Alternative N-terminal domains of PSD-95 and SAP97 govern activity-dependent regulation of synaptic AMPA receptor function

PSD-95 and SAP97 are scaffolding proteins that have been implicated in regulating AMPA receptor incorporation and function at synapses. Gain- and loss-of-function approaches, however, have generated conflicting results. To minimize adaptations during development and potential dominant-negative effects of overexpression, we have combined silencing of endogenous PSD-95 in mature neurons with heterologous expression of specific SAP97 or PSD-95 isoforms. We find that both PSD-95 and SAP97 contain alternative N termini expressing either double cysteines that normally are palmitoylated (alpha-isoforms) or an L27 domain (beta-isoforms). Whereas alpha-isoforms of PSD-95 and SAP97 influence AMPA receptor-mediated synaptic strength independent of activity, the effects of beta-isoforms are regulated by activity in a CaMKII-dependent manner. Importantly, the synaptic effects of the beta-isoforms are masked by the endogenous alpha-isoform of PSD-95. These results demonstrate that the different N termini of the predominant endogenous forms of PSD-95 (alpha-isoform) and SAP97 (beta-isoform) govern their role in regulating synaptic function.     

Enantiomer separation of amino acids in immunoaffinity micro LC-MS

Chiral immunoaffinity microbore columns were directly interfaced with MS detection, and the effect of column length and temperature on the enantiomer separation of a number of underivatized aromatic and aliphatic amino acids was investigated utilizing an antibody chiral stationary phase that had been prepared by immobilizing a monoclonal anti-D-amino acid antibody onto silica. The stronger affinity of the antibody towards aromatic and bulky amino acids allowed separation of such analytes in a 0.75 x 150 mm column, while an increase in column length enabled separation of more weakly bound compounds. The strength of interaction between chiral selector and analytes could be modulated conveniently by lowering the temperature. For the first time, simultaneous enantiomer separation of mixtures of amino acids was achieved on antibody-based chiral stationary phases using extracted ion chromatograms.     

Characterization of the divergent wound-healing responses occurring in the pathergy reaction and normal healthy volunteers

Beh?et's disease (BD) is a multisystem inflammatory disorder of unknown etiology characterized by recurrent oral and genital ulcerations and uveitis, with varying other manifestations associated with vascular inflammation. A unifying feature of BD inflammation is the skin pathergy reaction (SPR), a nonspecific tissue hyperreactivity to minor trauma involving epithelial disruption. This study compared skin responses to needle prick in BD patients and normal healthy volunteers. Two study groups, each consisting of 10 BD patients with SPR(+) and 6 controls, were evaluated using either immunohistochemistry or quantitative real-time PCR to measure inflammatory cell and cytokine levels in biopsy specimens obtained serially from independent sites at 0, 8, and 48 h after needle prick. We found similar cellular infiltration patterns in response to needle prick in BD patients and controls between 0 and 8 h. Further development of this immune response was limited in skin of normal control subjects, with stable or decreased inflammatory mediators observed at 48 h. In contrast, in BD-derived skin specimens, increased influxes of mature dendritic cells, monocytes, and lymphocytes, including T regulatory cells, were noted by 48 h. Similarly, increases in cytokines (IFN-gamma, IL-12 p40, IL-15), chemokines (MIP3-alpha, IP-10, Mig, and iTac), and adhesion molecules (ICAM-1, VCAM-1) were noted at 48 h in the skin of BD patients with SPR(+) but not in the skin of normal controls. These results suggest that, in contrast to the self-limited inflammation associated with epithelial disruption of normal skin, BD patients experience marked cellular influxes into the injury site, leading to an exaggerated lymphoid Th1-type response.     

Ferri-bacillibactin uptake and hydrolysis in Bacillus subtilis

Upon iron limitation, Bacillus subtilis secretes the catecholic trilactone (2,3-dihydroxybenzoate-glycine-threonine)3 siderophore bacillibactin (BB) for ferric iron scavenging. Here, we show that ferri-BB uptake is mediated by the FeuABC transporter and that YuiI, a novel trilactone hydrolase, catalyses ferri-BB hydrolysis leading to cytosolic iron release. Among several Fur-regulated ABC transport mutants, only DeltafeuABC exhibited impaired growth during iron starvation. Quantification of intra- and extracellular (ferri)-BB in iron-depleted DeltafeuABC cultures revealed a fourfold increase of the extracellular siderophore concentration, confirming a blocked ferri-BB uptake in the absence of FeuABC. Ferri-BB was found to bind selectively to the periplasmic binding protein FeuA (Kd = 57 +/- 1 nM), proving high-affinity transport of the iron-charged siderophore. During iron starvation, a DeltayuiI mutant displayed impaired growth and strong intracellular (30-fold) and extracellular (6.5-fold) (ferri)-BB accumulation. Kinetic studies in vitro revealed that YuiI hydrolyses both BB and ferri-BB. While BB hydrolysis led to strong accumulation of the tri- and dimeric reaction intermediates, ferri-BB hydrolysis yielded exclusively the monomeric reaction product and occurred with a 25-fold higher catalytic efficiency than BB single hydrolysis. Thus, ferri-BB was the preferred substrate of the YuiI esterase whose gene locus was designated besA.     

Isolation of targeted AAV2 vectors from novel virus display libraries

Random peptide ligands displayed on viral capsids are emerging tools for selection of targeted gene transfer vectors even without prior knowledge of the potential target cell receptor. We have previously introduced adeno-associated viral (AAV)-displayed peptide libraries that ensure encoding of displayed peptides by the packaged AAV genome. A major limitation of these libraries is their contamination with wild-type (wt) AAV. Here we describe a novel and improved library production system that reliably avoids generation of wt AAV by use of a synthetic cap gene. Selection of targeted AAV vectors from wt-containing and the novel wt-free libraries on cell types with different permissivity for wt AAV2 replication suggested the superiority of the wt-free library. However, from both libraries highly specific peptide sequence motifs were selected which improved transduction of cells with moderate or low permissivity for AAV2 replication. Strong reduction of HeLa cell transduction compared to wt AAV2 and only low level transduction of non-target cells by some selected clones showed that not only the efficiency but also the specificity of gene transfer was improved. In conclusion, our study validates and improves the unique potential of virus display libraries for the development of targeted gene transfer vectors.     

Evaluation of novel fluorogenic substrates for the detection of glycosidases in Escherichia coli and enterococci

AIMS: Enzyme substrates based on 4-methylumbelliferone are widely used for the detection of Escherichia coli and enterococci in water, by detection of beta-glucuronidase and beta-glucosidase activity respectively. This study aimed to synthesize and evaluate novel umbelliferone-based substrates with improved sensitivity for these two enzymes. METHODS AND RESULTS: A novel beta-glucuronide derivative based on 6-chloro-4-methylumbelliferone (CMUG) was synthesized and compared with 4-methylumbelliferyl-beta-D-glucuronide (MUG) using 42 strains of E. coli in a modified membrane lauryl sulfate broth. Over 7 h of incubation, the fluorescence generated from the hydrolysis of CMUG by E. coli was over twice that from MUG, and all of the 38 glucuronidase-positive strains generated a higher fluorescence with CMUG compared with MUG. Neither substrate caused inhibition of bacterial growth in any of the tested strains. Four beta-glucosidase substrates were also synthesized and evaluated in comparison with 4-methylumbelliferyl-beta-D-glucoside (MU-GLU) using 42 strains of enterococci in glucose azide broth. The four substrates comprised beta-glucoside derivatives of umbelliferone-3-carboxylic acid and its methyl, ethyl and benzyl esters. Glucosides of the methyl, ethyl and benzyl esters of umbelliferone-3-carboxylic acid, were found to be superior to MU-GLU for the detection of enterococci, especially after 18 h of incubation, while umbelliferone-3-carboxylic acid-beta-D-glucoside was inferior. However, the variability in detectable beta-glucosidase activity among the different strains of enterococci in short-term assays using the three carboxylate esters (7 h incubation) may compromise their use for rapid detection and enumeration of these faecal indicator bacteria. CONCLUSIONS: The beta-glucuronidase substrate CMUG appears to be a more promising detection system than the various beta-glucosidase substrates tested. SIGNIFICANCE AND IMPACT OF THE STUDY: The novel substrate CMUG showed enhanced sensitivity for the detection of beta-glucuronidase-producing bacteria such as E. coli, with a clear potential for application in rapid assays for the detection of this indicator organism in natural water and other environmental samples.     

High content cellular screening

Over the past few years, high content screening has firmly established itself as a high-throughput technology for the analysis of microscopy-based cellular assays. In particular, it has opened new areas of cell biology for the large-scale analysis of cellular phenotypes and has enabled the application of increasingly sophisticated assays for large-scale genetic and compound screening, benefiting both the academic and pharmaceutical research environment.     

Cisplatin mediates selective downstream hydrolytic cleavage of Met-(Gly)(n)-His segments (n=1,2) in methionine- and histidine-containing peptides: the role of ammine loss trans to the initial Pt-S(Met) anchor in facilitating amide hydrolysis

The pH- and time-dependent reactions of the antitumor drug cisplatin, cis-[PtCl(2)(NH(3))(2)], with the methionine- and histidine-containing pentapeptides Ac-Met-Gly-His-Gly-Gly-OH, Ac-Met-Gly-Gly-His-Gly-OH and Ac-Gly-Met-Gly-His-Gly-OH (Gly=glycyl, Met=L-methionyl, His=L-histidyl) at 313K have been investigated by high performance liquid chromatography, mass spectrometry and nuclear magnetic resonance. Cisplatin mediates a rapid "downstream" hydrolytic cleavage of the Met-Gly amide bond in weakly acid solution (pH < or =5) for all three peptides, leading to release of H-Gly-His-Gly-Gly-OH, H-Gly-Gly-His-Gly-OH and H-Gly-His-Gly-OH, respectively, and formation of kappa(2)S,N(M) chelate complexes of the methionine-containing residuals Ac-Met-OH or Ac-Gly-Met-OH. An alternative reaction pathway affords tridentate kappa(3)S,N(M),N(imidazole) macrochelates of the original pentapeptide following ammine loss. The downstream cleavage pathway is competitive with the likewise cisplatin-mediated upstream cleavage of the Ac-Gly linkage in the pentapeptide Ac-Gly-Met-Gly-His-Gly-OH. This leads to formation of both the kappa(3)S,N(M),N(G1) complex of H-Gly-Met-Gly-His-Gly-OH due to upstream cleavage and the analogous tridentate complex for H-Gly-Met-OH due to initial downstream loss of H-Gly-His-Gly-OH followed by upstream loss of acetic acid. As downstream cleavage is not observed for Ac-(Gly)(2)-Met-(Gly)(2)-OH under similar conditions, it may be concluded that rapid histidine imidazole substitution of the ammine ligand in trans-position to an anchoring methionine S atom must assist hydrolytic cleavage of the Met-Gly amide bond.     

Thermodynamic characterization of an engineered tetracycline-binding riboswitch

Riboswitches reflect a novel concept in gene regulation that is particularly suited for technological adaptation. Therefore, we characterized thermodynamically the ligand binding properties of a synthetic, tetracycline (tc)-binding RNA aptamer, which regulates gene expression in a dose-dependent manner when inserted into the untranslated region of an mRNA. In vitro, one molecule of tc is bound by one molecule of partially pre-structured and conformationally homogeneous apo-RNA. The dissociation constant of 770 pM, as determined by fluorimetry, is the lowest reported so far for a small molecule-binding RNA aptamer. Additional calorimetric analysis of RNA point mutants and tc derivatives identifies functional groups crucial for the interaction and including their respective enthalpic and entropic contributions we can propose detailed structural and functional roles for certain groups. The conclusions are consistent with mutational analyses in vivo and support the hypothesis that tc-binding reinforces the structure of the RNA aptamer, preventing the scanning ribosome from melting it efficiently.