Variations in the kinetic response of several different phosphate-dependent glutaminase isozymes during acute metabolic acidosis

Summary We describe the kinetic modifications to mitochondrial-membrane-bound phosphate-dependent glutaminase in various types of rat tissue brought about by acute metabolic acidosis. The activity response of phosphate-dependent glutaminase to glutamine was sigmoidal, showing positive co-operativity, the Hill coefficients always being higher than 2. The enzyme from acidotic rats showed increased activity at subsaturating concentrations of glutamine in kidney tubules, as might be expected, but not in brain, intestine or liver tissues. Nevertheless, when brain and intestine from control rats were incubated in plasma from acutely acidotic rats enzyme activity increased at 1 mM glutamine in the same way as in kidney cortex. The enzyme from liver tissue remained unaltered. S_0.5 and n_H values decreased significantly in kidney tubules, enterocytes and brain slices preincubated in plasma from acidotic rats. The sigmoidal curves of phosphate-dependent glutaminase shifted to the left without any significant changes in V_max. The similar response of phosphate-dependent glutaminase to acute acidosis in the kidney, brain and intestine confirms the fact that enzymes from these tissues are kinetically identical and reaffirms the presence of an ammoniagenic factor in plasma, either produced or concentrated in the kidneys of rats with acute acidosis.Abbreviations Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid - EDTA NN-1,2-Ethane-diylbis [N-(carboxymethyl)glycyne] - Tris 2-amino-2-hydroxymethyl-1,3-propanediol - PDG phosphate dependent glutaminase Publication No. 145 from Drogas, Tóxicos Ambientales y Metabolismo Celular Research Group. Department of Biochemistry and Molecular Biology, University of Granada, Spain     

Erratum to: Interactions Between Chronic Stress and Chronic Consumption of Caffeine on the Enzymatic Antioxidant System

Neurochemical Research -     

Tolerance,arsenic uptake,and oxidative stress in Acacia farnesiana under arsenate-stress

Acacia farnesiana is a shrub widely distributed in soils heavily polluted with arsenic in Mexico. However, the mechanisms by which this species tolerates the phytotoxic effects of arsenic are unknown. This study aimed to investigate the tolerance and bioaccumulation of As by A. farnesiana seedlings exposed to high doses of arsenate (AsV) and the role of peroxidases (POX) and glutathione S-transferases (GST) in alleviating As-stress. For that, long-period tests were performed in vitro under different AsV treatments. A. farnesiana showed a remarkable tolerance to AsV, achieving a half-inhibitory concentration (IC₅₀) of about 2.8 mM. Bioaccumulation reached about 940 and 4380 mg As·kg^?1 of dry weight in shoots and roots, respectively, exposed for 60 days to 0.58 mM AsV. Seedlings exposed to such conditions registered a growth delay during the first 15 days, when the fastest As uptake rate (117 mg kg^?1 day^?1) occurred, coinciding with both the highest rate of lipid peroxidation and the strongest up-regulation of enzyme activities. GST activity showed a strong correlation with the As bioaccumulated, suggesting its role in imparting AsV tolerance. This study demonstrated that besides tolerance to AsV, A. farnesiana bioaccumulates considerable amounts of As, suggesting that it may be useful for phytostabilization purposes.     

Weeds as crops: The value of maize field weeds in the valley of Toluca, Mexico

Maize field weeds or agrestals are widely used in central Mexico as potherbs (quelites) and forage. This work presents quantitative data on these uses from the village of San Bartolo del Llano, Municipio de Ixtlahuaca, Valley of Toluca, an area with a relatively intensive, semicommercial agriculture. We interviewed 24 families of the village and 10 vendors at the market of Ixtlahuaca regularly during one rainy season (1995) on type and quantity of weed use. Also, the weed vegetation was surveyed and we interviewed 49 farmers on their farming practices and on costs. All of the 74 weed species found in maize fields were useful as forage, potherb, medicinal, or ornamental. Within the village, 11 species were eaten; an average family consumed 4.5 kg of wild potherbs per month during the rainy season. In Ixtlahuaca, 2150 kg of 10 species were sold, worth 3054 pesos (US $611). For quantity and gross economic value, forage was much more important. On the average, 1 ha of maize field produced a harvest of 1.5 t of green forage, worth about 25% of the gross value of the maize harvest, and 55% of its net value. The combination of maize with forage weeds for stabled animals constitutes an interesting integrated farming system. The weeds increase the useful biomass of the field, improve nutrition of the farmers, do not reduce the yield of the main crop, as the fields are kept weed free during the critical period, and provide erosion control, shade, and green manure.     

Scientific societies fostering inclusivity through speaker diversity in annual meeting programming: a call to action

Scientific societies aiming to foster inclusion of scientists from underrepresented (UR) backgrounds among their membership often delegate primary responsibility for this goal to a diversity-focused committee. The National Science Foundation has funded the creation of the Alliance to Catalyze Change for Equity in STEM Success (ACCESS), a meta-organization bringing together representatives from several such STEM society committees to serve as a hub for a growing community of practice. Our goal is to coordinate efforts to advance inclusive practices by sharing experiences and making synergistic discoveries about what works. ACCESS has analyzed the approaches by which member societies have sought to ensure inclusivity through selection of annual meeting speakers. Here we discuss how inclusive speaker selection fosters better scientific environments for all and identify challenges and promising practices for societies striving to maximize inclusivity of speakers in their scientific programming.     

Continuous-flow enrichment of a strain ofErwinia carotovora having specificity for highly methylated pectin

A pectinolytic bacterium was isolated from a mixed microbial population by means of a chemostat enrichment procedure. The bacterium, which was identified asErwinia carotovora, grew only on highly methylated pectin and produced a pectin lysase which released unsaturated monomer and dimer from 71% esterified citrus pectin. The pectin lyase was inducible only by pectins having a high methyl content and in pectin-limited chemostats its synthesis passed through a maximum at a dilution rate close to 0.04h^-1.     

Evidence for morphine and morphine-like alkaloid responses resistant to naloxone blockade in the rat vas deferens.

The application of morphine or surrogates to the isolated rat vas deferens maintained at 37° C in Tyrode solution, produced an increase in the electrically induced muscular twitch. In contrast, leucine enkephalin or D-alanine₂methionine enkephalinamide produced a dose-dependent inhibition of the muscular twitch. The effect of morphine and derivatives was not antagonized by naloxone, but the depression caused by the opiate pentapeptides or β-Endorphin was readily antagonized and reversed by naloxone. Tolerance developed to the $\text{in vitro}$ effect of morphine; vasa deferentia obtained from tolerant-dependent rats were about six times less sensitive to the effect of morphine and about five times less sensitive to the depression caused by leucine enkephalin as compared to their respective paired, placebo implanted control rats.     

Molecularly imprinted chitosan-genipin hydrogels with recognition capacity toward o-xylene

A molecularly imprinted material was developed from hydrogels of chitosan (CS) cross-linked with genipin (GNP) using o-xylene as the template molecule. Gelling time, mechanical, and diffusion properties of CS-GNP hydrogels were initially investigated to establish optimal conditions to prepare molecularly imprinted hydrogels (MIHs). The elastic modulus was found to be directly proportional to the degree of cross-linking (R = moles of genipin/moles of glucosamine) while the diffusion of water, as monitored by magnetic resonance imaging, decreased with R. CS-GNP hydrogels of varying R were imprinted with o-xylene (MIH(o-xylene)). The adsorption capacity of o-xylene by MIH(o-xylene) was greater than the corresponding control hydrogels, particularly at R = 0.25. Freundlich isotherms yielded a better fitness than Langmuir ones and afforded n and Q(max)values of 2.55 and 103.3 mg/g, respectively. The imprinted hydrogel showed the highest adsorption capacity for o-xylene; however, the material was not highly selective as it also exhibited the capacity to adsorb m- and p-xylene isomers. In turn, the MIH(o-xylene) showed a low adsorption when 2-fluorotoluene was used in rebinding experiments, suggesting that molecular recognition by the binding sites is influenced by the electronic and steric properties of the analyte molecule, thus effectively confirming the imprinting effect within the MIH(o-xylene) network. This work opens the possibility to future development of materials with the capacity to adsorb o-xylene analogue molecules such as contaminants bearing chlorinated aromatic structures.     

Unravelling Glucan Recognition Systems by Glycome Microarrays Using the Designer Approach and Mass Spectrometry

Glucans are polymers of d-glucose with differing linkages in linear or branched sequences. They are constituents of microbial and plant cell-walls and involved in important bio-recognition processes, including immunomodulation, anticancer activities, pathogen virulence, and plant cell-wall biodegradation. Translational possibilities for these activities in medicine and biotechnology are considerable. High-throughput micro-methods are needed to screen proteins for recognition of specific glucan sequences as a lead to structure–function studies and their exploitation. We describe construction of a “glucome” microarray, the first sequence-defined glycome-scale microarray, using a “designer” approach from targeted ligand-bearing glucans in conjunction with a novel high-sensitivity mass spectrometric sequencing method, as a screening tool to assign glucan recognition motifs. The glucome microarray comprises 153 oligosaccharide probes with high purity, representing major sequences in glucans. Negative-ion electrospray tandem mass spectrometry with collision-induced dissociation was used for complete linkage analysis of gluco-oligosaccharides in linear “homo” and “hetero” and branched sequences. The system is validated using antibodies and carbohydrate-binding modules known to target α- or β-glucans in different biological contexts, extending knowledge on their specificities, and applied to reveal new information on glucan recognition by two signaling molecules of the immune system against pathogens: Dectin-1 and DC-SIGN. The sequencing of the glucan oligosaccharides by the MS method and their interrogation on the microarrays provides detailed information on linkage, sequence and chain length requirements of glucan-recognizing proteins, and are a sensitive means of revealing unsuspected sequences in the polysaccharides.Glucan polysaccharides are polymers of d-glucose with differing linkages in linear or branched sequences. They occur as storage materials in animals, secreted virulence factors of bacteria, and conserved structural components of cell walls of yeasts, fungi, some bacteria, and plants. Polysaccharides of this type are of considerable interest in biology, medicine, and biotechnology and are acknowledged for their immunostimulatory, anticancer, and health-promoting activities (1, 2); for their elicitor activities in defense responses and signaling in plants (3); and for acting as functional ingredients in human nutrition (4). Unraveling recognition systems that mediate these activities is highly desirable as a lead to effective translational applications.Recognition systems involving glucan polysaccharides include those in mammals, such as recognition of fungal β-glucans by Dectin-1, the major receptor of the innate immune system against fungal pathogens (5), and by natural or vaccine-induced protective antifungal antibodies (6, 7); also recognition of mycobacterial α-glucan by the innate immune receptor DC-SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin) (8); those in insects, such as the Drosophila Gram-negative binding protein 3 (GNBP3) sensor protein, which binds β-glucans (9); and those in bacteria, such as Brucella abortus, where cyclic β-glucans can serve as virulence factors (10).Another important class of glucan-recognizing proteins comprises noncatalytic carbohydrate-binding modules (CBMs)¹ of bacterial glycoside hydrolases that mediate association with substrate and increase catalytic activity, likely through a targeting mechanism or by driving enzyme specificity (11, 12). Notable examples are CBMs of bacterial cellulolytic enzymes that promote enzymatic deconstruction of intact plant cell walls and that are of industrial significance in the biofuel and bioprocessing sectors (13, 14) and CBMs of rumen or commensal human microbiota with roles in animal and human health (14, 15). CBMs also have roles in other systems: for example, CBM-containing enzymes as virulence factors of bacterial pathogens (16) and CBM-containing human laforin that regulates glycogen metabolism and for which mutations can lead to neurodegenerative disease (17). The number of putative glucan-binding CBMs that have been identified and classified in the Carbohydrate-Active enZyme (CAZy) database (http://www.cazy.org) is expanding, but relatively few have been experimentally investigated for details of carbohydrate binding and fine specificity (11).Searching for and assigning the specificities of glucan-recognizing proteins has thus become increasingly important. It is desirable to have high-throughput and sensitive micro-methods to screen for and characterize ligands for structure–function studies toward effective exploitation in modern therapeutic, nutritional, agricultural, and biofuel-related technologies. Carbohydrate microarrays have served to advance knowledge on specificities of diverse carbohydrate-recognition systems (18–22). Where the desired oligosaccharide probes are unavailable, microarrays need to be generated from ligand-bearing glycomes (23). Using a prototype of such designer microarrays of neoglycolipid (NGL)-probes (23) derived from oligosaccharide fragments of glucans rich in β1,3- or β1,6-linked sequences, we showed that linear β1,3-linked glucose sequences with degree of polymerization (DP) 10 or longer are bound by Dectin-1 (24). Recognition of other types of glucan sequences by Dectin-1 and the applicability of microarrays of diverse gluco-oligosaccharide sequences to other glucan-recognizing proteins required investigation. Cummings, Smith, and colleagues have developed the shotgun strategy (20) to create glycome-scale “gangliome” and “human milk glycome” microarrays. In the shotgun microarrays, the printed probes may not be sequence-defined before array construction and require metadata-assisted glycan sequencing (MAGS), which combines MS analysis (25), binding data with glycan-binding proteins or antibodies, and exoglycosidase treatment after printing (26, 27).Mass spectrometry has become a primary technique in carbohydrate structural analysis (28), and electrospray mass spectrometry (ESI-MS) has been used to provide sequence and partial linkage information on various types of oligosaccharides (29–33). For neutral oligosaccharides, we have found that tandem MS with collision-induced dissociation (CID-MS/MS) in the negative-ion mode is particularly useful and have successfully applied for oligosaccharide chain and blood-group typing (34, 35) and for branching pattern analysis (36).This is because that some important linkages at certain monosaccharide residues can be unambiguously determined with high sensitivity without the need for derivatization and anion complexation as previously recognized, e.g. in the area of gluco-oligosaccharides, Cl⁻-anion adduction has been used to determine sequences of tetrasaccharides of dextran (37).Here, we describe a strategy using the designer approach combined with negative-ion ESI-CID-MS/MS for constructing a microarray of sequence-defined gluco-oligosaccharides representing major sequences in glucans (glucome microarray) as a tool for screening glucan-recognizing proteins and assigning their recognition motifs (Fig. 1). We selected a comprehensive panel of glucan polysaccharides isolated from plants, fungi, and bacteria with different sequences to represent the glucome. We used finely tuned chemical and enzymatic methods to partially depolymerize the polysaccharides and prepare gluco-oligosaccharide fragments with different chain lengths (up to DP-13 or DP-16). We developed a ESI-CID-MS/MS method that enables linkage and sequence determination of linear or branched gluco-oligosaccharides at high-sensitivity and applied this to the sequencing of oligosaccharide fragments prepared. These sequence-defined gluco-oligosaccharides were then converted into NGL probes and used for construction of the microarray. The oligosaccharides encompassed linear sequences with homo (single) linkages: 1,2-, 1,3-, 1,4-, or 1,6- with α or β configurations; and hetero (multiple) linkages: 1,3-, 1,4, or 1,6-; also branched oligosaccharide sequences with 1,3 and 1,6-linkages.Open in a separate windowFig. 1.Neoglycolipid (NGL)-based designer glucome microarray with mass spectrometry as a tool to assign carbohydrate ligands in glucan recognition. Ligand-bearing glucan polysaccharides, described in supplemental Fig. S1 and Table S1, were selected as sources of gluco-oligosaccharides for construction of the microarray. A total of 121 gluco-oligosaccharide fractions were obtained with different DP after partial depolymerization of polysaccharides and fractionation. ESI-CID-MS/MS method was developed using gluco-oligosaccharides with known sequences and applied to determination of sequences of oligosaccharide fragments from polysaccharides. Gluco-oligosaccharides were converted to NGL probes for microarray construction and interrogation with the glucan-recognizing proteins described in supplemental Table S2.To our knowledge, this is the first sequence-defined glycome-scale microarray constructed. We used 12 selected proteins (antibodies and CBMs) known to target α- or β-glucans to validate the approach. We then applied the microarray analysis to Dectin-1 and DC-SIGN, which revealed new insights into the specificities of these signaling molecules of the innate immune system.