Effects of food deprivation on refuge use and dispersal in juvenile North Sea houting Coregonus oxyrinchus under experimental conditions

This study tested the influence of energetic state on refuge use and dispersal in juvenile North Sea houting Coregonus oxyrinchus in an artificial stream. Food-deprived fish spent more time outside refuges than well-fed fish; however, the well-fed fish initiated dispersal faster than the food-deprived fish. The results may indicate state-dependent refuge use and dispersal in C. oxyrinchus.     

Characterization of a Glutamate Transporter Operon,glnQHMP, in Streptococcus mutans and Its Role in Acid Tolerance

Glutamate contributes to the acid tolerance response (ATR) of many Gram-negative and Gram-positive bacteria, but its role in the ATR of the oral bacterium Streptococcus mutans is unknown. This study describes the discovery and characterization of a glutamate transporter operon designated glnQHMP (Smu.1519 to Smu.1522) and investigates its potential role in acid tolerance. Deletion of glnQHMP resulted in a 95% reduction in transport of radiolabeled glutamate compared to the wild-type UA159 strain. The addition of glutamate to metabolizing UA159 cells resulted in an increased production of acidic end products, whereas the glnQHMP mutant produced less lactic acid than UA159, suggesting a link between glutamate metabolism and acid production and possible acid tolerance. To investigate this possibility, we conducted a microarray analysis with glutamate and under pH 5.5 and pH 7.5 conditions which showed that expression of the glnQHMP operon was downregulated by both glutamate and mild acid. We also measured the growth kinetics of UA159 and its glnQHMP-negative derivative at pH 5.5 and found that the mutant doubled at a much slower rate than the parent strain but survived at pH 3.5 significantly better than the wild type. Taken together, these findings support the involvement of the glutamate transporter operon glnQHMP in the acid tolerance response in S. mutans.Streptococcus mutans is 1 of over 700 bacterial species commonly found in the oral environment (1). Its ability to rapidly metabolize dietary carbohydrates to acid end products causes demineralization of the tooth enamel, leading to caries formation (19). Acidogenicity (the ability to produce acid end products via glycolysis) and aciduricity (the ability to survive and grow in acidic environments) are two important virulence factors of S. mutans. Maintenance of a pH gradient across the cell membrane by increasing intracellular pH by 0.5 to 1.0 relative to the extracellular pH (ΔpH) when exposed to a low pH environment is critical for the survival of S. mutans at low pH. This is primarily accomplished by acid-induced mechanisms that facilitate proton extrusion via the proton-translocating ATPase (5, 20) and by acid end product efflux (8, 12). S. mutans also possesses an acid tolerance response (ATR) mechanism, whereby preexposure to sublethal pH environments (e.g., pH 5.5) affords protection from killing under lethal pH values as low as pH 3.0 (7). This adaptive process is characterized by increased acid resistance (4), increased glycolytic capacities (20), and increased proton-translocating enzyme F₁F₀-ATPase activity (44). The ATR is enhanced by sugar starvation and the addition of amino acids (48), the addition of potassium ions (12), growth in biofilms, and activity of multiple two-component signal transduction systems that include the ComDE, HK11/RR11 (also designated LiaS/LiaR), VicKR, CiaHR, LevSR, ScnKR, and HK1037/RR1038 (6, 17, 31, 32, 46).Previously, Noji et al. and Sato et al. described a glutamate/aspartate transporter in S. mutans (38, 45). Those researchers showed that the presence of potassium ions was required for transport and that, in environments of pH 6.0 or below, the activity of the H⁺-ATPase system was required (38, 45). Potassium ions are the main cations in plaque (50), and potassium uptake is associated with intracellular pH homeostasis in S. mutans (24, 35). In addition, expression of several genes involved in the glutamate synthesis pathway (icd, citZ, and acn) are downregulated under low pH (10), suggesting a link between glutamate metabolism, potassium levels, and aciduricity in S. mutans. Since acid tolerance is an important virulence property of S. mutans, we aimed to investigate a possible link between glutamate uptake and acid resistance in this oral pathogen. In bacteria, intracellular glutamate and glutamine levels are closely linked with nitrogen metabolism of the cell. Glutamine is synthesized from glutamate and ammonium, which is a major way for cells to assimilate the nitrogen required for biosynthesis of all amino acids, thus affecting protein synthesis and the structural and functional integrity of the cell. Notably, nitrogen metabolism, especially glutamine metabolism, has been linked to virulence in a number of microorganisms, including Streptococcus pneumoniae (26, 42), Staphylococcus aureus (41), Candida albicans (33), and Pseudomonas aeruginosa (51). Glutamate uptake and metabolism are known to be involved in the ATR of Gram-negative bacteria such as Escherichia coli via the use of glutamate decarboxylase and the glutamate/gamma-amino butyrate (glutamate/GABA) antiporter (9). Similarly, the homologous proteins of these systems in Lactococcus lactis, encoded by the gadBC genes, were shown to assist in a glutamate-dependent acid-resistance mechanism in that Gram-positive bacterium (44).In this study, we searched the S. mutans UA159 genome for potential glutamine transporter operons. We constructed a deletion mutant (SmuGLT) of the glnQHMP operon (Smu.1519 to Smu.1522) and confirmed its role as a glutamate transporter. The inability of SmuGLT to take up glutamate resulted in a general growth deficiency, especially at pH 5.5, as well as an increased tolerance to acid. Results from this study provide insight into the ATR of S. mutans, including a potential link between glutamate metabolism and acid resistance in S. mutans.     

Manganese(II) complexes of di-2-pyridinylmethylene-1,2-diimine di-Schiff base ligands: Structures and reactivity

Manganese(II) complexes [Mn(L)X₂] were prepared and characterized, where L is a neutral di-Schiff base ligand incorporating pyridylimine donor arms, including (1R,2R)-N,N′-bis(2-pyridylmethylidene)-1,2-diphenylethylenediimine (L¹), (1R,2R)-N,N′-bis(6-methyl-2-pyridylmethylidene)-1,2-cyclohexyldiimine (L²), or (1R,2R)-, (1S,2S)- or racemic N,N′-bis(2-pyridylmethylidene)-1,2-cyclohexyldiimine (L³), and X⁻ =  or Cl⁻. Product complexes were structurally characterized, specifically including [Mn(R,R-L¹)(NCCH₃)₃](ClO₄)₂, [Mn(R,R-L²)(OH₂)₂](ClO₄)₂ and racemic [Mn(L³)Cl₂]. The first of these complexes features a heptacoordinate ligand field in a distorted pentagonal bipyramid, and the latter two are hexacoordinate, but retain equatorially monovacant pentagonal bipyramidal structures. Complexes [Mn(L³)X₂] (X⁻ = Cl⁻, ) were reacted with the primary phosphine FcCH₂PH₂ (Fc = -C₅H₄FeC₅H₅), H₂O and ethyldiazoacetate (EDA). The first two substrates prompted reactivity at a single ligand imine bond, resulting in hydrophosphination and hydrolysis, respectively. Complexes of the derivative ligands were also structurally characterized. Evidence for EDA activation was obtained by electrospray ionization mass spectrometry, but catalytic carbene transfer was not obtained.     

Synthesis, structural studies and solubility properties of zinc(II), nickel(II) and copper(II) complexes of bulky tris(triazolyl)borate ligands

Tris(triazolyl)borate (Ttz) ligands are sterically similar to tris(pyrazolyl)borate (Tp) but complexes of Ttz show improved solubility in water and alcohols due to their propensity for forming hydrogen bonds. Recently developed bulky tris(triazolyl)borate ligands can produce four and five coordinate transition metal complexes and serve as models for enzyme active sites in an aqueous environment. Herein we report the synthesis of such complexes, i.e. (Ttz^tBu,Me)ZnCl, (Ttz^tBu,Me)ZnBr, (Ttz^tBu,Me)NiCl, and (Ttz^tBu,Me)CuCl, which were analyzed by X-ray crystallographic and spectroscopic methods [Ttz^tBu,Me = tris(3-t-butyl-5-methyl-1,2,4-triazolyl)borate]. (Ttz^tBu,Me)ZnCl crystallizes as two different polymorphs with cubic and monoclinic symmetry. Both polymorphs of (Ttz^tBu,Me)ZnCl and (Ttz^tBu,Me)ZnBr have tetrahedral zinc atoms whereas the geometries at the metal in (Ttz^tBu,Me)NiCl and (Ttz^tBu,Me)CuCl are distorted tetrahedral. All complexes are methanol soluble and they also dissolve in methanol/water mixtures with up to 60% water.     

Analysis of posttranslational modifications of proteins by tandem mass spectrometry

Protein activity and turnover is tightly and dynamically regulated in living cells. Whereas the three-dimensional protein structure is predominantly determined by the amino acid sequence, posttranslational modification (PTM) of proteins modulates their molecular function and the spatial-temporal distribution in cells and tissues. Most PTMs can be detected by protein and peptide analysis by mass spectrometry (MS), either as a mass increment or a mass deficit relative to the nascent unmodified protein. Tandem mass spectrometry (MS/MS) provides a series of analytical features that are highly useful for the characterization of modified proteins via amino acid sequencing and specific detection of posttranslationally modified amino acid residues. Large-scale, quantitative analysis of proteins by MS/MS is beginning to reveal novel patterns and functions of PTMs in cellular signaling networks and biomolecular structures.     

Genetic ablation of zyxin causes Mena/VASP mislocalization, increased motility, and deficits in actin remodeling

Focal adhesions are specialized regions of the cell surface where integrin receptors and associated proteins link the extracellular matrix to the actin cytoskeleton. To define the cellular role of the focal adhesion protein zyxin, we characterized the phenotype of fibroblasts in which the zyxin gene was deleted by homologous recombination. Zyxin-null fibroblasts display enhanced integrin-dependent adhesion and are more migratory than wild-type fibroblasts, displaying reduced dependence on extracellular matrix cues. We identified differences in the profiles of 75- and 80-kD tyrosine-phosphorylated proteins in the zyxin-null cells. Tandem array mass spectrometry identified both modified proteins as isoforms of the actomyosin regulator caldesmon, a protein known to influence contractility, stress fiber formation, and motility. Zyxin-null fibroblasts also show deficits in actin stress fiber remodeling and exhibit changes in the molecular composition of focal adhesions, most notably by severely reduced accumulation of Ena/VASP proteins. We postulate that zyxin cooperates with Ena/VASP proteins and caldesmon to influence integrin-dependent cell motility and actin stress fiber remodeling.