Comparison of PCR, electrochemical enzyme-linked immunosorbent assays, and the standard culture method for detecting salmonella in meat products

An electrochemical enzyme-linked immunosorbent assay (ELISA) coupled with flow injection analysis (ELISA-FIA) and a PCR-based method using ST11 and ST15 primers for detecting salmonellae in meat were evaluated in comparison with the International Organization for Standardization (ISO) culture method. The methods were applied to experimentally contaminated and naturally contaminated meat samples. The results showed that both ELISA-FIA and PCR allowed detection of salmonella in a product contaminated with a low number of the microorganisms (1 to 10 salmonellae/25 g) after only 5 h of incubation of preenrichment broth, and they were just as effective as the ISO method.     

Activity of LKB1 and AMPK-related kinases in skeletal muscle: effects of contraction, phenformin, and AICAR

Activation of AMP-activated protein kinase (AMPK) by exercise and metformin is beneficial for the treatment of type 2 diabetes. We recently found that, in cultured cells, the LKB1 tumor suppressor protein kinase activates AMPK in response to the metformin analog phenformin and the AMP mimetic drug 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). We have also reported that LKB1 activates 11 other AMPK-related kinases. The activity of LKB1 or the AMPK-related kinases has not previously been studied in a tissue with physiological relevance to diabetes. In this study, we have investigated whether contraction, phenformin, and AICAR influence LKB1 and AMPK-related kinase activity in rat skeletal muscle. Contraction in situ, induced via sciatic nerve stimulation, significantly increased AMPKalpha2 activity and phosphorylation in multiple muscle fiber types without affecting LKB1 activity. Treatment of isolated skeletal muscle with phenformin or AICAR stimulated the phosphorylation and activation of AMPKalpha1 and AMPKalpha2 without altering LKB1 activity. Contraction, phenformin, or AICAR did not significantly increase activities or expression of the AMPK-related kinases QSK, QIK, MARK2/3, and MARK4 in skeletal muscle. The results of this study suggest that muscle contraction, phenformin, or AICAR activates AMPK by a mechanism that does not involve direct activation of LKB1. They also suggest that the effects of excercise, phenformin, and AICAR on metabolic processes in muscle may be mediated through activation of AMPK rather than activation of LKB1 or the AMPK-related kinases.     

Structural determinants involved in the activation and regulation of G protein-coupled receptors: lessons from the alpha1-adrenegic receptor subtypes

The aim of a large number of studies on G protein-coupled receptors was centered on understanding the structural basis of their main functional properties. Here, we will briefly review the results obtained on the alpha1-adrenergic receptor subtypes belonging to the rhodopsin-like family of receptors. These findings contribute, on the one hand, to further understand the molecular basis of adrenergic transmission and, on the other, to provide some generalities on the structure-functional relationship of G protein-coupled receptors.     

Structural insights into the regulation of PDK1 by phosphoinositides and inositol phosphates

3-phosphoinositide-dependent protein kinase-1 (PDK1) phosphorylates and activates many kinases belonging to the AGC subfamily. PDK1 possesses a C-terminal pleckstrin homology (PH) domain that interacts with PtdIns(3,4,5)P3/PtdIns(3,4)P2 and with lower affinity to PtdIns(4,5)P2. We describe the crystal structure of the PDK1 PH domain, in the absence and presence of PtdIns(3,4,5)P3 and Ins(1,3,4,5)P4. The structures reveal a 'budded' PH domain fold, possessing an N-terminal extension forming an integral part of the overall fold, and display an unusually spacious ligand-binding site. Mutagenesis and lipid-binding studies were used to define the contribution of residues involved in phosphoinositide binding. Using a novel quantitative binding assay, we found that Ins(1,3,4,5,6)P5 and InsP6, which are present at micromolar levels in the cytosol, interact with full-length PDK1 with nanomolar affinities. Utilising the isolated PDK1 PH domain, which has reduced affinity for Ins(1,3,4,5,6)P5/InsP6, we perform localisation studies that suggest that these inositol phosphates serve to anchor a portion of cellular PDK1 in the cytosol, where it could activate its substrates such as p70 S6-kinase and p90 ribosomal S6 kinase that do not interact with phosphoinositides.     

Synthesis and GABA receptor potency of 3-thiomethyl-4-(hetero)aryl-5-amino-1-phenylpyrazoles

A convenient synthetic route to novel 4-arylpyrazoles is described. The potential for insecticidal activity through GABA channel blockage by this series of compounds, as well as their selectivity for insect versus mammalian receptors, are explored through in vitro and in vivo assays.     

A common open representation of mass spectrometry data and its application to proteomics research

A broad range of mass spectrometers are used in mass spectrometry (MS)-based proteomics research. Each type of instrument possesses a unique design, data system and performance specifications, resulting in strengths and weaknesses for different types of experiments. Unfortunately, the native binary data formats produced by each type of mass spectrometer also differ and are usually proprietary. The diverse, nontransparent nature of the data structure complicates the integration of new instruments into preexisting infrastructure, impedes the analysis, exchange, comparison and publication of results from different experiments and laboratories, and prevents the bioinformatics community from accessing data sets required for software development. Here, we introduce the 'mzXML' format, an open, generic XML (extensible markup language) representation of MS data. We have also developed an accompanying suite of supporting programs. We expect that this format will facilitate data management, interpretation and dissemination in proteomics research.     

Crystal structure of MO25 alpha in complex with the C terminus of the pseudo kinase STE20-related adaptor

Mouse protein 25 alpha (MO25 alpha) is a 40-kDa protein that, together with the STE20-related adaptor-alpha (STRAD alpha) pseudo kinase, forms a regulatory complex capable of stimulating the activity of the LKB1 tumor suppressor protein kinase. The latter is mutated in the inherited Peutz-Jeghers cancer syndrome (PJS). MO25 alpha binds directly to a conserved Trp-Glu-Phe sequence at the STRAD alpha C terminus, markedly enhancing binding of STRAD alpha to LKB1 and increasing LKB1 catalytic activity. The MO25 alpha crystal structure reveals a helical repeat fold, distantly related to the Armadillo proteins. A complex with the STRAD alpha peptide reveals a hydrophobic pocket that is involved in a unique and specific interaction with the Trp-Glu-Phe motif, further supported by mutagenesis studies. The data represent a first step toward structural analysis of the LKB1-STRAD-MO25 complex, and suggests that MO25 alpha is a scaffold protein to which other regions of STRAD-LKB1, cellular LKB1 substrates or regulatory components could bind.     

Vitamin E or coenzyme Q10 administration is not fully advantageous for heart mitochondrial function in diabetic goto kakizaki rats

The heart is one of the organs affected during the later stages of diabetes. Mitochondrial function has already been proposed to be affected during the course of diabetes. Nevertheless, little information is known concerning the impact of antioxidants in heart mitochondria of a milder model for diabetes, such as the Goto-Kakizaki (GK) rat, where mitochondrial function appears ameliorated. The objective of this work was to test if injections of Vitamin E and Coenzyme Q10, alone and in combination, were able to modify mitochondrial performance in the hearts of GK rats. Several aspects of mitochondrial function were measured, such as the respiratory control ratio and the electric potential, as well as the mitochondrial accumulation of Vitamin E and Coenzymes Q9 and Q10. We observed that only Vitamin E appeared to have a positive impact on the mitochondrial phosphorylation efficiency and on mitochondrial performance, namely on the ability to generate the electric transmembrane potential in the presence of supra-physiological calcium concentrations. Vitamin E administration also increased the mitochondrial concentration of Coenzyme Q10. None of the treatments was able to reverse the diabetic phenotype in GK rats. We conclude that in this model of mild hyperglycemia, administration of antioxidants may have a marginal positive impact on mitochondrial function.     

FAPPs control Golgi-to-cell-surface membrane traffic by binding to ARF and PtdIns(4)P

The molecular mechanisms underlying the formation of carriers trafficking from the Golgi complex to the cell surface are still ill-defined; nevertheless, the involvement of a lipid-based machinery is well established. This includes phosphatidylinositol 4-phosphate (PtdIns(4)P), the precursor for phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). In yeast, PtdIns(4)P exerts a direct role, however, its mechanism of action and its targets in mammalian cells remain uncharacterized. We have identified two effectors of PtdIns(4)P, the four-phosphate-adaptor protein 1 and 2 (FAPP1 and FAPP2). Both proteins localize to the trans-Golgi network (TGN) on nascent carriers, and interact with PtdIns(4)P and the small GTPase ADP-ribosylation factor (ARF) through their plekstrin homology (PH) domain. Displacement or knockdown of FAPPs inhibits cargo transfer to the plasma membrane. Moreover, overexpression of FAPP-PH impairs carrier fission. Therefore, FAPPs are essential components of a PtdIns(4)P- and ARF-regulated machinery that controls generation of constitutive post-Golgi carriers.