Acid-base physiology response to ocean acidification of two ecologically and economically important holothuroids from contrasting habitats, Holothuria scabra and Holothuria parva

Sea cucumbers are dominant invertebrates in several ecosystems such as coral reefs, seagrass meadows and mangroves. As bioturbators, they have an important ecological role in making available calcium carbonate and nutrients to the rest of the community. However, due to their commercial value, they face overexploitation in the natural environment. On top of that, occurring ocean acidification could impact these organisms, considered sensitive as echinoderms are osmoconformers, high-magnesium calcite producers and have a low metabolism. As a first investigation of the impact of ocean acidification on sea cucumbers, we tested the impact of short-term (6 to 12 days) exposure to ocean acidification (seawater pH 7.7 and 7.4) on two sea cucumbers collected in SW Madagascar, Holothuria scabra, a high commercial value species living in the seagrass meadows, and H. parva, inhabiting the mangroves. The former lives in a habitat with moderate fluctuations of seawater chemistry (driven by day-night differences) while the second lives in a highly variable intertidal environment. In both species, pH of the coelomic fluid was significantly negatively affected by reduced seawater pH, with a pronounced extracellular acidosis in individuals maintained at pH 7.7 and 7.4. This acidosis was due to an increased dissolved inorganic carbon content and pCO2 of the coelomic fluid, indicating a limited diffusion of the CO2 towards the external medium. However, respiration and ammonium excretion rates were not affected. No evidence of accumulation of bicarbonate was observed to buffer the coelomic fluid pH. If this acidosis stays uncompensated for when facing long-term exposure, other processes could be affected in both species, eventually leading to impacts on their ecological role.

Investigación técnica y económica sobre desinfección de aguas residuales por sistemas de oxidación

Un incremento de la demanda del agua, junto con el aumento de la contaminación, ha provocado que hoy en día la reutilización de las aguas depuradas sea necesaria, pero la reutilización de aguas debe garantizar y minimizar los posibles riesgos sanitarios y medioambientales que su práctica pueda provocar. En España estos parámetros se encuentran regulados por el RD 1620/2007 relativo al régimen jurídico de la reutilización de las aguas depuradas. Las aguas regeneradas son aguas que ya han sido sometidas a un tratamiento de depuración, y a las cuales se aplica un posterior tratamiento adicional o complementario que permita adecuar su calidad al uso al que vaya a destinarse. Siendo requeridos para los distintos reúsos procesos de desinfección, uno de los principales sistemas utilizados es el cloro, debido a su sencilla aplicación y costos bajos, sin tomar en cuenta la posible formación de compuestos organohalogenados potencialmente cancerígenos. Es por esto que surge la necesidad de aplicar distintos sistemas de oxidación objeto de estudio en esta tesis, como el dióxido de cloro estabilizado, ozono y los procesos avanzados de oxidación (Advanced Oxidation Processes, AOP), ozono/peróxido y uv/peróxido. En esta tesis se investiga los rendimientos que pueden alcanzar estos sistemas en la eliminación de los ácidos húmicos y los fenoles, siendo las principales sustancias formadoras de subproductos de la desinfección, así mismo, se considera necesario garantizar la desinfección del agua a través del estudio de tres grupos de microrganismos, los coliformes totales, e. coli y enterococos, siendo un punto importante el posible recrecimiento microbiológico debido a una desinfección escasa, por la permanencia en el agua de los compuestos antes mencionados, o por alguna fuente de alimento que pudieran encontrar en el sistema de distribución. Lo más importante será la calidad que se pueda alcanzar con estos desinfectantes, con el fin de obtener agua para los distintos reúsos que existen en la actualidad. Y así no limitar los alcances que puede tener la reutilización de las aguas residuales. Basándose en lo antes mencionado se procedió a realizar la caracterización del agua del rio Manzanares, con el fin de determinar la cantidad de ácidos húmicos disueltos y fenoles, obteniendo valores bajos, se decidió incorporar a las muestras de rio 5 mg/L de estos compuestos, con el fin de observar de que manera podrían interferir en la desinfección de esta agua. De esta forma se logran obtener resultados óptimos de los sistemas de desinfección estudiados, siendo el Ozono un oxidante eficiente en la desinfección de los microrganismos y en la eliminación de ácidos húmicos y fenoles con tiempos de contacto cortos, mostrando deficiencias al permitir el recrecimiento de los coliformes totales. Del sistema de oxidación avanzada UV/Peróxido se determino como un eficiente desinfectante para garantizar la inexistencia de rebrotes, al paso del tiempo. Así mismo se concluye que tiene buenos rendimientos en la eliminación del ácido húmico y los fenoles. An increase in water demand, coupled with increasing pollution, has caused today reuse of treated water is necessary, but must ensure water reuse and minimize potential health and environmental risks that their practice is cause. In Spain these parameters are regulated by Royal Decree 1620/2007 on the legal regime of the reuse of treated water. The reclaimed water is water that has already been subjected to a depuration treatment, which is applied as a subsequent further treatment that will bring quality to the use to which is to be delivered. As required for various reuses disinfection processes, one of the main systems used is chlorine, due to its simple implementation and low costs, without taking into account the possible formation of potentially carcinogenic halogenated organic compounds. That is why there is a need to apply different oxidation systems studied in this thesis, as stabilized chlorine dioxide, ozone and advanced oxidation processes (AOP), ozone/peroxide and UV/peroxide. This thesis investigates the rates can reach these systems in removing humic acids and phenols, the main substances forming disinfection byproducts, likewise, it is considered necessary to ensure water disinfection through the study of three groups of microorganisms, total coliform, e. coli and enterococci, the important point being a possible regrowth due to microbiological disinfection scarce, the water remaining on the aforementioned compounds, or a food source which may be found in the distribution system. The most important quality is that achievable with these disinfectants, with the water to obtain various reuses that exist today. And thus not limit the scope that can be reuse of wastewater. Based on the above we proceeded to perform characterization Manzanares river water, in order to determine the quantity of dissolved humic acids and phenols, obtaining low values, it was decided to incorporate river samples 5 mg / L of these compounds, in order to observe how they might interfere with the disinfection of the water. Thus optimum results are achieved for disinfection systems studied, being efficient ozone oxidant in the disinfection of microorganisms and the removal of humic acids and phenols with short contact times, showing gaps to allow regrowth total coliforms. Advanced oxidation system UV / peroxide were determined as an efficient disinfectant to ensure the absence of volunteers, the passage of time. Also it is concluded that has good yields in removing humic acid and phenols.

Estudio de la digestión anaerobia en dos fases para el tratamiento de las aguas residuales de despulpe del beneficiado húmedo del café

Debido al aumento de los estándares de calidad exigidos internacionalmente, así como por una mayor presión sobre la industria mediante legislaciones ambientales más rigurosas, el sector cafetalero está obligado a buscar, a través de la investigación, un sistema adecuado de tratamiento para las aguas residuales generadas en el beneficiado húmedo del café. En este trabajo se evaluó el funcionamiento de la digestión anaerobia para el tratamiento de las aguas residuales de despulpe. Para ello, se utilizaron dos sistemas anaerobios, uno en una etapa (UASB), y otro con separación de fases (2PUASB). Se investigó el efecto en la digestión anaerobia de tres cargas orgánicas volumétricas (OLR) y de las dos configuraciones de reactor usadas. Los valores de OLR de operación en el sistema UASB variaron en un intervalo de 3.6-4.1 kgCOD m-3 d-1, con una tasa de recirculación del efluente de 1.0. El sistema 2PUASB fue alimentado con OLR similares a las que se emplearon en el sistema en una etapa. El reactor de acidificación fue cargado a 11.0 kgCOD m-3 d-1, mientras que en el reactor metanogénico varió en el intervalo de 2.6-4.67 kgCOD m-3 d-1. El uso de reactores UASB en una etapa y en dos fases, bajo las mismas condiciones de operación ya descritas, propiciaron el logro de una eficiencia de degradación de COD total superior al 75% y al 85% para la COD soluble, respectivamente. Sin embargo, el sistema en dos fases mostró mejores resultados en el tratamiento de este tipo de agua residual, no solo en cuanto a eficiencia de eliminación de la carga orgánica contaminante así como una menor concentración de ácidos grasos volátiles (VFA) en el efluente. Obtenidas las mejores condiciones de trabajo, fue evaluada la separación de fases bajo el efecto de la recirculación. Los grupos de fermentaciones producidos fueron similares a los obtenidos en el experimento sin recirculación, indicando que está última no afectó la composición relativa de los VFA del reactor anaerobio, por lo que no cambió el patrón de degradación del residuo. Una tasa de recirculación de 1.0 del efluente del reactor metanogénico al reactor acidogénico mejoró significativamente el proceso, ya que se incrementó la conversión de los VFA (31%), la eliminación de la fracción total y soluble del residuo tratado (6.5%) y la reducción del consumo de alcalinizante (39%); manteniendo similares producciones de metano. El uso de la digestión anaerobia en dos fases demostró una mejora en la estabilidad del proceso y un incremento de la eficiencia de operación y de la producción de metano, respectivamente.Tesis Doctoral Yans Guardia Puebla Abstract ix ABSTRACT Due to the increase of quality standards internationally demanded, as well as for a greater pressure on the industry by means of more rigorous environmental legislations, the coffee sector is forced to search, through the research, an appropriated treatment system for coffee wet wastewaters generated. In this work the performance of the anaerobic digestion for the coffee wet wastewater treatment was evaluated. For it, two anaerobic systems, one in single-stage (UASB), and another with two-phase (2PUASB) were used. The effect in the anaerobic digestion of three organic loading rates (OLR) and of two reactor configurations used was investigated. OLR operation values in UASB system varied in an interval of 3.6-4.1 kgCOD m-3 d-1, with a recycle rate of the effluent of 1.0. 2PUASB system was fed with OLR similar to those that were used in the reactor in a stage. The acidification reactor was loaded to 11.0 kgCOD m-3 d-1, whereas in the methanogenic reactor varied in the interval of 2.6-4.67 kgCOD m-3 d-1. The use of single-stage and two-phase UASB reactors, under the same operation conditions already before described, a total COD removal efficiency of 75% and 85% for the soluble COD removal efficiency, respectively, was achieved. However, two-phase system showed better results in the treatment of this wastewater type, not only as for removal efficiency of loading organic polluting as well as a smaller volatile fatty acid (VFA) concentration in the effluent. Obtained the best work conditions, the two-phase system under the effect of the recycle was evaluated. Fermentations groups produced were similar to those obtained in the experiment without recycle, indicating that it last one do not affect the relative composition of VFA of the anaerobic reactor, for that reason the degradation pattern of the residue does not change. A recycle rate of 1.0 of the effluent of the methanogenic reactor to the acidogenic reactor improved the process significantly, since it was increased the VFA conversion (31%), the removal of total and soluble fraction of the residue treated (6.5%) and the decrease of the alkalinity consumption (39%); maintaining similar methane productions. The use of the two-phase anaerobic digestion demonstrated to an improvement in the stability of the process and an increase of the operation efficiency and methane production, respectively.

Amino acid availability in ruminants of cereals and cereal co-products

Microbial corrected in situ estimatesof the ruminal undegraded fraction (RU) and intestinal effectivedigestibility (IED) of amino acids (AA), except tryptophan, of rye, wheat and corn grains, wheat bran, wheat and barley distilled dried grains and corn gluten feed were measured on three rumen- and duodenum-cannulated wethers using 15N-labelling techniques and considering ruminal rates of particle comminution and outflow.

Tratamiento de vinazas provenientes de etanol en un reactor de lecho fluidizado inverso

En el estado de Veracruz, al sur de México, se ubican empresas dedicadas a la obtención de etanol a partir de melaza de azúcar de caña. Las más pequeñas, tienen una producción promedio de 20,000 L de alcohol/día. Los efluentes de la producción de etanol incluyen agua de enfriamiento de condensadores, agua del lavado de tanques de fermentación y vinazas, estas últimas son los efluentes más contaminantes en las destilerías, por su concentración de material orgánico biodegradable y no biodegradable. Las vinazas se generan en grandes volúmenes, produciéndose de 12 a 15 litros de vinazas por cada litro de alcohol destilado. Estos efluentes se caracterizan por tener altas temperaturas, pH ácido y una elevada concentración de DQO así como de sólidos totales. La determinación de la biodegradabilidad anaerobia de un agua residual, permite estimar la fracción de DQO que puede ser transformada potencialmente en metano y la DQO recalcitrante que queda en el efluente. Para el desarrollo de una prueba de biodegradabilidad, es importante considerar diversos factores relacionados con la composición del agua a tratar, composición de los lodos y las condiciones bajo las cuales se lleva a cabo la prueba. La digestión anaerobia de aguas residuales industriales es comúnmente usada en todo el mundo, ofrece significativas ventajas para el tratamiento de efluentes altamente cargados. Los sistemas anaerobios de tratamiento de aguas residuales industriales incluyen tecnologías con biopelículas, estos sistemas de tratamiento anaerobio con biopelícula son una tecnología bien establecida para el tratamiento de efluentes industriales. El Reactor de Lecho Fluidizado Inverso Anaerobio (LFI) ha sido diseñado para el tratamiento de aguas residuales de alta carga, teniendo como ventajas el empleo de un soporte que proporciona una gran superficie y un bajo requerimiento de energía para la fluidización del lecho. En el presente trabajo, se lleva a cabo el análisis de un proceso de producción de etanol, identificando a los efluentes que se generan en el mismo. Se encuentra que el efluente final está compuesto principalmente por las vinazas provenientes del proceso de destilación. En la caracterización de las vinazas provenientes del proceso de producción de etanol a partir de melaza de azúcar de caña, se encontraron valores promedio de DQO de 193.35 gDQO/L, para los sólidos totales 109.78 gST/L y pH de 4.64. Así mismo, en esta investigación se llevó a cabo una prueba de biodegradabilidad anaerobia, aplicada a la vinaza proveniente de la producción de etanol. En la caracterización de los lodos empleados en el ensayo se obtiene una Actividad Metanogénica Especifica de 0.14 g DQO/gSSV.d. El porcentaje de remoción de DQO de la vinaza fue de 62.7%, obteniéndose una k igual a 0.031 h-1 y una taza de consumo de sustrato de 1.26 gDQO/d. El rendimiento de metano fue de 0.19 LCH4/g DQOremovida y el porcentaje de biodegradabilidad de 54.1%. El presente trabajo también evalúa el desempeño de un LFI, empleando Extendospher® como soporte y tratando efluentes provenientes de la producción de etanol. El reactor se arrancó en batch y posteriormente se operó en continuo a diferentes Cargas Orgánicas Volumétricas de 0.5, 1.0, 3.3, 6.8 y 10.4 g DQO/L.d. Además, se evaluaron diferentes Tiempos de Residencia Hidráulica de 10, 5 y 1 días. El sistema alcanzó las siguientes eficiencias promedio de remoción de DQO: 81% para la operación en batch; 58, 67, 59 y 50 % para las cargas de 0.5, 1.0, 3.3, 6.8 g DQO/L.d respectivamente. Para la carga de 10.4 g DQO/L.d, la eficiencia promedio de remoción de DQO fue 38%, en esta condición el reactor presentó inestabilidad y disminución del rendimiento de metano. La generación de metano inició hasta los 110 días de operación del reactor a una carga de 1.0 g DQO/L.d. El sistema alcanzó un rendimiento de metano desde 0.15 hasta 0.34 LCH4/g DQO. Durante la operación del reactor a una carga constante de 6.4 g DQO/L.d, y un TRH de 1 día, se alcanzó una eficiencia promedio de remoción de DQO de 52%. In the state of Veracruz, to the south of Mexico, there are located companies dedicated to the production of ethanol from molasses of cane sugar. The smallest, have a average production of 20,000 L ethanol/day. The effluent of production of ethanol include water of condensers, water originated from the cleanliness of tanks of fermentation and vinasses, the above mentioned are more effluent pollutants in the distilleries, for the poor organic matter degradability. The vinasses are generated in high volumes, producing from 12 to 15 L of vinasses per every liter of distilled ethanol. These effluent are characterized by its high temperature, pH acid and a high concentration of DQO as well as high concentration of TS. The determination of the anaerobic degradability of a waste water, it allows to estimate the fraction of DQO that can be transformed potentially into methane and the recalcitrant DQO that stays in the effluent. For the development of degradability test, it is important to consider factors related to the composition of the water to be treated, composition of the sludge and the conditions under which the test is carried out. The anaerobic digestion of industrial wastes water is used commonly in the whole world, it offers significant advantages for the treatment of effluent highly loaded. The anaerobic treatment of industrial wastes water include technologies with biofilms, this anaerobic treatment whit biofilms systems, is a well-established technology for treatment of industrial effluents. The Anaerobic Inverse Fluidized Bed Reactor (IFBR) has been developed to provide biological treatment of high strength organic wastewater for their large specific surface and their low energy requirements for fluidization. In this work, there is carried out the analysis of a process of production of ethanol, identifying the effluent ones that are generated in the process. One determined that the effluent end is composed principally by the vinasses originated from the process of distillation. In the characterization of the vinasses originated from the process of production of ethanol from cane sugar molasses, there were average values of DQO of 193.35 gDQO/L, average values of solid of 109.78 gST/L and pH of 4.64. In this investigation there was carried out a anaerobic degradability test of the vinasses generated in the production of ethanol. In the characterization of the sludge used in the essay, the specific methanogenic activity (SMA) was 0.14 gDQO/gSSV.d. The average removal of DQO of the vinasses was 62.7 %, k equal to 0.031 h-1 was obtained one and a rate of removal substrate of 1.26 gDQO/d. The methane yield was 0.19 LCH4/gDQO removed and the anaerobic biodegradability was a 54.1 %. This study describes the performance of IFBR with Extendospher®, for the treatment of vinasses. The start-up was made in batch, increasing gradually the Organic Load Rate (OLR): 0.5, 1.0, 3.3, 6.8 and 10.4 g COD/L.d. Different Hydraulic Retention Times (HRT) were evaluated: 10, 5 and 1 days. During the operation in batch, the COD removal obtained was of 81 %, and for OLR of 0.5, 1.0, 3.3, 6.8 g COD/L.d the removal obtained was 58, 67, 59 and 50 % respectively. For a maximum OLR of 10.4 g COD/L.d, the COD removal was 38 %, and the system presented instability and decrease of the yield methane. The methane production initiated after 110 days of the start-up of the IFBR, to organic load rate of 1.0 g COD/L.d. The system reached values in the methane yield from 0.15 up to 0.34 LCH4/g CODremoved, for the different organic load rates. During the operation to a constant OLR of 6.4 g COD/L.d, and a HRT of 1 day, the Anaerobic Inverse Fluidized Bed Reactor reached a maximum efficiency of removal of 52 %.

Sunflower meal and spring pea ruminal degradation protection using malic acid or orthophosphoric acid-heat treatments

The effects of solutions of malic or orthophosphoric acids (0.752 Eqg/kg of feed) and heat to protect proteins of sunflower meal (SFM) and spring pea (SP) against ruminal degradation were studied using particle transit, 15N infusion, in situ and electrophoretic techniques. Three wethers fitted with rumen and duodenum cannulae were successively fed three isoproteic diets including SFM and SP, untreated or treated with malic or orthophosphoric acids. Incubations of tested meals were only performed while feeding the respective diet. Estimates of the ruminally undegraded fraction (RU) and its intestinal digestibility of dry matter, organic matter (only for RU), crude protein and starch (only in SP) were obtained considering ruminal microbial contamination and particle comminution and outflow rates. When corrected for microbial contamination, estimates of RU and intestinal digestibility decreased in all tested fractions for both feeds. All RU estimates increased with the protective treatments, whereas intestinal digestibility-dry matter also increased in SFM. Low intestinal digestibility-crude protein values suggested the presence of antitrypsin factors in SP. Protective treatments of both feeds led to consistent increases in the intestinal digested fraction of dry matter and crude protein, being only numerically different for SP-starch (60.5% as average). However, treatments also reduced the organic matter fermentation, which may decrease ruminal microbial protein synthesis. Electrophoretic studies showed albumin disappearance in both SFM and SP, whereas changes in other RU proteins were more pronounced in SP than SFM.

Genetic control of abscisic acid biosynthesis in maize

Abscisic acid (ABA), an apocarotenoid synthesized from cleavage of carotenoids, regulates seed maturation and stress responses in plants. The viviparous seed mutants of maize identify genes involved in synthesis and perception of ABA. Two alleles of a new mutant, viviparous14 (vp14), were identified by transposon mutagenesis. Mutant embryos had normal sensitivity to ABA, and detached leaves of mutant seedlings showed markedly higher rates of water loss than those of wild type. The ABA content of developing mutant embryos was 70% lower than that of wild type, indicating a defect in ABA biosynthesis. vp14 embryos were not deficient in epoxy-carotenoids, and extracts of vp14 embryos efficiently converted the carotenoid cleavage product, xanthoxin, to ABA, suggesting a lesion in the cleavage reaction. vp14 was cloned by transposon tagging. The VP14 protein sequence is similar to bacterial lignostilbene dioxygenases (LSD). LSD catalyzes a double-bond cleavage reaction that is closely analogous to the carotenoid cleavage reaction of ABA biosynthesis. Southern blots indicated a family of four to six related genes in maize. The Vp14 mRNA is expressed in embryos and roots and is strongly induced in leaves by water stress. A family of Vp14-related genes evidently controls the first committed step of ABA biosynthesis. These genes are likely to play a key role in the developmental and environmental control of ABA synthesis in plants.

Removal of LIF (leukemia inhibitory factor) results in increased vitamin A (retinol) metabolism to 4-oxoretinol in embryonic stem cells

Retinoids, vitamin A (retinol) and its metabolic derivatives, are required for normal vertebrate development. In murine embryonic stem (ES) cells, which remain undifferentiated when cultured in the presence of LIF (leukemia inhibitory factor), little metabolism of exogenously added retinol takes place. After LIF removal, ES cells metabolize exogenously added retinol to 4-hydroxyretinol and 4-oxoretinol and concomitantly differentiate. The conversion of retinol to 4-oxoretinol is a high-capacity reaction because most of the exogenous retinol is metabolized rapidly, even when cells are exposed to physiological (≈1 μM) concentrations of retinol in the medium. No retinoic acid or 4-oxoRA synthesis from retinol was detected in ES cells cultured with or without LIF. The cytochrome P450 enzyme CYP26 (retinoic acid hydroxylase) is responsible for the metabolism of retinol to 4-oxoretinol, and CYP26 mRNA is greatly induced (>15-fold) after LIF removal. Concomitant with the expression of CYP26, differentiating ES cells grown in the absence of LIF activate the expression of the differentiation marker gene FGF-5 whereas the expression of the stem cell marker gene FGF-4 decreases. The strong correlation between the production of polar metabolites of retinol and the differentiation of ES cells upon removal of LIF suggests that one important action of LIF in these cells is to prevent retinol metabolism to biologically active, polar metabolites such as 4-oxoretinol.

Misactivated amino acids translocate at similar rates across surface of a tRNA synthetase

Certain aminoacyl-tRNA synthetases have a second active site that destroys (by hydrolysis) errors of amino acid activation. For example, isoleucyl-tRNA synthetase misactivates valine (to produce valyl adenylate or Val-tRNAIle) at its active site. The misactivated amino acid is then translocated to an editing site located >25 Å away. The role of the misactivated amino acid in determining the rate of translocation is not known. Valyl-tRNA synthetase, a close homolog of isoleucyl-tRNA synthetase, misactivates threonine, α-aminobutyrate, and cysteine. In this paper, we use a recently developed fluorescence-energy-transfer assay to study translocation of misactivated threonine, α-aminobutyrate, and cysteine. Although their rates of misactivation are clearly distinct, their rates of translocation are similar. Thus, the rate of translocation is independent of the nature of the misactivated amino acid. This result suggests that the misactivated amino acid per se has little or no role in directing translocation.

Orthogonal combinatorial mutagenesis: a codon-level combinatorial mutagenesis method useful for low multiplicity and amino acid-scanning protocols

We describe here a method to generate combinatorial libraries of oligonucleotides mutated at the codon-level, with control of the mutagenesis rate so as to create predictable binomial distributions of mutants. The method allows enrichment of the libraries with single, double or larger multiplicity of amino acid replacements by appropriate choice of the mutagenesis rate, depending on the concentration of synthetic precursors. The method makes use of two sets of deoxynucleoside-phosphoramidites bearing orthogonal protecting groups [4,4′-dimethoxytrityl (DMT) and 9-fluorenylmethoxycarbonyl (Fmoc)] in the 5′ hydroxyl. These phosphoramidites are divergently combined during automated synthesis in such a way that wild-type codons are assembled with commercial DMT-deoxynucleoside-methyl-phosphoramidites while mutant codons are assembled with Fmoc-deoxynucleoside-methyl-phosphoramidites in an NNG/C fashion in a single synthesis column. This method is easily automated and suitable for low mutagenesis rates and large windows, such as those required for directed evolution and alanine scanning. Through the assembly of three oligonucleotide libraries at different mutagenesis rates, followed by cloning at the polylinker region of plasmid pUC18 and sequencing of 129 clones, we concluded that the method performs essentially as intended.

Prediction of protein deamidation rates from primary and three-dimensional structure

A method for the quantitative estimation of instability with respect to deamidation of the asparaginyl (Asn) residues in proteins is described. The procedure involves the observation of several simple aspects of the three-dimensional environment of each Asn residue in the protein and a calculation that includes these observations, the primary amino acid residue sequence, and the previously reported complete set of sequence-dependent rates of deamidation for Asn pentapeptides. This method is demonstrated and evaluated for 23 proteins in which 31 unstable and 167 stable Asn residues have been reported and for 7 unstable and 63 stable Asn residues that have been reported in 61 human hemoglobin variants. The relative importance of primary structure and three-dimensional structure in Asn deamidation is estimated.

pH-Regulated Leaf Cell Expansion in Droughted Plants Is Abscisic Acid Dependent1

Elongation rates of barley (Hordeum vulgare L. cv Hanna) leaves decreased with decreasing soil water content, whereas the pH of xylem sap increased from 5.9 to 6.9 over 6 d as the soil dried. The reduction in leaf-elongation rate (LER) was correlated with the increase in sap pH. Artificial sap buffered to different pH values was fed via the subcrown internode to derooted seedlings. Although leaves elongated at in planta rates when fed artificial sap at a well-watered pH of 6.0, LER declined with increasing sap pH. This effect persisted in the light and in the dark. pH had no effect on the relative water content or the bulk abscisic acid (ABA) concentration of the growing zone of these leaves. LERs of the ABA-deficient mutant Az34 were uniformly high over the pH range tested, whereas those of its isogenic wild-type cultivar Steptoe were reduced as the artificial sap pH was increased from 6.0 to 7.0. However, supplying a well-watered concentration of ABA (3 × 10−8 m) in the artificial xylem sap restored the pH response of the Az34 mutant. The results suggest that increased xylem sap pH acts as a drought signal to reduce LER via an ABA-dependent mechanism.

Effects of Xylem pH on Transpiration from Wild-Type and flacca Tomato Leaves1 : A Vital Role for Abscisic Acid in Preventing Excessive Water Loss Even from Well-Watered Plants

The pH of xylem sap from tomato (Lycopersicon esculentum) plants increased from pH 5.0 to 8.0 as the soil dried. Detached wild-type but not flacca leaves exhibited reduced transpiration rates when the artificial xylem sap (AS) pH was increased. When a well-watered concentration of abscisic acid (0.03 μm) was provided in the AS, the wild-type transpirational response to pH was restored to flacca leaves. Transpiration from flacca but not from wild-type leaves actually increased in some cases when the pH of the AS was increased from 6.75 to 7.75, demonstrating an absolute requirement for abscisic acid in preventing stomatal opening and excessive water loss from plants growing in many different environments.