Decreased expression of surfactant Protein-C and CD74 in alveolar epithelial cells during influenza virus A(H1N1)pdm09 and H3N2 infection.

The primary replication site of Influenza A virus (IAV) is type II alveolar epithelial cells (AECII), which are central to normal lung function and present important immune functions. Surfactant components are synthesized primarily by AECII, which play a crucial role in host defense against infection. The aim of this study was to analyze if the impact of influenza infection is differential between A(H1N1)pdm09 and A/Victoria/3/75 (H3N2) on costimulatory molecules and ProSP-C expression in AECII from BALB/c mice infected and A549 cell line infected with both strains. Pandemic A(H1N1)pdm09 and A/Victoria/3/75 (H3N2) were used to infect BALB/c mice and the A549 cell line. We evaluated the surface expression of co-stimulatory molecules (CD45/CD31/CD74/ProSP-C) in AECII and A549 cell lines. Our results showed a significant decrease in ProSP-C+ CD31- CD45- and CD74+ CD31- CD45- expression in AECII and A549 cell line with the virus strain A(H1N1)pdm09 versus A/Victoria/3/75 (H3N2) and controls (non-infection conditions). Our findings indicate that changes in the expression of ProSP-C in AECII and A549 cell lines in infection conditions could result in dysfunction leading to decreased lung compliance, increased work of breathing and increased susceptibility to injury.

Study of duplicated galU genes in Rhodococcus jostii and a putative new metabolic node for glucosamine-1P in rhodococci.

BACKGOUND: Studying enzymes that determine glucose-1P fate in carbohydrate metabolism is important to better understand microorganisms as biotechnological tools. One example ripe for discovery is the UDP-glucose pyrophosphorylase enzyme from Rhodococcus spp. In the R. jostii genome, this gene is duplicated, whereas R. fascians contains only one copy. METHODS: We report the molecular cloning of galU genes from R. jostii and R. fascians to produce recombinant proteins RjoGalU1, RjoGalU2, and RfaGalU. Substrate saturation curves were conducted, kinetic parameters were obtained and the catalytic efficiency (kcat/Km) was used to analyze enzyme promiscuity. We also investigated the response of R. jostii GlmU pyrophosphorylase activity with different sugar-1Ps, which may compete for substrates with RjoGalU2. RESULTS: All enzymes were active as pyrophosphorylases and exhibited substrate promiscuity toward sugar-1Ps. Remarkably, RjoGalU2 exhibited one order of magnitude higher activity with glucosamine-1P than glucose-1P, the canonical substrate. Glucosamine-1P activity was also significant in RfaGalU. The efficient use of the phospho-amino-sugar suggests the feasibility of the reaction to occur in vivo. Also, RjoGalU2 and RfaGalU represent enzymatic tools for the production of (amino)glucosyl precursors for the putative synthesis of novel molecules. CONCLUSIONS: Results support the hypothesis that partitioning of glucosamine-1P includes an uncharacterized metabolic node in Rhodococcus spp., which could be important for producing diverse alternatives for carbohydrate metabolism in biotechnological applications. GENERAL SIGNIFICANCE: Results presented here provide a model to study evolutionary enzyme promiscuity, which could be used as a tool to expand an organism's metabolic repertoire by incorporating non-canonical substrates into novel metabolic pathways.

Rational design of nitrofuran derivatives: Synthesis and valuation as inhibitors of Trypanosoma cruzi trypanothione reductase.

The rational design and synthesis of a series of 5-nitro-2-furoic acid analogues are presented. The trypanocidal activity against epimastigote forms of Trypanosoma cruzi and the toxic effects on human HeLa cells were tested. Between all synthetic compounds, three of thirteen had an IC50 value in the range of Nfx, but compound 13 exhibited an improved effect with an IC50 of 1.0 ± 0.1 µM and a selective index of 70 in its toxicity against HeLa cells. We analyzed the activity of compounds 8, 12 and 13 to interfere in the central redox metabolic pathway in trypanosomatids, which is dependent of reduced trypanothione as the major pivotal thiol. The three compounds behaved as better inhibitors of trypanothione reductase than Nfx (Ki values of 118 µM, 61 µM and 68 µM for 8, 12 and 13, respectively, compared with 245 µM for Nfx), all following an uncompetitive enzyme inhibition pattern. Docking analysis predicted a binding of inhibitors to the enzyme-substrate complex with binding energy calculated in-silico that supports such molecular interaction.

UDPglucose pyrophosphorylase from Xanthomonas spp. Characterization of the enzyme kinetics, structure and inactivation related to oligomeric dissociation.

The genes encoding for UDPglucose pyrophosphorylase in two Xanthomonas spp. were cloned and overexpressed in Escherichia coli. After purification to electrophoretic homogeneity, the recombinant proteins were characterized, and both exhibited similar structural and kinetic properties. They were identified as dimeric proteins of molecular mass 60kDa, exhibiting relatively high specific activity ( approximately 80Units/mg) for UDPglucose synthesis. Both enzymes utilized UTP or TTP as substrate with similar affinity. The purified Xanthomonas enzyme was inactivated after dilution into the assay medium. Studies of crosslinking with the bifunctional lysyl reagent bisuberate suggest that inactivation occurs by enzyme dissociation to monomers. UTP effectively protects the enzyme against inactivation, from which a dissociation constant of 15microM was calculated for the interaction substrate-enzyme. The UTP binding to the enzyme would induce conformational changes in the protein, favoring the subunits interaction to form an active dimer. This view was reinforced by protein modeling of the Xanthomonas enzyme on the basis of the prokaryotic UDPglucose pyrophosphorylase crystallographic structure. The in silico approach pointed out two main critical regions in the enzyme involved in subunit-subunit interaction: the region surrounding the catalytic-substrate binding site and the C-term.

ADPglucose pyrophosphorylase's N-terminus: structural role in allosteric regulation.

We studied the functional role of the Escherichia coli ADPglucose pyrophosphorylase's N-terminus in allosteric regulation, and the particular effects caused by its length. Small truncated mutants were designed, and those lacking up to 15-residues were active and highly purified for further kinetic analyses. Ndelta3 and Ndelta7 did not change the kinetic parameters with respect to the wild-type. Ndelta11 and Ndelta15 enzymes were insensitive to allosteric regulation and highly active in the absence of the activator. Co-expression of two polypeptides corresponding to the N- and C-termini generated an enzyme with activation properties lower than those of the wild-type [C.M. Bejar, M.A. Ballicora, D.F. Gómez Casati, A.A. Iglesias, J. Preiss, The ADPglucose pyrophosphorylase from Escherichia coli comprises two tightly bound distinct domains, FEBS Lett. 573 (2004) 99-104]. Here, we characterized a Ndelta15 co-expression mutant, in which the allosteric regulation was restored to wild-type levels. Unusual allosteric effects caused by either an N-terminal truncation or co-expression of individual domains may respond to structural changes favoring an up-regulated or a down-regulated conformation rather than specific activator or inhibitor sites' disruption.

Preliminary study on the efficacy and tolerability of newer anticonvulsants in a population of epileptic patients.

OBJECTIVE: To study the efficacy and safety of newer antiepileptic drugs. SUBJECTS AND METHODS: Clinical records of 461 epileptic patients attending the Consorcio General Hospital, Valencia, Spain, were reviewed. Demographic data, adverse reactions and clinical outcome were recorded. RESULTS: One hundred and five patients experienced a total of 151 adverse drug reactions to antiepileptic medications. Adverse drug reactions occurred in the central nervous system (54.9%), skin (17.0%), gastrointestinal tract (13.2%), liver (4.9%), mouth (4.4%) and others (5.6%). The newer anticonvulsants were withdrawn in 19.1% of patients because of side effects, while older drugs were withdrawn in 9.3% of patients. Of the 461 patients, 78 (17.4%) experienced a > or = 50% reduction in seizure frequency when one of the newer anticonvulsants was added to their therapy. Older anticonvulsants were better tolerated than newer drugs. Tiagabine was the worst tolerated of all the drugs. CONCLUSION: Our findings show that patients with simple partial secondary generalized epilepsy had a greater benefit when a newer anticonvulsant was added to the treatment regimen.

Identification of functionally important amino-terminal arginines of Agrobacterium tumefaciens ADP-glucose pyrophosphorylase by alanine scanning mutagenesis.

Treatment of the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase with the arginyl reagent phenylglyoxal resulted in complete desensitization to fructose 6-phosphate (F6P) activation, and partial desensitization to pyruvate activation. The enzyme was protected from desensitization by ATP, F6P, pyruvate, and phosphate. Alignment studies revealed that this enzyme contains arginine residues in the amino-terminal region that are relatively conserved in similarly regulated ADP-glucose pyrophosphorylases. To functionally evaluate the role(s) of these arginines, alanine scanning mutagenesis was performed to generate the following enzymes: R5A, R11A, R22A, R25A, R32A, R33A, R45A, and R60A. All of the enzymes, except R60A, were successfully expressed and purified to near homogeneity. Both the R5A and R11A enzymes displayed desensitization to pyruvate, partial activation by F6P, and increased sensitivity to phosphate inhibition. Both the R22A and R25A enzymes exhibited reduced V(max) values in the absence of activators, lower apparent affinities for ATP and F6P, and reduced sensitivities to phosphate. The presence of F6P restored R22A enzyme activity, while the R25A enzyme exhibited only approximately 1.5% of the wild-type activity. The R32A enzyme displayed an approximately 11.5-fold reduced affinity for F6P while exhibiting behavior identical to that of the wild type with respect to pyruvate activation. Both the R33A and R45A enzymes demonstrated a higher activity than the wild-type enzyme in the absence of activators, no response to F6P, partial activation by pyruvate, and desensitization to phosphate inhibition. These altered enzymes were also insensitive to phenylglyoxal. The data demonstrate unique functional roles for these arginines and the presence of separate subsites for the activators.

Characterization of Rhizobium loti strains from the Salado River Basin.

Thirty indigenous rhizobia strains, isolated from Lotus tenuis in the area of Chascomús and other regions of the Salado River Basin (Argentina), were characterized based on generation time, acid production, carbon utilization, protein profile, and molecular characterization by restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes amplified by the polymerase chain reaction (PCR). The results indicated that native rhizobia isolates from the Chascomús area are predominantly fast and intermediate-growers. The unclassified rhizobia examined by PCR-RFLP were found to be closely related to the reference strains of validly described Rhizobium species.

Measurement of the glycogen synthetic pathway in permeabilized cells of cyanobacteria.

A simple, rapid and reliable procedure for permeabilizing cyanobacterial cells and measuring the glycogen synthetic pathway in situ, is presented. Cells from Anabaena sp. strain PCC 7120 were permeabilized with a mixture of toluene:ethanol (1:4 v/v). Fluorescence microscopy of cells incubated with fluorescein diacetate showed Anabaena non-permeabilized cells as green fluorescents, whereas permeabilized (viable) cells exhibited the intrinsic red fluorescence. Labelled alpha-1,4-glucan was recovered when permeabilized cells were incubated with the substrates of ADP-glucose pyrophosphorylase or glycogen synthase. The kinetic and regulatory properties of both enzymes could be reproduced in situ. The simplicity of the procedure and the ability to measure in situ glucan fluxes show the methodology as useful for studying the intracellular regulation of storage polysaccharides in a photosynthetic prokaryote.

Ultrasensitivity in (supra)molecularly organized and crowded environments.

The ultrasensitive response of biological systems is a more sensitive one than that expected from the classical hyperbola of Michaelis-Menten kinetics, and whose physiological relevance depends upon the range of variation of substrate or effector for which ultrasensitivity is observed. Triggering and modulation of the ultrasensitive response in enzymatic and cellular systems are reviewed. Several demonstrations of ultrasensitive behavior in cellular systems and its impact on the amplification properties in signalling cascades and metabolic pathways are also highlighted. It is shown that ubiquitous cytoskeletal proteins may up- or downmodulate ultrasensitivity under physico-chemical conditions resembling those predominant in cells.

Kinetic and structural analysis of the ultrasensitive behaviour of cyanobacterial ADP-glucose pyrophosphorylase.

The kinetic and (supra)molecular properties of the ultrasensitive behaviour of ADP-glucose pyrophosphorylase (AGPase) from Anabaena PCC 7120 (a cyanobacterium) were exhaustively studied. The response of the enzyme toward the allosteric activator 3-phosphoglycerate (3PGA) occurs with ultrasensitivity as a consequence of the cross-talk with the inhibitor P(i). Molecular 'crowding' renders AGPase more sensitive to the interplay between the allosteric regulators and, consequently, enhances the ultrasensitive response. In crowded media, and when orthophosphate is present, the activation kinetics of the enzyme with 3PGA proceed with increased co-operativity and reduced affinity toward the activator. Under conditions of ultrasensitivity, the enzyme's maximal activation takes place in a narrow range of 3PGA concentrations. Moreover, saturation kinetics of the enzyme with respect to its substrates, glucose 1-phosphate and ATP, were different at low or high 3PGA levels in crowded media. Only under the latter conditions did AGPase exhibit discrimination between low or high levels of the activator, which increased the affinity toward the substrates and the maximal activity reached by the enzyme. Studies of fluorescence emission of tryptophan residues, fourth-derivative spectroscopy and size-exclusion chromatography indicated that the ultrasensitive behaviour is correlated with intramolecular conformational changes induced in the tertiary structure of the homotetrameric enzyme. The results suggest a physiological relevance of the ultrasensitive response of AGPase in vivo, since the enzyme could be subtly sensing changes in the levels of allosteric regulators and substrates, and thus determining the flux of metabolites toward synthesis of storage polysaccharides.

Studies on the effect of temperature on the activity and stability of cyanobacterial ADP-glucose pyrophosphorylase.

The effect of temperature on the activity and stability of ADPglucose pyrophosphorylase from Anabaena PCC 7120 was studied. Experimental optima temperatures were found around 37-40 degrees C or 42-45 degrees C, depending on the absence or the presence of allosteric effectors in the assay medium, respectively. In the range of temperature where the enzyme is stable, curved Arrhenius plots were obtained, indicating a transition temperature between 9 and 12 degrees C. Since these results were observed for both the forward and reverse reaction, with two different sets of substrates and two entirely different assay procedures, it seems unlikely that the effect can be on any component of the system other than the enzyme itself. Results suggest that cyanobacterial ADPglucose pyrophosphorylase undergoes conformational changes at different temperatures, rendering structures with different catalytic efficiencies. The different structures of the enzyme were visualized by emission fluorescence. ADPglucose pyrophosphorylase was irreversibly inactivated when exposed to temperatures above 40 degrees C. Inactivation was dependent on temperature and followed first order kinetics. The substrate, ATP, and the allosteric effectors, 3PGA and Pi, effectively protected the enzyme against thermal inactivation. Protection afforded by ATP was affected by MgCl2. These results suggest that the binding of the effectors to the enzyme resulted in conformational changes of the protein, rendering structures more stable to temperature treatments. Similar structures could be adopted by the enzyme in different environments, since the higher stability was observed in media containing either high ionic strength or high hydrophobicity.

Ultrasensitive glycogen synthesis in Cyanobacteria.

Cyanobacter ADPglucose pyrophosphorylase exhibits a ultrasensitive response in activity towards its allosteric effector 3-phosphoglycerate, elicited by orthophosphate and polyethyleneglycol-induced molecular crowding. The ultrasensitive response was observed either when the enzyme operates in the zero or first order region for its physiological substrates. The ultrasensitivity exhibited maximal amplification factors of 15-19-fold with respect to 1% of the maximal system velocity. Only a 2.4-3.8-fold increase in 3PGA concentration was necessary to augment the flux from 10% to 90% through AGPase as compared with 200-fold required for the control. The results are discussed in terms of finely tuned regulatory mechanisms of polysaccharide synthesis in oxygenic photosynthetic organisms.

Cloning and expression of the glgC gene from Agrobacterium tumefaciens: purification and characterization of the ADPglucose synthetase.

The gene encoding ADPglucose synthetase (EC 2.7.7.27) from Agrobacterium tumefaciens was isolated and expressed in Escherichia coli. The recombinant protein was purified to electrophoretic homogeneity in steps including ion-exchange and hydrophobic chromatography. The same purification procedure was utilized to purify ADPglucose synthetase from A. tumefaciens cells. The enzymes from the two sources were purified and characterized and were found to have identical kinetic, regulatory, and structural properties. In polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, only one polypeptide band of 50 kDa was detected. In immunoblotting following electrophoresis, the 50-kDa band reacted with antibodies raised against the Escherichia coli ADPglucose synthetase; there was no reaction with antibodies raised against the spinach enzyme. The immunoreactivity of the A. tumefaciens ADPglucose synthetase was confirmed in antibody neutralization assays. Using gel filtration, the native enzyme was shown to be a tetramer. Fructose 6-phosphate and pyruvate were the most effective activators of the enzyme; maximal activation was observed in the ADPglucose synthesis direction, in which the enzyme was activated about ninefold by fructose 6-phosphate and fivefold by pyruvate. Both activators increased the affinity of the enzyme for the substrates ATP and glucose 1-phosphate. Inorganic orthophospate, ADP, AMP, and pyridoxal phosphate behaved as inhibitors of the enzyme. The distinctive regulatory properties of the enzyme from A. tumefaciens are compared with those of two enterobacterial enzymes and discussed in the context of their deduced amino acid sequences.

Structure-function relationships of cyanobacterial ADP-glucose pyrophosphorylase. Site-directed mutagenesis and chemical modification of the activator-binding sites of ADP-glucose pyrophosphorylase from Anabaena PCC 7120.

Chemical modification studies of spinach leaf ADP-glucose pyrophosphorylase with pyridoxal-P have shown that a highly conserved lysyl residue near the C terminus might be involved in the binding of 3-P-glycerate, the allosteric activator. Site-directed mutagenesis of the corresponding residue (Lys419) of the Anabaena enzyme was performed to determine the role of this conserved residue. Replacing Lys419 with either arginine, alanine, glutamine, or glutamic acid produced mutant enzymes with apparent affinities for 3-P-glycerate, 25-150-fold lower than that of the wild-type enzyme. The mutant enzymes, however, were still activated to a great extent at higher concentrations of activator suggesting that an additional site or residue was involved in the binding of the activator. These mutations caused lesser or no effect on the kinetic constants for the substrates and inhibitor, P(i), as well as on the catalytic efficiency and thermal stability. The results suggest that both the charge and size of lysine residue 419 are required for the proper binding of 3-P-glycerate. Chemical modification of the Anabaena wild-type enzyme with pyridoxal-P indicated that Lys419 was the only lysyl residue modified. We further performed the same experiment on the K419R mutant enzyme and found another lysyl residue, Lys382, was modified. Reductive phosphopyridoxylation of the wild-type and K419R enzymes caused dramatic alteration in allosteric properties. The activator, 3-P-glycerate, and inhibitor, P(i), protected the enzyme from reductive phosphopyridoxylation. The modified enzymes were more active in the absence of 3-P-glycerate and less sensitive to P(i) inhibition.

Characterization of the kinetic, regulatory, and structural properties of ADP-glucose pyrophosphorylase from Chlamydomonas reinhardtii.

ADP-glucose pyrophosphorylase (ADP-Glc PPase) from Chlamydomonas reinhardtii cells was purified over 2000-fold to a specific activity of 81 units/mg protein, and its kinetic and regulatory properties were characterized. Inorganic orthophosphate and 3-phosphoglycerate were the most potent inhibitor and activator, respectively. Rabbit antiserum raised against the spinach leaf ADP-Glc PPase (but not the one raised against the enzyme from Escherichia coli) inhibited the activity of the purified algal enzyme, which migrated as a single protein band in native polyacrylamide gel electrophoresis. Two-dimensional and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicate that the enzyme from C. reinhardtii is composed of two subunits with molecular masses of 50 and 53 kD, respectively. The molecular mass of the native enzyme is estimated to be 210 kD. Antisera raised against the spinach leaf holoenzyme and against the 51-kD spinach subunit cross-reacted with both subunits of the algal ADP-Glc PPase in immunoblot hybridization, but the cross-reaction was stronger for the 50-kD algal subunit than for the 53-kD subunit. No cross-reaction was observed when antiserum raised against the spinach leaf pyrophosphorylase 54-kD subunit was used. These results suggest that the ADP-Glc PPase from C. reinhardtii is a heterotetrameric protein, since the enzyme from higher plants and its two subunits are structurally more related to the small subunit of the spinach leaf enzyme than to its large subunit. This information is discussed in the context of the possible evolutionary changes leading from the bacterial ADP-Glc PPase to the cyanobacterial and higher plant enzymes.

Expression of the potato tuber ADP-glucose pyrophosphorylase in Escherichia coli.

cDNA clones encoding the putative mature forms of the large and small subunits of the potato tuber ADP-glucose pyrophosphorylase have been expressed separately and together in an Escherichia coli B mutant deficient in ADP-glucose pyrophosphorylase activity. Expression of both subunits from compatible vectors resulted in restoration of ADP-glucose pyrophosphorylase activity. Maximal enzyme activity required both subunits. The expressed ADP-glucose pyrophosphorylase was purified and characterized. The recombinant enzyme exhibited catalytic and allosteric kinetic properties very similar to the enzyme purified from potato tuber. The expressed enzyme activity was neutralized by incubation with antibodies raised against potato tuber and spinach leaf ADP-glucose pyrophosphorylases but not with anti-Escherichia coli enzyme serum. 3-Phosphoglycerate was the most efficient activator and its effect was increased by dithiothreitol. In the ADP-glucose synthesis direction, 3-phosphoglycerate activated the recombinant enzyme nearly 100-fold in the presence of dithiothreitol, with an A0.5 value of 57 microM. The recombinant ADP-glucose pyrophosphorylase was less sensitive to P(i) inhibition and more sensitive to heat denaturation than the potato tuber enzyme. Results suggest that bacterial expression of potato tuber cDNAs could be used to study the role and interaction of the subunits of the native ADP-glucose pyrophosphorylase.

The role of inorganic phosphate in the regulation of C4 photosynthesis.

Inorganic phosphate participates in many fundamental processes within the plant cell. Its broad influence on plant metabolism is related to such key operations as metabolite transport, enzyme regulation and carbohydrate metabolism in general. This review discusses these topics with special emphasis on the role assigned to this ubiquitous anion within the C4 pathway of photosynthesis.

Molecular cloning and expression of the gene encoding ADP-glucose pyrophosphorylase from the cyanobacterium Anabaena sp. strain PCC 7120.

Previous studies have indicated that ADP-glucose pyrophosphorylase (ADPGlc PPase) from the cyanobacterium Anabaena sp. strain PCC 7120 is more similar to higher-plant than to enteric bacterial enzymes in antigenicity and allosteric properties. In this paper, we report the isolation of the Anabaena ADPGlc PPase gene and its expression in Escherichia coli. The gene we isolated from a genomic library utilizes GTG as the start codon and codes for a protein of 48,347 Da which is in agreement with the molecular mass determined by SDS-PAGE for the Anabaena enzyme. The deduced amino acid sequence is 63, 54, and 33% identical to the rice endosperm small subunit, maize endosperm large subunit, and the E. coli sequences, respectively. Southern analysis indicated that there is only one copy of this gene in the Anabaena genome. The cloned gene encodes an active ADPGlc PPase when expressed in an E. coli mutant strain AC70R1-504 which lacks endogenous activity of the enzyme. The recombinant enzyme is activated and inhibited primarily by 3-phosphoglycerate and Pi, respectively, as is the native Anabaena ADPGlc PPase. Immunological and other biochemical studies further confirmed the recombinant enzyme to be the Anabaena enzyme.