shape increase, whereas size-based classifications do not reveal any clear trend. This means that steep slopes and cirque-type glaciers are less sensitive to changes in glacier area. Moreover, the volumes of the total population of Svalbard glaciers calculated according to partitioning in subgroups by size and slope are smaller (by 1-4%) than that obtained considering all glaciers without partitioning into subgroups, whereas the volumes calculated according to partitioning in subgroups by shape are 3-5% larger. We also did multivariate experiments attempting to optimally predict the volume of Svalbard glaciers from a combination of different predictors. Our results show that a simple power-type V-A model explains 98.6% of the variance. Only the predictor glacier length provides statistical significance when used in addition to the predictor glacier area, though the coefficient of determination decreases as compared with the simpler V-A model. The predictor elevation range did not provide any additional information when used in addition to glacier area. Our estimates of the volume of the entire population of Svalbard glaciers using the different scaling relationships that we have derived along this thesis range within 6890-8106 km3, with estimated relative errors in total volume of the order of 6.6-8.1% The average value of all of our estimates, which could be used as a best estimate for the volume, is 7,504 km3. In terms of sea-level equivalent (SLE), our volume estimates correspond to a potential contribution to sea-level rise within 17-20 mm SLE, averaging 19 _ 2 mm SLE, where the quoted error corresponds to our estimated relative error in volume. For comparison, the estimates using the V-A scaling relations found in the literature range within 13-26 mm SLE, averaging 20 _ 2 mm SLE, where the quoted error represents the standard deviation of the different estimates.

Non-destructive measurement techniques for taper equation development: a study case in the Spanish Northern Iberian Range

In recent years, the technology for measuring the diameter and height of standing trees has improved significantly. These enhancements allow estimation of the volume of standing trees using stem taper equations, which traditionally have been constructed with data from felled trees, in an accurate and economically feasible way. A nondestructive method was evaluated with data from 38 pines and was validated with data from another 38 pines, both in the Northern Iberian Range (Spain). The electronic dendrometer Criterion RD1000 (Laser Technology Inc.) and the laser hypsometer TruPulse (Laser Technology Inc.) were used due to their accuracy and interoperability. The methodology was valid (unbiased and precise) measuring from a distance similar to the height of the tree. In this distance, statistical criteria and plots based on the residuals showed no clear advantage in volume estimation with models fitted with data from destructive methods against models fitted with data from the proposed non-destructive technique. This methodology can be considered useful for individual volume estimation and for developing taper equations.

Residual stress distribution in ferritic to austenitic steel joints made by laser welding,

In this study, autogenous laser welding was used to join thin plates of low carbon ferritic and austenitic stainless steel. Due to the differences in the thermo-physical properties of base metals, this kind of weld exhibits a complex microstructure, which frequently leads to an overall loss of joint quality. Four welded samples were prepared by using different sets of processing parameters, with the aim of minimizing the induced residual stress field. The dissimilar austenitic-ferritic joints obtained under all welding conditions were uniform and free of defects. Variations in beam position did not influence the weld geometiy, which is a typical keyhole welding. Microstructural characterization and residual strain scanning (by neutron diffraction) were used to assess the features of the joints. By varying laser beam power density and by displacing the laser beam towards the carbon steel side, an optimum combination of processing parameters was found.

Nitrogen fixation by native Bradyrhizobia in symbiosis with Lupinus mariae-josephae requires a T3SS encoding a NopE-like effector

Several bradyrhizobial isolates from L. mariae-josephae root nodules [1] contain a type III secretion system (T3SS) within a cluster of about 30 genes. Among those genes, ttsI codes for the transcriptional activator of the system. Mutation of ttsI resulted in the formation of white, non-fixing nodules with the natural legume host, L. mariae-josephae. The T3SS cluster also contains a gene coding for a NopE-like protein. NopE proteins have been demonstrated to be effectors in the Bradyrhizobium-soybean symbiosis [2] and belong to a small group of poorly characterized proteins from plant-associated bacteria that contain one or two autocleavage motifs known as DUF1521 (Schirrmeister et al. 2011). The amino acid sequence of a NopE-like protein in the L. mariae-josephae strain LmjC contains just one autocatalytic motif. This is unlike NopE1 and NopE2 proteins secreted by the T3SS of B. japonicum, that contain two motifs [3]. The autocleavage of LmjC NopE protein was analyzed after expression in E. coli and purification. Two protein fragments of the predicted sizes appeared in the presence of Ca2+, Cu2+, Cd2+, Zn2+ and Mn2+ cations. In contrast, autocleavage did not take place in the presence of Ni2+, Co2+ or Mg2+. Site-directed mutagenesis of the DUF1521 motif in LmjC NopE abolished self-cleavage in vitro. Symbiotic competence of a NopE- mutant with the L. mariae-josephae host was not affected. Possible roles of NopE are discussed.

Reproducing the Bolund experiment in wind tunnel

The Bolund experiment has been reproduced in a neutral boundary layer wind tunnel (WT) at scale 1:115 for two Reynolds numbers. All the results have been obtained for an incoming flow from the 270o wind direction (transect B in the Bolund experiment jargon). Vertical scans of the velocity field are obtained using non-time resolved two components particle image velocimetry. Time-resolved velocity time series with a three component hot-wire probe have been also measured for transects at 2 and 5 m height and in the vertical transects at met masts M6, M3 and M8 locations. Special attention has been devoted to the detailed characterization of the inflow in order to reduce uncertainties in future comparisons with other physical and numerical simulations. Emphasis is placed on the analysis of spectral functions of the undisturbed flow and those of the flow above the island. The result?s reproducibility and trustworthiness have been addressed through redundancy measurements using particle image velocimetry, two and three components hot-wire anemometry. The bias in the prediction of the mean speed is similar to the one reported during the Bolund experiment by the physical modellers. However, certain reduction of the bias in the estimation of the turbulent kinetic energy is achieved. TheWT results of spectra and cosprectra have revealed a behaviour similar to the full-scale measurements in some relevant locations, showing that WT modelling can contribute to provide valid information about these important structural loading factors.

Uncertainty in ellipse fitting using a flatbed scanner: development and experimental verification

In the field of dimensional metrology, the use of optical measuring machines requires the handling of a large number of measurement points, or scanning points, taken from the image of the measurand. The presence of correlation between these measurement points has a significant influence on the uncertainty of the result. The aim of this work is the development of an estimation procedure for the uncertainty of measurement in a geometrically elliptical shape, taking into account the correlation between the scanning points. These points are obtained from an image produced using a commercial flat bed scanner. The characteristic parameters of the ellipse (coordinates of the center, semi-axes and the angle of the semi-major axis with regard to the horizontal) are determined using a least squares fit and orthogonal distance regression. The uncertainty is estimated using the information from the auto-correlation function of the residuals and is propagated through the fitting algorithm according to the rules described in Evaluation of Measurement Data—Supplement 2 to the ‘Guide to the Expression of Uncertainty in Measurement’—Extension to any number of output quantities. By introducing the concept of cut-off length, it can be observed how it is possible to take into account the presence of the correlation in the estimation of uncertainty in a very simple way while avoiding underestimation.

Estudio y análisis de la retracción de hormigones normales mediante una base de datos de ensayos unificada

El presente trabajo de investigación se plantea el estudio del fenómeno de la retracción. El fenómeno de la retracción, al igual que otras propiedades del hormigón, ha sido ampliamente divulgado, pero de un tiempo a esta parte, debido a la aparición de nuevos tipos de hormigones (autocompactantes, de altas resistencias, reciclados, etc.) se ha dejado en un segundo plano. Este segundo plano no significa que haya pasado al olvido, pues en los “nuevos hormigones” se toman los modelos de estimación de los hormigones normales como base para estimar sus propiedades, siendo, por este motivo, muy necesario reflexionar sobre si los modelos que estamos empleando son apropiados o no lo son. Este trabajo pretende, mediante la confección de una base de datos que unifica y amplía la del profesor Bazant, realizar un análisis estadístico de los datos que actualmente tenemos a nuestra disposición, y que conforman un total de 802 ensayos que abarcan desde el fenómeno el entumecimiento hasta la retracción total, pasando por la retracción autógena, con un intervalo de tiempo que abarca desde 1958 hasta 2013. Además, este trabajo realiza una revisión de los siete modelos de cálculo más empleados a la hora de estimar la deformación por retracción, analizándolos minuciosamente y superponiéndolos con los datos reales de la base anteriormente nombrada. Como conclusión, se demuestra que los modelos estudiados estiman razonablemente bien el fenómeno de la retracción, si bien no se llegan a discriminar todas las variables con la precisión esperada, siendo alguno de ellos conservador. En este sentido, se abre la puerta a insistir en un estudio más afinado de la retracción del hormigón, por dos motivos: los actuales modelos sirven de base para la estimación de los anteriormente mencionados nuevos hormigones y porque la introducción y desarrollo de aditivos que sean capaces de disminuir este fenómeno deben asentarse ambos sobre una base más sólida y precisa. Por último, deja la puerta abierta para que otros trabajos de investigación puedan realizarse, bien con la retracción, bien con otras propiedades, como el módulo de deformación, la adquisición de resistencias, etc., pues se adjunta como anexo toda la base completa con más de 20.000 datos que pueden ser de gran utilidad. The main objective of this research study is to evaluate the phenomenon of shrinkage in concrete. The phenomenon of shrinkage, as well as other properties of concrete, has been widely studied, but for a time, and due to the emergence of new types of concrete (self-compacting, high strength, recycled, etc.) it has been left in the background. Having moved to second plane did not mean that it have been abandoned, since in the "new concrete" these properties are calculated using the estimation models developed for conventional concrete, and it is for this reason, why it is important and necessary to reflect whether these models we are using are appropriate or are not. This work seeks to unify and bring up-to-date a database of experimental shrinkage results. The database is based on existing databases, especially on the last version of the database created by Professor Bazant. The database includes a total of 802 experimental results, and include results for total shrinkage, autogenous shrinkage and swelling, and over experimental studies for 1958 to 2013. The database serves as a basis for a complex and detailed statistical analysis of the data. In addition, seven commonly used shrinkage estimation models are presented and evaluated. These models are used for a thoroughly comparison analysis between the estimated results obtained using these models and the actual experimental data included in the unified database. In conclusion, it is shown that the models estimate reasonably well the shrinkage strains, although they fail to discriminate adequately some of the influencing variables, resulting in some of them being too conservative in their estimation. In this sense, the results of this work emphasis the importance and the need of addition research on the shrinkage of concrete. This is especially important considering that the existing models provide a basis for estimating the shrinkage in "new concretes", and that the increase use of shrinkage reducing additives should be based and supported by solid research in this field. Finally, this study includes an extensive and updated database that can serve for further research on shrinkage. The full database, developed as part of this work, which includes more than 20,000 data, is included in the annex of the study.

Glycerin and lecithin inclusion in diets for brown egg-layinghens: Effects on egg production and nutrient digestibility

The effects of the inclusion of raw glycerin (GLYC) and lecithin in the diet on egg production,egg quality and total tract apparent retention (TTAR) of dietary components was studied inbrown egg-laying hens from 23 to 51 wk of age. The experimental design was completelyrandomized with six diets combined as a 2 × 3 factorial with two levels of GLYC (0 vs.70 g/kg) and three animal fat to lecithin ratios (40:0, 20:20 and 0:40 g/kg). Each treatmentwas replicated eight times and the experimental unit was a cage with ten hens. Productionwas recorded by replicate every 28-d period and cumulatively. For the entire experiment,the inclusion of GLYC in the diet hindered feed conversion ratio per kilogram of eggs (2.071vs. 2.039; P < 0.05) but did not affect any of the other production or egg quality traits studied.The replacement of animal fat by lecithin (40:0, 20:20 and 0:40 g/kg) increased egg weight(60.1, 60.7 and 61.8 g, respectively; P < 0.001) and egg mass production (56.8, 57.5 and58.8 g/d, respectively; P < 0.01) and improved yolk color as measured by the DSM colorfan (9.2, 9.2 and 9.5, respectively; P < 0.001) and feed conversion ratio per kilogram of eggs(2.072, 2.068 and 2.027, respectively; P < 0.05). Feed intake, egg production and body weightgain, however, were not affected. The inclusion of GLYC in the diet did not affect nutrientretention but lecithin inclusion improved TTAR of dry matter (P < 0.05), organic matter(P < 0.05), ether extract (P < 0.001) and gross energy (P < 0.001). In summary, the inclusionof 70 g glycerol/kg diet hindered feed conversion ratio per kilogram of eggs but did notaffect any other production or digestibility trait. The replacement of animal fat by lecithinimproved egg weight, egg yolk color and nutrient digestibility. Consequently, lecithin canbe used as a lipid source in laying hen diets with beneficial effects on egg production

Comparison of fermentation characteristics and bacterial diversity in the rumen of sheep and batch cultures of rumen microorganisms

The objective of the current study was to assess how closely batch cultures (BC) of rumen microorganisms can mimic the dietary differences in fermentation characteristics found in the rumen, and to analyse changes in bacterial diversity over the in vitro incubation period. Four ruminally and duodenally cannulated sheep were fed four diets having forage : concentrate ratios (FCR) of 70 : 30 or 30 : 70, with either alfalfa hay or grass hay as forage. Rumen fluid from each sheep was used to inoculate BC containing the same diet fed to the donor sheep, and the main rumen fermentation parameters were determined after 24 h of incubation. There were differences between BC and sheep in the magnitude of most measured parameters, but BC detected differences among diets due to forage type similar to those found in sheep. In contrast, BC did not reproduce the dietary differences due to FCR found in sheep for pH, degradability of neutral detergent fibre and total volatile fatty acid (VFA) concentrations. There were differences between systems in the magnitude of most determined parameters and BC showed higher pH values and NH3–N concentrations, but lower fibre degradability and VFA and lactate concentrations compared with sheep. There were significant relationships between in vivo and in vitro values for molar proportions of acetate, propionate and butyrate, and the acetate : propionate ratio. The automated ribosomal intergenic spacer analysis (ARISA) of 16S ribosomal deoxyribonucleic acid showed that FCR had no effect on bacterial diversity either in the sheep rumen fluid used as inoculum (IN) or in BC samples. In contrast, bacterial diversity was greater with alfalfa hay diets than those with grass hay in the IN, but was unaffected by forage type in the BC. Similarity index between the bacterial communities in the inocula and those in the BC ranged from 67·2 to 74·7%, and was unaffected by diet characteristics. Bacterial diversity was lower in BC than in the inocula with 14 peaks out of a total of 181 detected in the ARISA electropherograms never appearing in BC samples, which suggests that incubation conditions in the BC may have caused a selection of some bacterial strains. However, each BC sample showed the highest similarity index with its corresponding rumen IN, which highlights the importance of using rumen fluid from donors fed a diet similar to that being incubated in BC when conducting in vitro experiments.

Influence of glycerin and lecithin inclusion in the diet on liver characteristics and lipid fraction in the serum of brownegg laying hens at 55 week of age

The effects of the inclusion of raw glycerin (GLYC) and raw lecithin, in the diet (23 to 55 wk) on liver characteristics and various serum lipid fractions were studied in brown egg-laying hens at 55 wk of age. The control diets were based on corn, soybean meal, and 4% supplemental fat and contained 2,750 kcal AMEn/kg, 16.5% CP, and 0.73% digestible Lys. The diets were arranged as a 2 × 3 factorial with 2 levels of GLYC (0 and 7%) and 3 animal fat to lecithin ratios (4:0, 2:2, and 0:4%). Each treatment was replicated 8 times and the experimental unit was a cage with 10 hens. At 55 wk of age, 2 hens per cage replicate were randomly selected, weighed individually, and slaughtered by CO2 inhalation. Liver was immediately removed and weighed and the color recorded by spectrophotometry. In addition, blood samples from one bird per replicate were collected from the wing vein and the concentration of total cholesterol, low and high density lipoprotein cholesterol, and triglycerides were determined. The data were analyzed as a completely randomized design and the main effects of GLYC and lecithin content of the diet and the interactions were determined. No interactions between GLYC and lecithin content of the diets were detected for any of the variables studied. Liver characteristics and serum lipid traits were not affected by the inclusion of GLYC in the diet. The substitution of animal fat by lecithin, however, reduced the redness (a* 14.9 to 13.8) and yellowness (b* 8.60 to 7.20) values of the liver (P < 0.05) but did not affect the content of serum lipid fractions. It is concluded that the inclusion of GLYC and lecithin in the diet did not affect liver size or serum lipid fraction. However, the inclusion of lecithin reduced the a* and b* value of the liver

In vivo evidence that erythropoietin protects neurons from ischemic damage

Erythropoietin (EPO) produced by the kidney and the liver (in fetuses) stimulates erythropoiesis. In the central nervous system, neurons express EPO receptor (EPOR) and astrocytes produce EPO. EPO has been shown to protect primary cultured neurons from N-methyl-d-aspartate (NMDA) receptor-mediated glutamate toxicity. Here we report in vivo evidence that EPO protects neurons against ischemia-induced cell death. Infusion of EPO into the lateral ventricles of gerbils prevented ischemia-induced learning disability and rescued hippocampal CA1 neurons from lethal ischemic damage. The neuroprotective action of exogenous EPO was also confirmed by counting synapses in the hippocampal CA1 region. Infusion of soluble EPOR (an extracellular domain capable of binding with the ligand) into animals given a mild ischemic treatment that did not produce neuronal damage, caused neuronal degeneration and impaired learning ability, whereas infusion of the heat-denatured soluble EPOR was not detrimental, demonstrating that the endogenous brain EPO is crucial for neuronal survival. The presence of EPO in neuron cultures did not repress a NMDA receptor-mediated increase in intracellular Ca2+, but rescued the neurons from NO-induced death. Taken together EPO may exert its neuroprotective effect by reducing the NO-mediated formation of free radicals or antagonizing their toxicity.

Absence of cytokine receptor-dependent specificity in red blood cell differentiation in vivo

Erythropoietin (EPO) is required for red blood cell development, but whether EPO-specific signals directly instruct erythroid differentiation is unknown. We used a dominant system in which constitutively active variants of the EPO receptor were introduced into erythroid progenitors in mice. Chimeric receptors were constructed by replacing the cytoplasmic tail of constitutively active variants of the EPO receptor with tails of diverse cytokine receptors. Receptors linked to granulocyte or platelet production supported complete erythroid development in vitro and in vivo, as did the growth hormone receptor, a nonhematopoietic receptor. Therefore, EPOR-specific signals are not required for terminal differentiation of erythrocytes. Furthermore, we found that cellular context can influence cytokine receptor signaling.

Identification of an intramolecular interaction between small regions in type V adenylyl cyclase that influences stimulation of enzyme activity by Gsα

Using the full-length and two engineered soluble forms (C1-C2 and Cla-C2) of type V adenylyl cyclase (ACV), we have investigated the role of an intramolecular interaction in ACV that modulates the ability of the α subunit of the stimulatory GTP-binding protein of AC (Gsα) to stimulate enzyme activity. Concentration–response curves with Gsα suggested the presence of high and low affinity sites on ACV, which interact with the G protein. Activation of enzyme by Gsα interaction at these two sites was most apparent in the C1a-C2 form of ACV, which lacks the C1b region (K572–F683). Yeast two-hybrid data demonstrated that the C1b region interacted with the C2 region and its 64-aa subdomain, C2I. Using peptides corresponding to the C2I region of ACV, we investigated the role of the C1b/C2I interaction on Gsα-mediated stimulation of C1-C2 and full-length ACV. Our data demonstrate that a 10-aa peptide corresponding to L1042–T1051 alters the profile of the activation curves of full-length and C1-C2 forms of ACV by different Gsα concentrations to mimic the activation profile observed with C1a-C2 ACV. The various peptides used in our studies did not alter forskolin-mediated stimulation of full-length and C1-C2 forms of ACV. We conclude that the C1b region of ACV interacts with the 10-aa region (L1042–T1051) in the C2 domain of the enzyme to modulate Gsα-elicited stimulation of activity.

Multiple loop structures critical for ligand binding of the integrin α4 subunit in the upper face of the β-propeller mode 1

A non-I-domain integrin, α4β1, recognizes vascular cell adhesion molecule 1 (VCAM-1) and the IIICS portion of fibronectin. To localize regions of α4 critical for ligand binding, we swapped several predicted loops within or near the putative ligand-binding site of α4 (which spans repeats 2–5 of the seven N-terminal repeats) with the corresponding regions of α5. Swapping residues 112–131 in repeat 2, or residues 237–247 in repeat 4, completely blocked adhesion to immobilized VCAM-1 and connecting segment 1 (CS-1) peptide. However, swapping residues 40–52 in repeat 1, residues 151–164 in repeat 3, or residues 282–288 (which contain a putative cation binding motif) in repeat 5 did not affect or only slightly reduced adhesion to these ligands. The binding of several function-blocking antibodies is blocked by swapping residues 112–131, 151–164, and 186–191 (which contain previously identified residues critical for ligand binding, Tyr-187 and Gly-190). These results are consistent with the recently published β-propeller folding model of the integrin α4 subunit [Springer, T. A. (1997) Proc. Natl. Acad. Sci. USA 94, 65–72], in which seven four-stranded β-sheets are arranged in a torus around a pseudosymmetric axis. The regions of α4 critical for ligand binding are adjacent to each other and are located in the upper face, the predicted ligand-binding site, of the β-propeller model, although they are not adjacent in the primary structure.

Uridine Diphosphate–Glucose Transport into the Endoplasmic Reticulum of Saccharomyces cerevisiae: In Vivo and In Vitro Evidence

It has been proposed that synthesis of β-1,6-glucan, one of Saccharomyces cerevisiae cell wall components, is initiated by a uridine diphosphate (UDP)-glucose–dependent reaction in the lumen of the endoplasmic reticulum (ER). Because this sugar nucleotide is not synthesized in the lumen of the ER, we have examined whether or not UDP–glucose can be transported across the ER membrane. We have detected transport of this sugar nucleotide into the ER in vivo and into ER–containing microsomes in vitro. Experiments with ER-containing microsomes showed that transport of UDP–glucose was temperature dependent and saturable with an apparent Km of 46 μM and a Vmax of 200 pmol/mg protein/3 min. Transport was substrate specific because UDP–N-acetylglucosamine did not enter these vesicles. Demonstration of UDP–glucose transport into the ER lumen in vivo was accomplished by functional expression of Schizosaccharomyces pombe UDP–glucose:glycoprotein glucosyltransferase (GT) in S. cerevisiae, which is devoid of this activity. Monoglucosylated protein-linked oligosaccharides were detected in alg6 or alg5 mutant cells, which transfer Man9GlcNAc2 to protein; glucosylation was dependent on the inhibition of glucosidase II or the disruption of the gene encoding this enzyme. Although S. cerevisiae lacks GT, it contains Kre5p, a protein with significant homology and the same size and subcellular location as GT. Deletion mutants, kre5Δ, lack cell wall β-1,6 glucan and grow very slowly. Expression of S. pombe GT in kre5Δ mutants did not complement the slow-growth phenotype, indicating that both proteins have different functions in spite of their similarities.