Diagnostic Real-Time PCR for Detection of Salmonella in Food

A robust 5′ nuclease (TaqMan) real-time PCR was developed and validated in-house for the specific detection of Salmonella in food. The assay used specifically designed primers and a probe target within the ttrRSBCA locus, which is located near the Salmonella pathogenicity island 2 at centisome 30.5. It is required for tetrathionate respiration in Salmonella. The assay correctly identified all 110 Salmonella strains and 87 non-Salmonella strains tested. An internal amplification control, which is coamplified with the same primers as the Salmonella DNA, was also included in the assay. The detection probabilities were 70% when a Salmonella cell suspension containing 10³ CFU/ml was used as a template in the PCR (5 CFU per reaction) and 100% when a suspension of 10⁴ CFU/ml was used. A pre-PCR sample preparation protocol including a preenrichment step in buffered peptone water followed by DNA extraction-purification was applied when 110 various food samples (chicken rinses, minced meat, fish, and raw milk) were investigated for Salmonella. The diagnostic accuracy was shown to be 100% compared to the traditional culture method. The overall analysis time of the PCR method was approximately 24 h, in contrast to 4 to 5 days of analysis time for the traditional culture method. This methodology can contribute to meeting the increasing demand of quality assurance laboratories for standard diagnostic methods. Studies are planned to assess the interlaboratory performance of this diagnostic PCR method.     

AtPTR1 and AtPTR5 transport dipeptides in planta

Transporters for di- and tripeptides belong to the large and poorly characterized PTR/NRT1 (peptide transporter/nitrate transporter 1) family. A new member of this gene family, AtPTR5, was isolated from Arabidopsis (Arabidopsis thaliana). Expression of AtPTR5 was analyzed and compared with tissue specificity of the closely related AtPTR1 to discern their roles in planta. Both transporters facilitate transport of dipeptides with high affinity and are localized at the plasma membrane. Mutants, double mutants, and overexpressing lines were exposed to several dipeptides, including toxic peptides, to analyze how the modified transporter expression affects pollen germination, growth of pollen tubes, root, and shoot. Analysis of atptr5 mutants and AtPTR5-overexpressing lines showed that AtPTR5 facilitates peptide transport into germinating pollen and possibly into maturating pollen, ovules, and seeds. In contrast, AtPTR1 plays a role in uptake of peptides by roots indicated by reduced nitrogen (N) levels and reduced growth of atptr1 mutants on medium with dipeptides as the sole N source. Furthermore, overexpression of AtPTR5 resulted in enhanced shoot growth and increased N content. The function in peptide uptake was further confirmed with toxic peptides, which inhibited growth. The results show that closely related members of the PTR/NRT1 family have different functions in planta. This study also provides evidence that the use of organic N is not restricted to amino acids, but that dipeptides should be considered as a N source and transport form in plants.     

Tyrosine-rich conopeptides affect voltage-gated K+ channels

Two venom peptides, CPY-Pl1 (EU000528) and CPY-Fe1 (EU000529), characterized from the vermivorous marine snails Conus planorbis and Conus ferrugineus, define a new class of conopeptides, the conopeptide Y (CPY) family. The peptides have no disulfide cross-links and are 30 amino acids long; the high content of tyrosine is unprecedented for any native gene product. The CPY peptides were chemically synthesized and shown to be biologically active upon injection into both mice and Caenorhabditis elegans; activity on mammalian Kv1 channel isoforms was demonstrated using an oocyte heterologous expression system, and selectivity for Kv1.6 was found. NMR spectroscopy revealed that the peptides were unstructured in aqueous solution; however, a helical region including residues 12-18 for one peptide, CPY-Pl1, formed in trifluoroethanol buffer. Clones obtained from cDNA of both species encoded prepropeptide precursors that shared a unique signal sequence, indicating that these peptides are encoded by a novel gene family. This is the first report of tyrosine-rich bioactive peptides in Conus venom.     

Simplified screening for the detection of soluble fusion constructs expressed in <Emphasis Type="Italic">E. coli</Emphasis> using a modular set of vectors

Background  

The solubility of recombinant proteins expressed in bacteria is often disappointingly low. Several strategies have been developed to improve the yield and one of the most common strategies is the fusion of the target protein with a suitable partner. Despite several reports on the successful use of each of these carriers to increase the solubility of some recombinant proteins, none of them was always successful and a combinatorial approach seems more efficient to identify the optimal combination for a specific protein. Therefore, the efficiency of an expression system critically depends on the speed in the identification of the optimal combination for the suitable fusion candidate in a screening process. This paper describes a set of expression vectors (pETM) designed for rapid subcloning, expression and subsequent purification using immobilized metal affinity chromatography (IMAC).     

The zinc finger of the CSN-associated deubiquitinating enzyme USP15 is essential to rescue the E3 ligase Rbx1

The COP9 signalosome (CSN) is a conserved protein complex found in all eukaryotic cells and involved in the regulation of the ubiquitin (Ub)/26S proteasome system. It binds numerous proteins, including the Ub E3 ligases and the deubiquitinating enzyme Ubp12p, the S. pombe ortholog of human USP15. We found that USP15 copurified with the human CSN complex. Isolated CSN complex exhibited protease activity that deubiquitinated poly-Ub substrates and was completely inhibited by o-phenanthroline (OPT), a metal-chelating agent. Surprisingly, the recombinant USP15 was also not able to cleave isopeptide bonds of poly-Ub chains in presence of OPT. Detailed analysis of USP sequences led to the discovery of a novel zinc (Zn) finger in USP15 and related USPs. Mutation of a single conserved cysteine residue in the predicted Zn binding motif resulted in the loss of USP15 capability to degrade poly-Ub substrates, indicating that the Zn finger is essential for the cleavage of poly-Ub chains. Moreover, pulldown experiments demonstrated diminished binding of tetra-Ub to mutated USP15. Cotransfection of USP15 and the Ub ligase Rbx1 revealed that the wild-type deubiquitinating enzyme, but not the USP15 mutant with a defective Zn finger, stabilized Rbx1 toward the Ub system, most likely by reversing poly/autoubiquitination. In summary, a functional Zn finger of USP15 is needed to maintain a conformation essential for disassembling poly-Ub chains, a prerequisite for rescuing the E3 ligase Rbx1.     

Akt activates the mammalian target of rapamycin by regulating cellular ATP level and AMPK activity

The serine/threonine kinase Akt is an upstream positive regulator of the mammalian target of rapamycin (mTOR). However, the mechanism by which Akt activates mTOR is not fully understood. The known pathway by which Akt activates mTOR is via direct phosphorylation and inhibition of tuberous sclerosis complex 2 (TSC2), which is a negative regulator of mTOR. Here we establish an additional pathway by which Akt inhibits TSC2 and activates mTOR. We provide for the first time genetic evidence that Akt regulates intracellular ATP level and demonstrate that Akt is a negative regulator of the AMP-activated protein kinase (AMPK), which is an activator of TSC2. We show that in Akt1/Akt2 DKO cells AMP/ATP ratio is markedly elevated with concomitant increase in AMPK activity, whereas in cells expressing activated Akt there is a dramatic decrease in AMP/ATP ratio and a decline in AMPK activity. Currently, the Akt-mediated phosphorylation of TSC2 and the inhibition of AMPK-mediated phosphorylation of TSC2 are viewed as two separate pathways, which activate mTOR. Our results demonstrate that Akt lies upstream of these two pathways and induces full inhibition of TSC2 and activation of mTOR both through direct phosphorylation and by inhibition of AMPK-mediated phosphorylation of TSC2. We propose that the activation of mTOR by Akt-mediated cellular energy and inhibition of AMPK is the predominant pathway by which Akt activates mTOR in vivo.     

CD70 signaling is critical for CD28-independent CD8+ T cell-mediated alloimmune responses in vivo

The inability to reproducibly induce robust and durable transplant tolerance using CD28-B7 pathway blockade is in part related to the persistence of alloreactive effector/memory CD8(+) T cells that are less dependent on this pathway for their cellular activation. We studied the role of the novel T cell costimulatory pathway, CD27-CD70, in alloimmunity in the presence and absence of CD28-B7 signaling. CD70 blockade prolonged survival of fully mismatched vascularized cardiac allografts in wild-type murine recipients, and in CD28-deficient mice induced long-term survival while significantly preventing the development of chronic allograft vasculopathy. CD70 blockade had little effect on CD4(+) T cell function but prevented CD8(+) T cell-mediated rejection, inhibited the proliferation and activation of effector CD8(+) T cells, and diminished the expansion of effector and memory CD8(+) T cells in vivo. Thus, the CD27-CD70 pathway is critical for CD28-independent effector/memory CD8(+) alloreactive T cell activation in vivo. These novel findings have important implications for the development of transplantation tolerance-inducing strategies in primates and humans, in which CD8(+) T cell depletion is currently mandatory.     

Kinetic mechanism and catalysis of a native-state prolyl isomerization reaction

Folding of tendamistat is a rapid two-state process for the majority of the unfolded molecules. In fluorescence-monitored refolding kinetics about 8% of the unfolded molecules fold slowly (lambda=0.083s(-1)), limited by peptidyl-prolyl cis-trans isomerization. This is significantly less than expected from the presence of three trans prolyl-peptide bonds in the native state. In interrupted refolding experiments we detected an additional very slow folding reaction (lambda=0.008s(-1) at pH 2) with an amplitude of about 12%. This reaction is caused by the interconversion of a highly structured intermediate to native tendamistat. The intermediate has essentially native spectroscopic properties and about 2% of it remain populated in equilibrium after folding is complete. Catalysis by human cyclophilin 18 identifies this very slow reaction as a prolyl isomerization reaction. This shows that prolyl-isomerases are able to efficiently catalyze native state isomerization reactions, which allows them to influence biologically important regulatory conformational transitions. Folding kinetics of the proline variants P7A, P9A, P50A and P7A/P9A show that the very slow reaction is due to isomerization of the Glu6-Pro7 and Ala8-Pro9 peptide bonds, which are located in a region that makes strong backbone and side-chain interactions to both beta-sheets. In the P50A variant the very slow isomerization reaction is still present but native state heterogeneity is not observed any more, indicating a long-range destabilizing effect on the alternative native state relative to N. These results enable us to include all prolyl and non-prolyl peptide bond isomerization reactions in the folding mechanism of tendamistat and to characterize the kinetic mechanism and the energetics of a native-state prolyl isomerization reaction.     

Test for cooperativity in the early kinetic intermediate in lysozyme folding

During the folding of many proteins, collapsed globular states are formed prior to the native structure. The role of these states for the folding process has been widely discussed. Comparison with properties of synthetic homo and heteropolymers had suggested that the initial collapse represented a shift of the ensemble of unfolded conformations to more compact states without major energy barriers. We investigated the folding/unfolding transition of a collapsed state, which transiently populates early in lysozyme folding. This state forms within the dead-time of stopped-flow mixing and it has been shown to be significantly more compact and globular than the denaturant-induced unfolded state. We used the GdmCl-dependence of the dead-time signal change to characterize the unfolding transition of the burst phase intermediate. Fluorescence and far-UV CD give identical unfolding curves, arguing for a cooperative two-state folding/unfolding transition between unfolded and collapsed lysozyme. These results show that collapse leads to a distinct state in the folding process, which is separated from the ensemble of unfolded molecules by a significant energy barrier. NMR, fluorescence and small angle X-ray scattering data further show that some local interactions in unfolded lysozyme exist at denaturant concentrations above the coil-collapse transition. These interactions might play a crucial role in the kinetic partitioning between fast and slow folding pathways.     

Purification and characterization of an NADH oxidase from Eubacterium ramulus

An NADH oxidase from the strictly anaerobic Eubacterium ramuluswas purified to homogeneity. The enzyme is composed of two types of subunits with molecular masses of 40 and 30 kDa. The molecular mass of the native enzyme is 450 kDa according to gel filtration and PAGE analysis. Six to eight mol of FAD were found per mol of native enzyme. The NADH-specific enzyme was inhibited by N-bromosuccinimide and sulfhydryl reagents such as N-ethylmaleimide, CuCl(2) or ZnCl(2). The physiological function of the purified enzyme is unclear, but the demonstration of NADH-dependent O(2)-consumption suggests that it plays a role in the scavenging of oxygen.