Diagnostic application of padlock probes--multiplex detection of plant pathogens using universal microarrays

Padlock probes (PLPs) are long oligonucleotides, whose ends are complementary to adjacent target sequences. Upon hybridization to the target, the two ends are brought into contact, allowing PLP circularization by ligation. PLPs provide extremely specific target recognition, which is followed by universal amplification and microarray detection. Since target recognition is separated from downstream processing, PLPs enable the development of flexible and extendable diagnostic systems, targeting diverse organisms. To adapt padlock technology for diagnostic purposes, we optimized PLP design to ensure high specificity and eliminating ligation on non-target sequences under real-world assay conditions. We designed and tested 11 PLPs to target various plant pathogens at the genus, species and subspecies levels, and developed a prototype PLP-based plant health chip. Excellent specificity was demonstrated toward the target organisms. Assay background was determined for each hybridization using a no-target reference sample, which provided reliable and sensitive identification of positive samples. A sensitivity of 5 pg genomic DNA and a dynamic range of detection of 100 were observed. The developed multiplex diagnostic system was validated using genomic DNAs of characterized isolates and artificial mixtures thereof. The demonstrated system is adaptable to a wide variety of applications ranging from pest management to environmental microbiology.     

Suppression of protein kinase Cepsilon mediates 17beta-estradiol-induced neuroprotection in an immortalized hippocampal cell line

Although estrogens are neuroprotective in a variety of neuroprotection models, the precise underlying mechanisms are currently not well understood. Here, we examined the role of protein kinase C (PKC) in mediating estrogen-induced neuroprotection in the HT-22 immortalized hippocampal cell line. The neuroprotection model utilized calcein fluorescence to quantitate cell viability following glutamate insults. 17beta-Estradiol (betaE2) protected HT-22 cells when treatment was initiated before or after the glutamate insult. The inhibition of PKC by bis-indolylmaleimide mimicked and enhanced betaE2-induced neuroprotection. In contrast, the inhibition of specific PKC isozymes (alpha and beta) by Go6976, inhibition of 1-phosphatidylinositol 3 kinase by wortmannin, or inhibition of protein kinase A by H-89, did not alter cell viability, suggesting a specific involvement of PKC in an isozyme-dependent manner. We further examined whether estrogen interacts with PKC in a PKC isozyme-specific manner. Protein levels and activity of PKC isozymes (alpha, delta, epsilon, and zeta) were assessed by western blot analysis and radiolabeled phosphorylation assays respectively. Among the isozymes tested, betaE2 altered only PKCepsilon; it reduced the activity and membrane translocation of PKCepsilon in a manner that correlated with its protection against glutamate toxicity. Furthermore, betaE2 reversed the increased activity of membrane PKCepsilon induced by glutamate. These data suggest that the neuroprotective effects of estrogens are mediated in part by inhibition of PKCepsilon activity and membrane translocation.     

Adenovirus stimulates choline efflux by increasing expression of organic cation transporter-2

We examined the effect of wild-type human adenovirus (Ad5) on choline transport in murine lung epithelia (MLE) and in rodent primary alveolar type II cells. Cells were active in pH-sensitive, reversible transport of choline, a process blocked pharmacologically with phenoxybenzamine, an inhibitor of organic cation transporters (OCT). PCR products for the choline transporters, OCT-1 and OCT-2, were detected, but only OCT-2 protein was robustly expressed within MLE and primary alveolar epithelial cells. Ad5 produced a two- to threefold increase in choline efflux from cells, resulting in a significant reduction in intracellular choline content and its major product, phosphatidylcholine. Effects of Ad5 on choline efflux were inhibited with phenoxybenzamine, and choline efflux was attenuated by OCT-2 small interfering RNA. Adenovirus also produced a dose-dependent increase in immunoreactive OCT-2 levels concomitant with increased cellular OCT-2 steady-state mRNA. These results indicate that adenoviruses can significantly disrupt choline trafficking in lung epithelia by upregulating expression of an alveolar protein involved in organic cation transport.     

Identification of the cyclamate interaction site within the transmembrane domain of the human sweet taste receptor subunit T1R3

The artificial sweetener cyclamate tastes sweet to humans, but not to mice. When expressed in vitro, the human sweet receptor (a heterodimer of two taste receptor subunits: hT1R2 + hT1R3) responds to cyclamate, but the mouse receptor (mT1R2 + mT1R3) does not. Using mixed-species pairings of human and mouse sweet receptor subunits, we determined that responsiveness to cyclamate requires the human form of T1R3. Using chimeras, we determined that it is the transmembrane domain of hT1R3 that is required for the sweet receptor to respond to cyclamate. Using directed mutagenesis, we identified several amino acid residues within the transmembrane domain of T1R3 that determine differential responsiveness to cyclamate of the human versus mouse sweet receptors. Alanine-scanning mutagenesis of residues predicted to line a transmembrane domain binding pocket in hT1R3 identified six residues specifically involved in responsiveness to cyclamate. Using molecular modeling, we docked cyclamate within the transmembrane domain of T1R3. Our model predicts substantial overlap in the hT1R3 binding pockets for the agonist cyclamate and the inverse agonist lactisole. The transmembrane domain of T1R3 is likely to play a critical role in the interconversion of the sweet receptor from the ground state to the active state.     

Neurogenesis and neuronal communication on micropatterned neurochips

Neural networks are formed by accurate connectivity of neurons and glial cells in the brain. These networks employ a three-dimensional bio-surface that both assigns precise coordinates to cells during development and facilitates their connectivity and functionality throughout life. Using specific topographic and chemical features, we have taken steps towards the development of poly(dimethylsiloxane; PDMS) neurochips that can be used to generate and study synthetic neural networks. These neurochips have micropatterned structures that permit adequate cell positioning and support cell survival. Within days of plating, cells differentiate into neurons displaying excitability and communication, as evidenced by intracellular calcium oscillations and action potentials. The structural and functional capacities of such simple neural networks open up new opportunities to study synaptic communication and plasticity.     

Binding of cationic porphyrin to isolated DNA and nucleoprotein complex: quantitative analysis of binding forms under various experimental conditions

We studied the complex formation of tetrakis(4-N-methylpyridyl)porphyrin (TMPyP) with double stranded DNAs and T7 phage nucleoprotein complex. We analyzed the effect of base pair composition of DNA, the presence of capsid protein, and the composition of the microenvironment on the distribution of TMPyP between binding forms as determined by the decomposition of porphyrin absorption spectra. No difference was found in the amount of bound TMPyP between DNAs of various base compositions; however, the ratio of TMPyP binding forms depends on the AT/GC ratio. The presence of protein capsid opposes the binding of TMPyP to DNA. This behavior offers a possibility to investigate the protein capsid integrity due to the analysis of porphyrin binding. Increasing ionic strength of monovalent ions decreases the amount of bound porphyrin through the inhibition of intercalation, but does not influence the quantity of groove-binding forms when TMPyP interacts with isolated DNA. In the case of the nucleoprotein complex the groove-binding is also inhibited already at 140 mM ionic strength. The presence of 1 mM divalent cations (Mg(2+), Ca(2+), Cu(2+) and Ni(2+)) in a buffer solution of 70 mM ionic strength does not influence significantly the free to bound ration of TMPyP when it interacts with isolated DNA. The contribution of binding forms is remarkably different in Mg(2+)/Ca(2+) and Cu(2+)/Ni(2+) containing solutions. Transition metals significantly decrease the binding sites for intercalation in both DNA and nucleoprotein complex, but facilitate the groove-binding of TMPyP to isolated DNA.     

Structure of the O-polysaccharide of Proteus serogroup O34 containing 2-acetamido-2-deoxy-alpha-D-galactosyl phosphate

On mild acid degradation of the lipopolysaccharide of Proteus vulgaris O34, strain CCUG 4669, the O-polysaccharide was cleaved at a glycosyl-phosphate linkage that is present in the main chain. The resultant phosphorylated oligosaccharides and an alkali-treated lipopolysaccharide were studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, and the following structure of the branched tetrasaccharide phosphate repeating unit of the O-polysaccharide was established: [carbohydrate structure: see text]The O-polysaccharide of Proteus mirabilis strain TG 276 was found to have the same structure and, based on the structural and serological data, this strain was proposed to be classified into the same Proteus serogroup O34.     

Identification and characterization of structural domains of human ERp57: association with calreticulin requires several domains

The amino acid sequence of ERp57, which functions in the endoplasmic reticulum together with the lectins calreticulin and calnexin to achieve folding of newly synthesized glycoproteins, is highly similar to that of protein disulfide isomerase (PDI), but they have their own distinct roles in protein folding. We have characterized the domain structure of ERp57 by limited proteolysis and N-terminal sequencing and have found it to be similar but not identical to that of PDI. ERp57 had three major protease-sensitive regions, the first of which was located between residues 120 and 150, the second between 201 and 215, and the third between 313 and 341, the data thus being consistent with a four-domain structure abb'a'. Recombinant expression in Escherichia coli was used to verify the domain boundaries. Each single domain and a b'a' double domain could be produced in the form of soluble, folded polypeptides, as verified by circular dichroism spectra and urea gradient gel electrophoresis. When the ability of ERp57 and its a and a' domains to fold denatured RNase A was studied by electrospray mass analyses, ERp57 markedly enhanced the folding rate at early time points, although less effectively than PDI, but was an ineffective catalyst of the overall process. The a and a' domains produced only minor, if any, increases in the folding rate at the early stages and no increase at the late stages. Interaction of the soluble ERp57 domains with the P domain of calreticulin was studied by chemical cross-linking in vitro. None of the single ERp57 domains nor the b'a' double domain could be cross-linked to the P domain, whereas cross-linking was obtained with a hybrid ERpabb'PDIa'c polypeptide but not with ERpabPDIb'a'c, indicating that multiple domains are involved in this protein-protein interaction and that the b' domain of ERp57 cannot be replaced by that of PDI.