Electrochemical metal ion sensors. Exploiting amino acids and peptides as recognition elements

Amino acids and peptides are known to bind metal ions, in some cases very strongly. There are only a few examples of exploiting this binding in sensors. The review covers the current literature on the interaction of peptides and metals and the electrochemistry of bound metal ions. Peptides may be covalently attached to surfaces. Of particular interest is the attachment to gold via sulfur linkages. Sulfur-containing peptides (eg cysteine) may be adsorbed directly, while any amino group can be covalently attached to a carboxylic acid-terminated thiol. Once at a surface, the possibility for using the attached peptide as a sensor for metal ions becomes realised. Results from the authors&rsquo; laboratory and elsewhere have shown the potential for selective monitoring of metal ions at ppt levels. Examples of the use of poly-aspartic acid and the copper binding peptide Gly-Gly-His for detecting copper ions are given.<br /

Light-Triggered Myosin Activation for Probing Dynamic Cellular Processes

Shining light on myosin: The incorporation of a caging group onto the essential phosphoserine residue of myosin by protein semisynthesis enables light-triggered activation of the protein (see picture). Caging eliminates the myosin activity, but exposure to 365 nm light restores its function to native levels. The caged protein can also be introduced into cells to facilitate studies of myosin with precise spatial and temporal resolution.American Heart Association (Fellowship)National Institutes of Health (U.S.) (NIH Cell Migration Consortium (GM064346))National Institute of General Medical Sciences (U.S.) (Biotechnology Training Grant

Relationships between Root Pathogen Resistance, Abundance and Expression of Pseudomonas Antimicrobial Genes, and Soil Properties in Representative Swiss Agricultural Soils

Strains of Pseudomonas that produce antimicrobial metabolites and control soilborne plant diseases have often been isolated from soils defined as disease-suppressive, i.e., soils, in which specific plant pathogens are present, but plants show no or reduced disease symptoms. Moreover, it is assumed that pseudomonads producing antimicrobial compounds such as 2,4-diacetylphloroglucinol (DAPG) or phenazines (PHZ) contribute to the specific disease resistance of suppressive soils. However, pseudomonads producing antimicrobial metabolites are also present in soils that are conducive to disease. Currently, it is still unknown whether and to which extent the abundance of antimicrobials-producing pseudomonads is related to the general disease resistance of common agricultural soils. Moreover, virtually nothing is known about the conditions under which pseudomonads express antimicrobial genes in agricultural field soils. We present here results of the first side-by-side comparison of 10 representative Swiss agricultural soils with a cereal-oriented cropping history for (i) the resistance against two soilborne pathogens, (ii) the abundance of Pseudomonas bacteria harboring genes involved in the biosynthesis of the antimicrobials DAPG, PHZ, and pyrrolnitrin on roots of wheat, and (iii) the ability to support the expression of these genes on the roots. Our study revealed that the level of soil disease resistance strongly depends on the type of pathogen, e.g., soils that are highly resistant to Gaeumannomyces tritici often are highly susceptible to Pythium ultimum and vice versa. There was no significant correlation between the disease resistance of the soils, the abundance of Pseudomonas bacteria carrying DAPG, PHZ, and pyrrolnitrin biosynthetic genes, and the ability of the soils to support the expression of the antimicrobial genes. Correlation analyses indicated that certain soil factors such as silt, clay, and some macro- and micronutrients influence both the abundance and the expression of the antimicrobial genes. Taken together, the results of this study suggests that pseudomonads producing DAPG, PHZ, or pyrrolnitrin are present and abundant in Swiss agricultural soils and that the soils support the expression of the respective biosynthetic genes in these bacteria to various degrees. The precise role that these pseudomonads play in the general disease resistance of the investigated agricultural soils remains elusive

Accelerated and Improved Quantification of Lymphocytic Choriomeningitis Virus (LCMV) Titers by Flow Cytometry

Lymphocytic choriomeningitis virus (LCMV), a natural murine pathogen, is a member of the Arenavirus family, may cause atypical meningitis in humans, and has been utilized extensively as a model pathogen for the study of virus-induced disease and immune responses. Historically, viral titers have been quantified by a standard plaque assay, but for non-cytopathic viruses including LCMV this requires lengthy incubation, so results cannot be obtained rapidly. Additionally, due to specific technical constraints of the plaque assay including the visual detection format, it has an element of subjectivity along with limited sensitivity. In this study, we describe the development of a FACS-based assay that utilizes detection of LCMV nucleoprotein (NP) expression in infected cells to determine viral titers, and that exhibits several advantages over the standard plaque assay. We show that the LCMV-NP FACS assay is an objective and reproducible detection method that requires smaller sample volumes, exhibits a ∼20-fold increase in sensitivity to and produces results three times faster than the plaque assay. Importantly, when applied to models of acute and chronic LCMV infection, the LCMV-NP FACS assay revealed the presence of infectious virus in samples that were determined to be negative by plaque assay. Therefore, this technique represents an accelerated, enhanced and objective alternative method for detection of infectious LCMV that is amenable to adaptation for other viral infections as well as high throughput diagnostic platforms

In Situ Photoactivation of a Caged Phosphotyrosine Peptide Derived from Focal Adhesion Kinase Temporarily Halts Lamellar Extension of Single Migrating Tumor Cells

Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, mediates integrin-based cell signaling by transferring signals regulating cell migration, adhesion, and survival from the extracellular matrix to the cytoplasm. Following autophosphorylation at tyrosine 397, FAK binds the Src homology 2 domains of Src and phosphoinositide 3-kinase, among several other possible binding partners. To further investigate the role of phosphorylated FAK in cell migration in situ, peptides comprising residues 391-406 of human FAK with caged phosphotyrosine 397 were synthesized. Although the caged phosphopeptides were stable to phosphatase activity, the free phosphopeptides showed a half-life of approximately 10-15 min in cell lysates. Migrating NBT-II cells (a rat bladder tumor cell line) were microinjected with the caged FAK peptide and locally photoactivated using a focused laser beam. The photoactivation of caged FAK peptide in 8-microm diameter spots over the cell body led to the temporary arrest of the leading edge migration within approximately 1 min of irradiation. In contrast, cell body migration was not inhibited. Microinjection of a non-caged phosphorylated tyrosine 397 FAK peptide into migrating NBT-II cells also led to lamellar arrest; however, this approach lacks the temporal control afforded by the caged phosphopeptides. Photoactivation of related phosphotyrosine peptides with altered sequences did not result in transient lamellar arrest. We hypothesize that the phosphorylated FAK peptide competes with the endogenous FAK for binding to FAK effectors including, but not limited to, Src and phosphoinositide 3-kinase, causing spatiotemporal misregulation and subsequent lamellar arrest

Biochemical evidence for an alternate pathway in N-linked glycoprotein biosynthesis

Asparagine-linked glycosylation is a complex protein modification conserved among all three domains of life. Herein we report the in vitro analysis of N-linked glycosylation from the methanogenic archaeon Methanococcus voltae. Using a suite of synthetic and semisynthetic substrates, we show that AglK initiates N-linked glycosylation in M. voltae through the formation of α-linked dolichyl monophosphate N-acetylglucosamine, which contrasts with the polyprenyl diphosphate intermediates that feature in both eukaryotes and bacteria. Notably, AglK has high sequence homology to dolichyl phosphate β-glucosyltransferases, including Alg5 in eukaryotes, suggesting a common evolutionary origin. The combined action of the first two enzymes, AglK and AglC, afforded an α-linked dolichyl monophosphate glycan that serves as a competent substrate for the archaeal oligosaccharyl transferase AglB. These studies provide what is to our knowledge the first biochemical evidence revealing that, despite the apparent similarity of the overall pathways, there are actually two general strategies to achieve N-linked glycoproteins across the domains of life.National Institutes of Health (U.S.) (Grant GM039334