Identification of key amino acid residues in the catalytic mechanism of diaminopropionate ammonialyase from Salmonella typhimurium

Diaminopropionate ammonialyase (DAPAL), a fold-typeII pyridoxal 5-phosphate-dependent enzyme, catalyzes the ,-elimination of diaminopropionate (DAP) to pyruvate and ammonia. DAPAL was able to utilize both d- and l-DAP as substrates with almost equal efficiency. Mutational analysis of functionally important residues such as Thr385, Asp125 and Asp194 was carried out to understand the mechanism by which the isomers are hydrolyzed. Further, the putative residues involved in the formation of disulfide bond Cys271 and Cys299 were also mutated. T385S, T385D sDAPAL were as active with dl-DAP as substrate as sDAPAL, whereas the later exhibited a threefold increase in catalytic efficiency with d-Ser as substrate. Further analysis of these mutants suggested that DAPAL might follow an anti-E-2 mechanism of catalysis that does not involve the formation of a quinonoid intermediate. Of the two mutants of Asp125, D125E showed complete loss of activity with d-DAP as substrate, whereas the reaction with l-DAP was not affected significantly, demonstrating that Asp125 was essential for abstraction of protons from the d-isomer. By contrast, mutational analysis of Asp194 showed that the residue may not be directly involved in proton abstraction from l-DAP. sDAPAL does not form a disulfide bond in solution, although the position of Cys299 and Cys271 in the modeled structure of sDAPAL favored the formation of a disulfide bond. Further, unlike eDAPAL, sDAPAL could be activated by monovalent cations. Mutation of the cysteine residues showed that Cys271 may be involved in coordinating the monovalent cation, as observed in the case of other fold-typeII enzymes.

Pt-6(II) nanoscopic cages with an organometallic backbone as sensors for picric acid

An organometallic building block 1,3,5-tris(4-trans-Pt(PEt3)(2)I(ethynyl)phenyl)benzene (1) incorporating Pt-ethynyl functionality has been synthesized and characterized. 2 + 3] self-assembly of its nitrate analogue 1,3,5-tris(4-trans-Pt(PEt3)(2)(ONO2)(ethynyl)phenyl)benzene (2) with ``clip'' type bidentate donors (L-1-L-3) separately afforded three trigonal prismatic architectures (3a-3c), respectively. All these prisms were characterized and their shapes/sizes are predicted through geometry optimization employing molecular mechanics universal force field (MMUFF) simulation. The extended p-conjugation including the presence of Pt-ethynyl functionality makes them electron rich as well as luminescent in nature. Macrocycles 3b and 3c exhibit fluorescence quenching in solution upon addition of picric acid PA], which is a common constituent of many explosives. Interestingly, the non-responsive nature of fluorescent intensity towards other electron-deficient nitro-aromatic explosives (NAEs) makes them promising selective sensors for PA with a detection limit predicted to be ppb level. Furthermore, solid-state quenching of fluorescent intensity of the thin film of 3b upon exposure to saturated vapor of picric acid has drawn special attention for infield applications.

Remarkable Regioisomer Control in the Hydrogel Formation from a Two-Component Mixture of Pyridine-End Oligo(p-phenylenevinylene)s and N-Decanoyl-L-alanine

N-Decanoyl-L-alanine (DA) was mixed with either colorless 4,4-bipyridine (BP) or various derivatives such as chromogenic oligo(p-phenylenevinylene) (OPV) functionalized with isomeric pyridine termini in specific molar ratios. This mixtures form salt-type gels in a water/ethanol (2:1, v/v) mixture. The gelation properties of these two-component mixtures could be modulated by variation of the position of the N atom of the end pyridyl groups in OPVs. The presence of acid-base interactions in the self-assembly of these two-component systems leading to gelation was probed in detail by using stoichiometry-dependent UV/Vis and FTIR spectroscopy. Furthermore, temperature-dependent UV/Vis and fluorescence spectroscopy clearly demonstrated a J-type aggregation mode of these gelator molecules during the sol-to-gel transition process. Morphological features and the arrangement of the molecules in the gels were examined by using scanning electron microscopy (SEM), atomic force microscopy (AFM), and X-ray diffraction (XRD) techniques. Calculation of the length of each molecular system by energy minimization in its extended conformation and comparison with the XRD patterns revealed that this class of gelator molecules adopts lamellar organizations. Rheological properties of these two-component systems provided clear evidence that the flow behavior could be modulated by varying the acid/amine ratio. Polarized optical microscopy (POM), differential scanning calorimetry (DSC), and XRD results revealed that the solid-phase behavior of such two-component mixtures (acid/base=2:1) varied significantly upon changing the proton-acceptor part from BP to OPV. Interestingly, the XRD pattern of these acid/base mixtures after annealing at their associated isotropic temperature was significantly different from that of their xerogels.

Differential Cocrystallization Behavior of Isomeric Pyridine Carboxamides toward Antitubercular Drug Pyrazinoic Acid

Pyrazinoic acid, the active form of the antitubercular pro-drug Pyrazinamide, is an amphiprotic molecule containing carboxylic acid and pyridine groups and therefore can form both salts and cocrystals with relevant partner molecules. Cocrystallization of pyrazinoic acid with isomeric pyridine carboxamide series resulted in a dimorphic mixed-ionic complex with isonicotinamide and in eutectics with nicotinamide and picolinamide, respectively. It is observed that with alteration of the carboxamide position, steric and electrostatic compatibility issues between molecules of the combination emerge and affect intermolecular interactions and supramolecular growth, thus leading to either cocrystal or eutectic for different pyrazinoic acid-pyridine carboxamide combinations. Intermolecular interaction energy calculations have been performed to understand the role of underlying energetics on the formation of cocrystal/eutectic in different combinations. On the other hand, two molecular salts with piperazine and cytosine and a gallic acid cocrystal of the drug were obtained, and their X-ray crystal structures were also determined in this work.

Identification of key amino acid residues in the catalytic mechanism of diaminopropionate ammonialyase from Salmonella typhimurium

Diaminopropionate ammonialyase (DAPAL), a fold-typeII pyridoxal 5-phosphate-dependent enzyme, catalyzes the ,-elimination of diaminopropionate (DAP) to pyruvate and ammonia. DAPAL was able to utilize both d- and l-DAP as substrates with almost equal efficiency. Mutational analysis of functionally important residues such as Thr385, Asp125 and Asp194 was carried out to understand the mechanism by which the isomers are hydrolyzed. Further, the putative residues involved in the formation of disulfide bond Cys271 and Cys299 were also mutated. T385S, T385D sDAPAL were as active with dl-DAP as substrate as sDAPAL, whereas the later exhibited a threefold increase in catalytic efficiency with d-Ser as substrate. Further analysis of these mutants suggested that DAPAL might follow an anti-E-2 mechanism of catalysis that does not involve the formation of a quinonoid intermediate. Of the two mutants of Asp125, D125E showed complete loss of activity with d-DAP as substrate, whereas the reaction with l-DAP was not affected significantly, demonstrating that Asp125 was essential for abstraction of protons from the d-isomer. By contrast, mutational analysis of Asp194 showed that the residue may not be directly involved in proton abstraction from l-DAP. sDAPAL does not form a disulfide bond in solution, although the position of Cys299 and Cys271 in the modeled structure of sDAPAL favored the formation of a disulfide bond. Further, unlike eDAPAL, sDAPAL could be activated by monovalent cations. Mutation of the cysteine residues showed that Cys271 may be involved in coordinating the monovalent cation, as observed in the case of other fold-typeII enzymes.

Subcutaneous fat composition of youthful and mature Canadian beef: emphasis on individual conjugated linoleic acid and trans-18:1 isomers

[EN]A comprehensive evaluation of the fatty acid composition of subcutaneous adipose tissue from beef cattle produced in western Canada was undertaken to determine if the current Canadian grading system is able to distinguish classes of animals with value added potential due to their fatty acid composition. Grades included youthful Canadian Yield Grade 1 A/AA beef, under (YUTM) and over (YOTM) 30 mo of age and the four mature grades (D1, D2, D2 and D4). Subcutaneous fat between the 12th and 13th ribs over the longissimus muscle was obtained from 18_21 animals per grade. Fatty acids were analyzed using a combination of silver-ion HPLC and GC with a highly polar 100 m column. There were no differences in total trans-18:1 content amongst grades, but adipose tissue from grade D1, D2 and D4 had more 11t-18:1 than YUTM (PB0.05), whereas adipose tissue from YUTM carcasses had more 10t-18:1 than all other grades (PB0.05). Adipose tissue from YUTM carcasses also had less total CLA (PB0.05) than the D grades, mainly due to a lower level of 9c,11t-CLA, but they had slightly more 7t,9c-CLA and 10t,12c-CLA (PB0.05). Adipose tissue from YOTM and D grades contained more n-3 fatty acids relative to YUTM (0.56% vs. 0.29%; PB0.05) and lower n-6:n-3 ratios (PB0.05). Overall, older animals (YOTM and D grades) had adipose tissue compositions with higher levels of fatty acids with reported health benefits. Taken together, these higher levels may provide opportunities for value added marketing if regulatory authorities allow claims for their enrichment based on demonstrated health benefits. Higher concentrations of beneficial fatty acids, however, need to be considered within the context of the complete fatty acid profile and it would be important to demonstrate their advantages in the presence of relatively high levels of saturated fatty acids.

Review: Trans-forming beef to provide healthier fatty acid profiles

[EN]Trans fatty acids are found naturally in foods, particularly in those derived from ruminant animals, such as beef and dairy cattle. Over the past few decades, human consumption of trans fatty acids has increased, but this has been mainly from products containing partially hydrogenated vegetable oils. The correlation of trans fatty acid consumption with diseases such as coronary heart disease has been cause for concern, and led to recommendations to reduce their consumption. Trans fatty acids, however, have differing effects on human health. Therefore, in foods produced from ruminant animals, it is important to know their trans fatty acid composition, and how to enrich or deplete fatty acids that have positive or negative health effects. This review will cover the analysis of trans fatty acids in beef, their origin, how to manipulate their concentrations, and give a brief overview of their health effects.

Evaluation of SP1001 (Pelargonic Acid) in Combination with Glyphosate on Cattail and Alligatorweed

Cattail (Typha latifolia L.) is a common and troublesome weed in shallow, freshwater environments throughout the United States. Alligatorweed (Alternanthera philoxeroides (Mart.)Griseb.), in spite of the introduction and success of several insects as biological controls, remains a troublesome we4ed in a a number of locations in the Southeast where there are frequent human disturbances (e.g., insecticide spraying, mechaniceal removal, etc.) and/or weather conditions that affect the life cycle of the insects (Kay1992, Vogt et al. 1992). Both of these weeds routinely are managed by foliar applications of the herbicide, glyphosate [N-(phosphonomethyl)glycine]. Regrowth and reinfestation of previously treated areas usually necessitates additional herbicide application during subsequent years. A new product that could enhance the activity of glyphosate on these weeds would be useful in their management. In 1997, SePRO Corp. initiated t4esting of an experimental compound, SP1001, to determine its efficacy either as a herbicide or as an adjuvant to boost the activity of glyphosate for use in aquatic sites. The objective of this study was to evaluate the potential for using SP1001 as an adjuvant to replace surfactants customarily used during application of glyphosate for control of cattail and alligatorweed.

Seasonal Changes in Chemical Composition of Eurasion Watermilfoil (Myriophyllum spicatum L.) and Water Temperature at Two Sites in northern California: implications for Herbivory

We compared seasonal changes in Eurasian watermilfoil (Myriophyllum spicatum L.) characteristics and water temperature for a shallow poind in Davis, CA, and the Truckee River, near Tahoe City, CA. Tissue C and N were 15% lower in plants from the Truckee River than in plants from the Davis pond. Seasonal fluctuations in tissue N were also different. Mean phenolic acid content of Truckee River palnts (162yM g-1) was less than those from the shallow pond (195 yM g-1). Phenolic acid content was positively related to tissue C for Truckee River and Davis pond plants and, tissue C:N ratio for Truckee River plants. Mean monthly water temperature (1990 to 1998) for the Truckee River site was less than 20 C. Water temperatures were warmer in August and September at this site. However, Eurasian watermilfoil collected during these months was characterized by lower levels of tissue N. During a 29-month period beginning January 1994, mean monthly water temperature for the Davis pond exceeded 20 C, only during July to September 1995. Tissue N was generally greater during summer for watermilfoil growing in the pond. These results imply that Eurasian watermilfoil biological control agents may have different developmental rates in these habitats, and thus different impacts on watermilfoil populations.

Influence of Dilute Acetic Acid Treatments on Survival of Monoecious Hydrilla Tubers in the Oregon House Canal, California

Hydrilla (Hydrilla verticillata (L.f.)Royle), a serious aquatic weed, reproduces through formation of underground tubers. To date, attacking this life-cycle stage has been problematic. The purpose of this study was to measure the impact of exposure to dilute acetic acid on monoecious hydrilla tubers under field conditions. In this field experiment, treatments were acetic acid concentration (0, 2.5, or 5%) and sediment condition (perforated or not perforated). Each of 60, 1x1 m plots (in the Oregon House Canal) were randomly assigned to one treatment. Two weeks after treatment, we collected three samples from each plot. One was washed over 2 mm wire mesh screens to separate tubers from sediment. Relative electrolyte leakage was measured for one tuber from each plot. Five additional tubers from each plot were placed in a growth chamber and sprouting monitored for four weeks. A second sample from each plot was placed in a plastic tub and placed in an outdoor tank, filled with water. These samples were monitored for tuber sprouting. Relative electrolyte leakage increased significantly for tubers exposed to 2.5% or 5% acetic acid. Effects on tubers in perforated sediment were reduced. Exposure to acetic acid inhibited tuber sprouting by 80 to 100%, in both chamber and outdoor tests. These results confirm findings from earlier laboratory/greenhouse experiments, and suggest that this approach may be useful in the management of hydrilla tuber banks in habitats where the water level can be lowered to expose the sediments.

Influence of Dilute Acetic Acid Treatments on American Pondweed Winter Buds in the Nevada Irrigation District, California

American pondweed ( Potamogeton nodosus Poir.) is commonly found in northern California irrigation canals. The purpose of this study was to test the hypothesis that exposure of American pondweed winter buds to dilute acetic acid under field conditions would result in reduced subsequent biomass.

The Role of the Subthalamic Nucleus in L-DOPA Induced Dyskinesia in 6-Hydroxydopamine Lesioned Rats

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Assessment of phytostabilization of Pb/Zn mine soils with organic amendments and a native metallicolous ecotype of Festuca Rubra l.: implications for amendment selection and recovery of soil health

La contaminación del suelo es una de las principales amenazas para los ecosistemas y la salud humana. Actualmente, desde un punto de vista tanto económico como ambiental, la fitoestabilización es la mejor tecnología para remediar suelos contaminados con elevadas concentraciones de metales como son los suelos mineros. La fitoestabilización asistida consiste en el empleo de plantas y enmiendas orgánicas y/o inorgánicas con el fin de reducir la movilidad y la biodisponibilidad de los contaminantes y recuperar la salud de suelo. En este trabajo se han realizado ensayos en microcosmos y en campo centrándonos en la salud del suelo minero contaminado con Pb y Zn durante un proceso de fitoestabilización empleando enmiendas orgánicas (purines vacunos, gallinaza, estiércol de oveja y lodos de papelera mezclados con gallinaza) y/o la especie metalífera Festuca rubra con el objetivo de (i) estudiar las interacciones suelo-enmienda responsables de los cambios inducidos por el proceso de quimioestabilización en las propiedades físicoquímicas y biológicas del suelo, (ii) evaluar la efectividad del proceso de fitoestabilización sobre suelos vegetados y de la revegetación sobre suelos desnudos (iii) valorar la idoneidad de distintos indicadores químicos y biológicos (parámetros microbianos y de la vegetación) para monitorizar la efectividad de la fitoestabilización asistida en términos de reducción de la biodisponibilidad de metales en el suelo, mejora de la vegetación y de la recuperación de la salud del suelo. La aplicación de enmiendas al suelo minero supone una entrada de materia orgánica y nutrientes que conduce a una disminución de la biodisponibilidad de metales, facilitando la colonización de las plantas y el crecimiento de la vegetación nativa, además de estimular la actividad microbiana del suelo. El pH del suelo es un factor crítico que condiciona la movilidad de los metales y la toxicidad del suelo. Las poblaciones microbianas de las enmiendas no modificaron la diversidad funcional de las comunidades microbianas nativas de la mina. Los purines vacunos y los lodos de papelera mezclados con gallinaza son los tratamientos más efectivos en el proceso de fitoestabilización asistida bajo condiciones de campo. La gallinaza fue el tratamiento que más estimuló el crecimiento de la vegetación nativa y la colonización en los suelos desnudos. El bioensayo de elongación radical de lechuga es un test sensible, sencillo y barato para evaluar la biodisponibilidad de metal y la ecotoxicidad del suelo. Los tocoferoles son biomarcadores de exposición a metales con potencial para su implementación en bioensayos de toxicidad. Este trabajo permite concluir que la población metalífera de F. rubra, combinada con enmiendas orgánicas, es una excelente candidata para los proyectos de fitoestabilización asistida. Además, la monitorización simultánea de los parámetros fisicoquímicos y microbiológicos del suelo y de su ecotoxicidad permite una evaluación adecuada de la salud del suelo, así como la selección de enmiendas apropiadas para el desarrollo de un proceso fitoestabilizador.

The locus coeruleus Is Directly Implicated in L-DOPA-Induced Dyskinesia in Parkinsonian Rats: An Electrophysiological and Behavioural Study

Despite being the most effective treatment for Parkinson's disease, L-DOPA causes a development of dyskinetic movements in the majority of treated patients. L-DOPA-induced dyskinesia is attributed to a dysregulated dopamine transmission within the basal ganglia, but serotonergic and noradrenergic systems are believed to play an important modulatory role. In this study, we have addressed the role of the locus coeruleus nucleus (LC) in a rat model of L-DOPA-induced dyskinesia. Single-unit extracellular recordings in vivo and behavioural and immunohistochemical approaches were applied in rats rendered dyskinetic by the destruction of the nigrostriatal dopamine neurons followed by chronic treatment with L-DOPA. The results showed that L-DOPA treatment reversed the change induced by 6-hydroxydopamine lesions on LC neuronal activity. The severity of the abnormal involuntary movements induced by L-DOPA correlated with the basal firing parameters of LC neuronal activity. Systemic administration of the LC-selective noradrenergic neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine did not modify axial, limb, and orolingual dyskinesia, whereas chemical destruction of the LC with ibotenic acid significantly increased the abnormal involuntary movement scores. These results are the first to demonstrate altered LC neuronal activity in 6-OHDA lesioned rats treated with L-DOPA, and indicate that an intact noradrenergic system may limit the severity of this movement disorder.

Nutritional and anti-nutritional analyses of Azolla africana Desv. and Spirodela polyrrhiza L. Schleiden as feedstuffs for fish production

The chemical composition of Azolla africana and Spirodela polyrrhiza cultivated in earthen ponds were determined. Crude protein contents of the samples were 28.9~c0.6 and 25.6~c0.2% dry matter for A. africana and S. polyrrhiza respectively. Dry matter, crude fibre and lipid contents of A. africana were higher (P<0.05) than values obtained for S. polyrrhiza. Mineral analyses showed that S. polyrrhiza contained higher levels of Na, S, Ca, Mg and Fe than A. africana. Except for Ca content in S. polyrrhiza, heavy metals (Ni and Zn) accumulation in Azolla were very high. There were no wide differences in the individual amino acid indexes except for methionine. Some anti-nutritional factors were determined. Cyanide, tannin and phytin contents of fresh weed samples were higher than sun-dried samples. A. africana contained more cyanide and tannin than S. polyrrhiza both in fresh and sun-dried forms