Amino Acids Trp Research Articles

Structural Basis for Carbohydrate Recognition and Anti-inflammatory Modulation by Gastrointestinal Nematode Parasite Toxascaris leonina Galectin

Toxascaris leonina galectin (Tl-gal) is a galectin-9 homologue protein isolated from an adult worm of the canine gastrointestinal nematode parasite, and Tl-gal-vaccinated challenge can inhibit inflammation in inflammatory bowel disease-induced mice. We determined the first X-ray structures of full-length Tl-gal complexes with carbohydrates (lactose, N-acetyllactosamine, lacto-N-tetraose, sialyllactose, and glucose). Bonds were formed on concave surfaces of both carbohydrate recognition domains (CRDs) in Tl-gal. All binding sites were found in the HXXXR and WGXEER motifs. Charged Arg61/Arg196 and Glu80/Glu215 on the conserved motif of Tl-gal N-terminal CRD and C-terminal CRD are critical amino acids for recognizing carbohydrate binding, and the residues can affect protein folding and structure. The polar amino acids His, Asn, and Trp are also important residues for the interaction with carbohydrates through hydrogen bonding. Hemagglutination activities of Tl-gal were inhibited by interactions with carbohydrates and mutations. We found that the mutation of Tl-gal (E80A/E215A) at the carbohydrate binding region induced protein aggregation and could be caused in many diseases. The short linker region between the N-terminal and C-terminal CRDs of Tl-gal was very stable against proteolysis and maintained its biological activity. This structural information is expected to elucidate the carbohydrate recognition mechanism of Tl-gal and improve our understanding of anti-inflammatory mediators and modulators of immune response.

435 - Metabolomic-Based Amino Acid and Biogenic Amine Plasmatic Profile of Brazilian Cri-du-Chat Patients

Cri-du-chat syndrome (CDCS) is a rare innate disease attributed to chromosome 5p deletion characterized by a cat-like cry, craniofacial malformation, and altered behavior of affected children. Here, UHPLC/MS and chemometrics were employed to analyze blood samples withdrawn from CDCS carriers (n=18) and normal parental subjects (n=18), all aged 0-34 years, aiming to set up a representative CDCS profile constructed from 33 targeted amino acids and biogenic amines. Methionine sulfoxide (MetO), nitrotyrosine and His were of particular concern with respect to CDCS redox balance. Increased serotonin (3-fold), methionine sulfoxide (2-fold), and Asp (50%) levels, and a little lower Orn (26%), citrulline (23%), Leu (29%), Val (20%), Ile (16%), Asn (17%), Gln (10%), Trp (15%), Thr (13%), His (12%), Phe (11%), Met (6%), and creatinine (35%) levels were found in the plasma of CDCS patients. Nitrotyrosine did not differ in normal and CDCS individuals, whereas the ratio Trp/Σtotal metabolites was 37% higher in the latter group. The accumulated metabolites may reflect disturbances in the redox balance, deficient purine biosynthesis, and altered behavior, whereas the amino acid abatement in the latter group may affect the homeostasis of the urea cycle, citric acid cycle, branched chain amino acid synthesis, Tyr and Trp metabolism and amino acid biosynthesis. The identification of enzymatic deficiencies leading to the amino acid burden in CDCS is further required for elucidating its molecular bases and eventually propose specific or mixed amino acid supplementation to newborn patients aiming to balance their metabolism. Support: FAPESP, CNPq, CAPES.

Ru(II)/bisphosphine/diimine/amino acid complexes: diastereoisomerism, cytotoxicity, and inhibition of tumor cell adhesion to collagen type I

We herein report the synthesis and characterization of Ru(II)/amino acid complexes with general formula [Ru(AA-H)(dppb)(4-mebipy)](PF6), where AA-H means the deprotonated amino acids Gly, Ala, Val, Met, Trp, Tyr, and Ser; dppb is 1,4-bis(diphenylphosphino)butane and 4-mebipy = 4,4′-dimethyl-2,2′-bipyridine. The complexes were characterized by 31P{1H}, 13C, and 1H NMR spectroscopy, as well as X-ray crystallographic analysis of [Ru(DL-Ala-H)(dppb)(4-mebipy)]+, suggesting the presence of diastereoisomers. The complexes exhibit IC50 values against breast tumor cells (MDA-MB-231) comparable with cisplatin. In addition, the Ru(II)-based complex with tryptophan inhibited tumor cell adhesion to collagen type I. Therefore, the use of ruthenium complexes containing amino acids can be an interesting tool for development of new therapeutic agents.

Side Chain Cyclized Aromatic Amino Acids: Great Tools as Local Constraints in Peptide and Peptidomimetic Design.

Constraining the conformation of flexible peptides is a proven strategy to increase potency, selectivity, and metabolic stability. The focus has mostly been on constraining the backbone dihedral angles; however, the correct orientation of the amino acid side chains (χ-space) that constitute the peptide pharmacophore is equally important. Control of χ-space utilizes conformationally constrained amino acids that favor, disfavor, or exclude the gauche (-), the gauche (+), or the trans conformation. In this review we focus on cyclic aromatic amino acids in which the side chain is connected to the peptide backbone to provide control of χ1- and χ2-space. The manifold applications for cyclized analogues of the aromatic amino acids Phe, Tyr, Trp, and His within peptide medicinal chemistry are showcased herein with examples of enzyme inhibitors and ligands for G protein-coupled receptors.

Structure-Function Analysis of the Two-Peptide Bacteriocin Plantaricin EF.

Plantaricin EF is a two-peptide bacteriocin that depends on the complementary action of two different peptides (PlnE and PlnF) to function. The structures of the individual peptides have previously been analyzed by nuclear magnetic resonance spectroscopy (Fimland, N. et al. (2008), Biochim. Biophys. Acta1784, 1711−171918555030), but the bacteriocin structure and how the two peptides interact have not been determined. All two-peptide bacteriocins identified so far contain GxxxG motifs. These motifs, together with GxxxG-like motifs, are known to mediate helix–helix interactions in membrane proteins. We have mutated all GxxxG and GxxxG-like motifs in PlnE and PlnF in order to determine if any of these motifs are important for antimicrobial activity and thus possibly for interactions between PlnE and PlnF. Moreover, the aromatic amino acids Tyr and Trp in PlnE and PlnF were substituted, and four fusion polypeptides were constructed in order to investigate the relative orientation of PlnE and PlnF in target cell membranes. The results obtained with the fusion polypeptides indicate that PlnE and PlnF interact in an antiparallel manner and that the C-terminus of PlnE and N-terminus of PlnF are on the outer part of target cell membranes and the N-terminus of PlnE and C-terminus of PlnF are on the inner part. The preference for an aromatic residue at position 6 in PlnE suggests a positioning of this residue in or near the membrane interface on the cells inside. Mutations in the GxxxG motifs indicate that the G5xxxG9 motif in PlnE and the S26xxxG30 motif in PlnF are involved in helix–helix interactions. Atomistic molecular dynamics simulation of a structural model consistent with the results confirmed the stability of the structure and its orientation in membranes. The simulation approved the anticipated interactions and revealed additional interactions that further increase the stability of the proposed structure.

The Yin and Yang of codon usage.

The genetic code is degenerate. With the exception of two amino acids (Met and Trp), all other amino acid residues are each encoded by multiple, so-called synonymous codons. Synonymous codons were initially presumed to have entirely equivalent functions, however, the finding that synonymous codons are not present at equal frequencies in genes/genomes suggested that codon choice might have functional implications beyond amino acid coding. The pattern of non-uniform codon use (known as codon usage bias) varies between organisms and represents a unique feature of an organism. Organism-specific codon choice is related to organism-specific differences in populations of cognate tRNAs. This implies that, in a given organism, frequently used codons will be translated more rapidly than infrequently used ones and vice versa A theory of codon-tRNA co-evolution (necessary to balance accurate and efficient protein production) was put forward to explain the existence of codon usage bias. This model suggests that selection favours preferred (frequent) over un-preferred (rare) codons in order to sustain efficient protein production in cells and that a given un-preferred codon will have the same effect on an organism's fitness regardless of its position within an mRNA's open reading frame. However, many recent studies refute this prediction. Un-preferred codons have been found to have important functional roles and their effects appeared to be position-dependent. Synonymous codon usage affects the efficiency/stringency of mRNA decoding, mRNA biogenesis/stability, and protein secretion and folding. This review summarizes recent developments in the field that have identified novel functions of synonymous codons and their usage.

A DFT and TDDFT investigation of interactions between pyrene and amino acids with cyclic side chains

A computational investigation of photoinduced charge transfer complex formation between pyrene (Py) and amino acids with cyclic side chains (Phenylalanine: Phe, Tyrosine: Tyr, Tryptophan: Trp, Histidine, His) has been carried out in gas phase and in water. Geometry optimizations were performed by density functional theory (DFT) at ωB97XD/6-311++G(d,p) level. Time-dependent density functional theory (TDDFT) was used to calculate the electronic transitions of molecules at B3LYP/6-311++G(d,p) level using the above ground-state geometries. Polarizable Continuum Model (PCM) and SMD are used for calculations in water. Analyses and comparisons have been performed for total electronic energies, complexation energies, free energy differences, solvation energies, excitation wavelengths, and HOMO–LUMO energy gaps of complexes in gas phase and in solution. The intermolecular distances between Py and amino acids increased in water compared to the gas phase for all studied with the exception of Py–Tyr system. The optimized complexes display an increasing complex stability in the order Trp>Tyr>Phe>His. Analyses of first excited singlet states have indicated charge transfers transitions between Py and amino acids His and Trp through π–π stacking in gas phase at 345nm and 393nm, respectively. Py–Trp system has also charge transfer (CT) in water at 389nm. Py–Trp systems have the most significant charge transfer between HOMO and LUMO (full CT, 70%). However, S0–S1 transition (393nm) has weaker dipole moment and oscillator strength than the other studied systems. Due to its charge transfer character, Py–Trp systems seem to be appropriate models to investigate and design bioorganic photosensitive materials.

Quantifying the π-Stacking Interactions in Nitroarene Binding Sites of Proteins.

Stacking interactions in nitroarene binding sites of proteins were studied through analyses of structures in the protein data bank (PDB), as well as DFT and ab initio computations applied to model systems. Stacked dimers of mono-, di-, and trinitrobenzene with the amino acid side chains histidine (His), phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp) were optimized at the B97-D/TZV(2d,2p) level of theory. Binding energies for the global minimum dimer geometries were further refined at the estimated CCSD(T)/aug-cc-pVTZ level of theory. The results show that the interactions between aromatic amino acids and nitroarenes are very strong (up to -14.6 kcal mol(-1)), and the regiochemistry of the nitro substituents plays a significant role in the relative monomer orientations and strength of the interaction. In contrast to model stacked benzene dimers, effects of nitro substituents in stacking complexes with aromatic amino acid side chains are not perfectly additive. This is attributed to direct interactions of the nitro substituents with functional groups in the amino acid side chain. Overall, the strength of stacking interactions with these nitrobenzenes follows the order Trp > Tyr > Phe ≈ His. We also analyzed nitroarene binding sites in the PDB. Out of 216 selected crystal structures containing nitroarene ligands, 191 have nearby aromatic residues, providing 65 examples of π-stacking interactions involving a nitroarene. Of these, the representations of the different aromatic amino acids (Trp > Tyr > Phe > His) are correlated with the strength of model complexes of nitroarenes, with the exception of His. B97-D computations applied to complexes extracted from these crystal structures reveal that π-stacking interactions between the nitroarene and aromatic amino acid side chains exhibit a broad range of strengths, with many contributing significantly to binding.

Exploring Strong Interactions in Proteins with Quantum Chemistry and Examples of Their Applications in Drug Design.

ObjectivesThree strong interactions between amino acid side chains (salt bridge, cation-π, and amide bridge) are studied that are stronger than (or comparable to) the common hydrogen bond interactions, and play important roles in protein-protein interactions.MethodsQuantum chemical methods MP2 and CCSD(T) are used in calculations of interaction energies and structural optimizations.ResultsThe energies of three types of amino acid side chain interactions in gaseous phase and in aqueous solutions are calculated using high level quantum chemical methods and basis sets. Typical examples of amino acid salt bridge, cation-π, and amide bridge interactions are analyzed, including the inhibitor design targeting neuraminidase (NA) enzyme of influenza A virus, and the ligand binding interactions in the HCV p7 ion channel. The inhibition mechanism of the M2 proton channel in the influenza A virus is analyzed based on strong amino acid interactions.Conclusion(1) The salt bridge interactions between acidic amino acids (Glu- and Asp-) and alkaline amino acids (Arg+, Lys+ and His+) are the strongest residue-residue interactions. However, this type of interaction may be weakened by solvation effects and broken by lower pH conditions. (2) The cation- interactions between protonated amino acids (Arg+, Lys+ and His+) and aromatic amino acids (Phe, Tyr, Trp and His) are 2.5 to 5-fold stronger than common hydrogen bond interactions and are less affected by the solvation environment. (3) The amide bridge interactions between the two amide-containing amino acids (Asn and Gln) are three times stronger than hydrogen bond interactions, which are less influenced by the pH of the solution. (4) Ten of the twenty natural amino acids are involved in salt bridge, or cation-, or amide bridge interactions that often play important roles in protein-protein, protein-peptide, protein-ligand, and protein-DNA interactions.

Indoleamine 2,3 Dioxygenase as a Potential Therapeutic Target in Huntington’s Disease

Within the past decade, there has been increasing interest in the role of tryptophan (Trp) metabolites and the kynurenine pathway (KP) in diseases of the brain such as Huntington’s disease (HD). Evidence is accumulating to suggest that this pathway is imbalanced in neurologic disease states. The KP diverges into two branches that can lead to production of either neuroprotective or neurotoxic metabolites. In one branch, kynurenine (Kyn) produced as a result of tryptophan (Trp) catabolism is further metabolized to neurotoxic metabolites such as 3-hydroxykunurenine (3-HK) and quinolinic acid (QA). In the other branch, Kyn is converted to the neuroprotective metabolite kynurenic acid (KA). The enzyme Indoleamine 2,3 dioxygenase (IDO1) catalyzes the conversion of Trp into Kyn, the first and rate-limiting enzymatic step of the KP. This reaction takes place throughout the body in multiple cell types as a required step in the degradation of the essential amino acid Trp. Studies of IDO1 in brain have focused primarily on a potential role in depression, immune tolerance associated with brain tumours, and multiple sclerosis; however the role of this enzyme in neurodegenerative disease has garnered significant attention in recent years. This review will provide a summary of the current understanding of the role of IDO1 in Huntington’s disease and will assess this enzyme as a potential therapeutic target for HD.

Structure-activity relationship of a series of antioxidant tripeptides derived from β-Lactoglobulin using QSAR modeling

Antioxidative peptides derived from food proteins have been screened and characterized in in vitro assays, and several quantitative structure–activity relationship (QSAR) models have been established to understand the structure–activity relationship of the selected antioxidant peptides. However, a systematic evaluation of each fragment that could be produced by a certain protein is still lacking. In this study, all the tripeptides that could possibly derive from bovine beta-lactoglobulin based on its amino acid sequence have been synthesized, and their antioxidant activities were measured using a ferric-reducing antioxidant power assay. Three of the peptides (i.e., LTC, CQC, and GTW) exhibited higher activities than that of glutathione. A QSAR model was also computed using multiple linear regression with divided physicochemical property scores descriptors, and the validity of the model was confirmed by cross-validation and Y-scrambling. The result of the QSAR modeling indicated that the electronic and hydrogen-bonding properties of the amino acids in the tripeptide sequences, as well as the steric properties of the amino acid residues at the C- and N-termini, played an important role in the antioxidant activities of the tripeptides. The antioxidant activities of the tripeptides were generally higher with Cys and Trp amino acid residues in the sequence. The structural characteristics we found may contribute to the further research of antioxidative mechanism.

Selective requirement of the shikimate pathway of Legionella pneumophila for intravacuolar growth within human macrophages but not within Acanthamoeba.

Legionella pneumophila utilizes the Dot/Icm type IV translocation system to proliferate within a vacuole in a wide variety of natural amoebal hosts and in alveolar macrophages of the human accidental host. Although L. pneumophila utilizes host amino acids as the main sources of carbon and energy, it is not known whether de novo synthesis of amino acids by intravacuolar L. pneumophila contributes to its nutrition. The aroB and aroE genes encode enzymes for the shikimate pathway that generates the aromatic amino acids Phe, Trp, and Tyr. Here we show the aroB and aroE mutants of L. pneumophila to be defective in growth in human monocyte-derived macrophages (hMDMs) but not in Acanthamoeba spp. The aroB and aroE mutants are severely attenuated in intrapulmonary proliferation in the A/J mouse model of Legionnaires' disease, and the defect is fully complemented by the respective wild-type alleles. The two mutants grow normally in rich media but do not grow in defined media lacking aromatic amino acids, and the growth defect is rescued by inclusion of the aromatic amino acids, which are essential for production of the pyomelanin pigment. Interestingly, supplementation of infected hMDMs with the three aromatic amino acids or with Trp alone rescues the intramacrophage defect of the aroE but not the aroB mutant. Therefore, the shikimate pathway of L. pneumophila is differentially required for optimal growth within human macrophages, which are auxotrophic for Trp and Phe, but is dispensable for growth within the Acanthamoeba spp. that synthesize the aromatic amino acids.

Adverse In utero and Postnatal Environments Promote Hepatic Microvesicular Steatosis in conjunction with Differential Alterations in Fatty Acid and Amino Acid Metabolism in Early Adulthood

We questioned whether an adverse in utero environment, resulting in low birth weight (LBW), and postnatal Western diet (WD) feeding interact in hepatic steatosis pathogenesis in young males, with particular attention focused upon differential alterations in lipid and amino acid metabolism. LBW guinea pigs, generated via uterine artery ablation, and normal birth weight (NBW) controls were fed either a WD or control diet (CD) after weaning. At 150 days of age, livers were harvested for histologic, molecular and biochemical assessments, gas chromatography and mass spectrometry. In LBW/CD animals, steatosis was absent. NBW/WD animals however, displayed a macrovesicular steatosis whereas LBW/WD animals exhibited a microvesicular steatosis. Hepatic carnitine palmitoyltransferase I and uncoupling protein 2 mRNA, long‐chain acylcarnitines (C16, C18 and C18:1) and amino acids (Asp, Phe, Tyr and Trp) were lower in LBW/WD vs. NBW/WD. Independent of birth weight, WD resulted in increased hepatic triglycerides, fatty acid translocase (CD36) mRNA, expression of lipogenic genes and proteins (FAS, ACC, and HK2), and metabolite‐based lipogenic parameters (C16:1/C16:0, C18:1/C18:0 and C16:0/C18:2n‐6). Hepatic medium chain acylcarnitine (C12), long‐chain acylcarnitines (C14, C14:1, C14‐OH, C16:1, C16‐OH, C18‐OH, C18:1‐OH and C18:3) and amino acid concentrations (Arg, Cys, Thr and Leu) were increased in WD animals. In conclusion, WD and LBW/WD induce hepatic steatosis accompanied with environment specific lipid and amino acid profile alterations. These data imply a less favorable liver health prognosis for adversely in utero grown offspring fed a WD postnatally.

Evaluation of novel 1-(4-(substituted)piperazin-1-yl)- 2-(phenylamino)ethanone derivatives as Falcipain-2 inhibitors

Background: Malaria, an infectious disease transmitted by mosquitoes, has affected the world since the beginning of recorded human history and it remains an ensconced global health challenge even today. Among the various proteases, expressed in the life cycle of parasite, cysteine protease falcipain-2 plays a pivotal role in parasite food assimilation and inhibition of this protease cause deleterious effects on the growth of parasite. Methods: Employing a ligand-based approach, 1-(4-(substituted)piperazin-1-yl)-2-(phenylamino)ethanone derivatives were designed and synthesized from the starting material piperazine in a sequence of reactions. Structural assignments are based on spectral data ( 1 H NMR, mass) and elemental analyses. The purity of the final compounds was confirmed by HPLC. The compounds were tested for their in vitro falcipain-2 inhibitor activity on recombinant falcipain-2 enzyme. Furthermore, molecular docking studies were performed using Glide 5.9 software to incur a precise picture of the active ligand at the atomic level which will be helpful in the discovery of new antimalarial drugs. Results: Among the screening results of seventeen novel entities, three compounds (6h, 6n and 6o) have showed good inhibitory activity and eleven compounds were showed weak to moderate inhibitor activity. Docking studies for these active analogues revealed that the amino acids Trp 206, Ile 85, Leu 84, Val 152 most commonly involved in hydrophobic interactions and Asn 173, Cys 42, Gln 36, amino acids involved in hydrogen bonding. Conclusion: The preliminary structure-activity relationships indicated that compound 6h, is the most potent compound from this series, and it can be used as a potential lead compound in the designing of new candidates to optimize the inhibitory potencies of this class of compounds, and potentially with potent antimalarial activity. Key words: Antimalarial, Falcipain-2, Ligand-based drug design, Plasmodium falciparum .

Impact of sodium chloride replacement by salt substitutes on the proteolysis and rheological properties of dry fermented sausages

The effect of a 50% reduction of NaCl and its replacement by KCl, CaCl2, and a blend of KCl and CaCl2 (1:1) on the proteolysis and rheological properties of dry fermented sausages was investigated. The reduction or replacement of NaCl by KCl did not cause changes in the electrophoretic profile and the addition of CaCl2 decreased the degradation of sarcoplasmic proteins during the manufacturing process. Samples with a 50% reduction of NaCl showed a higher content of the amino acids Arg, Glu, His, Val, Cys, Lys, and Trp, whereas the samples containing CaCl2 had a higher content of the amino acids Asp, Thr, Ala, Met, Leu, Ile, and Phe. The reduction or replacement of NaCl by KCl decreased sample firmness, whereas the addition of CaCl2 increased the hardness of the samples.

Tunable control of polyproline helix (PPII) structure via aromatic electronic effects: an electronic switch of polyproline helix.

Aromatic rings exhibit defined interactions via the unique aromatic π face. Aromatic amino acids interact favorably with proline residues via both the hydrophobic effect and aromatic–proline interactions, C−H/π interactions between the aromatic π face and proline ring C–H bonds. The canonical aromatic amino acids Trp, Tyr, and Phe strongly disfavor a polyproline helix (PPII) when they are present in proline-rich sequences because of the large populations of cis amide bonds induced by favorable aromatic–proline interactions (aromatic–cis-proline and proline–cis-proline–aromatic interactions). We demonstrate the ability to tune polyproline helix conformation and cis–trans isomerism in proline-rich sequences using aromatic electronic effects. Electron-rich aromatic residues strongly disfavor polyproline helix and exhibit large populations of cis amide bonds, while electron-poor aromatic residues exhibit small populations of cis amide bonds and favor polyproline helix. 4-Aminophenylalanine is a pH-dependent electronic switch of polyproline helix, with cis amide bonds favored as the electron-donating amine, but trans amide bonds and polyproline helix preferred as the electron-withdrawing ammonium. Peptides with block proline–aromatic PPXPPXPPXPP sequences exhibited electronically switchable pH-dependent structures. Electron-poor aromatic amino acids provide special capabilities to integrate aromatic residues into polyproline helices and to serve as the basis of aromatic electronic switches to change structure.

Effects of acivicin on growth, mycotoxin production and virulence of phytopathogenic fungi.

Acivicin is an inhibitor of γ-glutamyl transpeptidase and glutamine amidotransferase. When grown on a synthetic minimal agar medium, acivicin strongly inhibited the growth of Magnaporthe oryzae and Alternaria brassicicola, and to a lesser extent, Botrytis cinerea. However, only partial or marginal growth inhibition was observed with regard to Fusarium sporotrichioides and Fusarium graminearum. The growth retardation caused by acivicin was significantly alleviated by cultivating the fungus on a nutrient-rich medium. The inhibition of M. oryzae growth caused by 1 μmol l(-1) of acivicin on minimal agar medium was subdued by the addition of specific single amino acids, including His, a branched-chain amino acid (Leu, Ile or Val), an aromatic amino acid (Trp, Tyr or Phe), Met or Gln, at a concentration of 0·4 mmol l(-1). Trichothecene production by F. graminearum in trichothecene-inducing liquid medium was reduced significantly in the presence of acivicin despite its inability to inhibit growth in the trichothecene-inducing liquid medium. Foliar application of conidia in the presence of acivicin reduced the severity of rice blast disease caused by M. oryzae. These results suggest the usefulness of this modified amino acid natural product to mitigate agricultural problems caused by some phytopathogenic fungi. Significance and impact of the study: Fusarium head blight or scab disease and rice blast, caused by Fusarium graminearum and Magnaporthe oryzae, respectively, are major diseases of cereal crops that cause a significant loss of yield and deterioration in the quality of the grain. The present study investigated the effects of acivicin, a glutamine amino acid analog, on the physiology of various phytopathogenic fungi. Application of acivicin to a fungal culture and conidial suspension reduced mycotoxin production by the wheat scab fungus and the severity of rice blast, respectively. These results suggest the possibility that acivicin may serve as a lead compound to develop agricultural chemicals for the control of some plant diseases.

The gluconeogenesis pathway is involved in maintenance of enterohaemorrhagic Escherichia coli O157:H7 in bovine intestinal content.

Enterohaemorrhagic Escherichia coli (EHEC) are responsible for outbreaks of food- and water-borne illness. The bovine gastrointestinal tract (GIT) is thought to be the principle reservoir of EHEC. Knowledge of the nutrients essential for EHEC growth and survival in the bovine intestine may help in developing strategies to limit their shedding in bovine faeces thus reducing the risk of human illnesses. To identify specific metabolic pathways induced in the animal GIT, the transcriptome profiles of EHEC O157:H7 EDL933 during incubation in bovine small intestine contents (BSIC) and minimal medium supplemented with glucose were compared. The transcriptome analysis revealed that genes responsible for the assimilation of ethanolamine, urea, agmatine and amino acids (Asp, Thr, Gly, Ser and Trp) were strongly up-regulated suggesting that these compounds are the main nitrogen sources for EHEC in BSIC. A central role for the gluconeogenesis pathway and assimilation of gluconeogenic substrates was also pinpointed in EHEC incubated in BSIC. Our results suggested that three amino acids (Asp, Ser and Trp), glycerol, glycerol 3-phosphate, L-lactate and C4-dicarboxylates are important carbon sources for EHEC in BSIC. The ability to use gluconeogenic substrates as nitrogen sources (amino acids) and/or carbon sources (amino acids, glycerol and lactate) may provide a growth advantage to the bacteria in intestinal fluids. Accordingly, aspartate (2.4 mM), serine (1.9 mM), glycerol (5.8 mM) and lactate (3.6 mM) were present in BSIC and may represent the main gluconeogenic substrates potentially used by EHEC. A double mutant of E. coli EDL933 defective for phosphoenolpyruvate synthase (PpsA) and phosphoenolpyruvate carboxykinase (PckA), unable to utilize tricarboxylic acid (TCA) intermediates was constructed. Growth competition experiments between EHEC EDL933 and the isogenic mutant strain in BSIC clearly showed a significant competitive growth advantage of the wild-type strain further illustrating the importance of the gluconeogenesis pathway in maintaining EHEC in the bovine GIT.

Transcriptome profiling and physiological studies reveal a major role for aromatic amino acids in mercury stress tolerance in rice seedlings.

Mercury (Hg) is a serious environmental pollution threat to the planet. The accumulation of Hg in plants disrupts many cellular-level functions and inhibits growth and development, but the mechanism is not fully understood. To gain more insight into the cellular response to Hg, we performed a large-scale analysis of the rice transcriptome during Hg stress. Genes induced with short-term exposure represented functional categories of cell-wall formation, chemical detoxification, secondary metabolism, signal transduction and abiotic stress response. Moreover, Hg stress upregulated several genes involved in aromatic amino acids (Phe and Trp) and increased the level of free Phe and Trp content. Exogenous application of Phe and Trp to rice roots enhanced tolerance to Hg and effectively reduced Hg-induced production of reactive oxygen species. Hg induced calcium accumulation and activated mitogen-activated protein kinase. Further characterization of the Hg-responsive genes we identified may be helpful for better understanding the mechanisms of Hg in plants.

FKBP12.6 Activates RyR1: Investigating the Amino Acid Residues Critical for Channel Modulation

We have previously shown that FKBP12 associates with RyR2 in cardiac muscle and that it modulates RyR2 function differently to FKBP12.6. We now investigate how these proteins affect the single-channel behavior of RyR1 derived from rabbit skeletal muscle. Our results show that FKBP12.6 activates and FKBP12 inhibits RyR1. It is likely that both proteins compete for the same binding sites on RyR1 because channels that are preactivated by FKBP12.6 cannot be subsequently inhibited by FKBP12. We produced a mutant FKBP12 molecule (FKBP12E31Q/D32N/W59F) where the residues Glu31, Asp32, and Trp59 were converted to the corresponding residues in FKBP12.6. With respect to the functional regulation of RyR1 and RyR2, the FKBP12E31Q/D32N/W59F mutant lost all ability to behave like FKBP12 and instead behaved like FKBP12.6. FKBP12E31Q/D32N/W59F activated RyR1 but was not capable of activating RyR2. In conclusion, FKBP12.6 activates RyR1, whereas FKBP12 activates RyR2 and this selective activator phenotype is determined within the amino acid residues Glu31, Asp32, and Trp59 in FKBP12 and Gln31, Asn32, and Phe59 in FKBP12.6. The opposing but different effects of FKBP12 and FKBP12.6 on RyR1 and RyR2 channel gating provide scope for diversity of regulation in different tissues.