Expression of the selected adhesive molecules (cadherin E, CD44, LGAL3 and CA50) in papillary thyroid carcinoma

INTRODUCTION: The aim of the study was to determine the expression of selected adhesive molecules in papillary thyroid carcinoma. MATERIAL AND METHODS: 47 papillary thyroid carcinoma cases and 11 nonmalignant goiter cases were analyzed by immunohistochemistry. RESULTS: Galectin-3 (LGAL3) was a sensitive and specific marker, present in 91% of analyzed tumors and only in 5% of tumor margin. The presence of CA50 was 86% and 3% respectively with only 3% positive non-malignant cases. Cadherin E expression was noted in 91% of primary tumors, in 84% of the surrounding tissue and in 63% of non-malignant goiter. CD44 (DF1485) was observed in 89% of primary tumors and 48% of surrounding tissue; the reaction with BBA10 was more characteristic for metastases. CONCLUSIONS: Our study confirms the high diagnostic value of galectin-3 in papillary thyroid carcinoma and reveals the similar efficiency of CA50. CD44 (DF1485) expression in primary tumor is more intensive than in surrounding tissue, but the diagnostical inportance is not high because it is often observed in benign lesions. Using of BBA10 is more sensitive, but less specific. High expression of cadherin E in benign lesions impairs its diagnostical application in papillary thyroid cancer.     

Angiotensin II increases activity of the epithelial Na+ channel (ENaC) in distal nephron additively to aldosterone

Dietary salt intake controls epithelial Na+ channel (ENaC)-mediated Na+ reabsorption in the distal nephron by affecting status of the renin-angiotensin-aldosterone system (RAAS). Whereas regulation of ENaC by aldosterone is generally accepted, little is known about whether other components of RAAS, such as angiotensin II (Ang II), have nonredundant to aldosterone-stimulatory actions on ENaC. We combined patch clamp electrophysiology and immunohistochemistry in freshly isolated split-opened distal nephrons of mice to determine the mechanism and molecular signaling pathway of Ang II regulation of ENaC. We found that Ang II acutely increases ENaC Po, whereas prolonged exposure to Ang II also induces translocation of α-ENaC toward the apical membrane in situ. Ang II actions on ENaC Po persist in the presence of saturated mineralocorticoid status. Moreover, aldosterone fails to stimulate ENaC acutely, suggesting that Ang II and aldosterone have different time frames of ENaC activation. AT1 but not AT2 receptors mediate Ang II actions on ENaC. Unlike its effect in vasculature, Ang II did not increase [Ca2+]i in split-opened distal nephrons as demonstrated using ratiometric Fura-2-based microscopy. However, application of Ang II to mpkCCDc14 cells resulted in generation of reactive oxygen species, as probed with fluorescent methods. Consistently, inhibiting NADPH oxidase with apocynin abolished Ang II-mediated increases in ENaC Po in murine distal nephron. Therefore, we concluded that Ang II directly regulates ENaC activity in the distal nephron, and this effect complements regulation of ENaC by aldosterone. We propose that stimulation of AT1 receptors with subsequent activation of NADPH oxidase signaling pathway mediates Ang II actions on ENaC.     

Efficient tuning of potential parameters for liquid–solid interactions

Spherical and cylindrical water droplets on silicon surface are studied to tune the silicon–oxygen interaction. We use molecular dynamics simulations to estimate the contact angle of two different shaped droplets. We found that the cylindrical droplets are independent of the line tension as their three phases curvature is equal zero. Additionally, we compare an analytical model, taking into account or not the Tolman length and we show that for spherical small size droplets, this length is important to be included, in contrast to cylindrical droplets in which the influence of the Tolman length is negligible. We demonstrate that the usual convenient way to exclude linear tension in the general case can give wrong results. Here, we consider cylindrical droplets, since their contact angle does not depend on the droplet size in the range of few to 10ths of nanometres. The droplets are stabilised due to the periodic boundary conditions. This allows us to propose a new parameterisation for nanoscale droplets, which is independent the size of the droplets or its shape, minimising at the same time the calculation procedure. With the proposed methodology, we can extract the epsilon parameter of the interaction potential between a liquid and a solid from the nanoscaled molecular simulation with only as input the macrosized experimental wetting angle for a given temperature.     

Phosphorylation of ribosomal protein S6 differentially affects mRNA translation based on ORF length

Phosphorylation of Ribosomal Protein S6 (RPS6) was the first post-translational modification of the ribosome to be identified and is a commonly-used readout for mTORC1 activity. Although the cellular and organismal functions of RPS6 phosphorylation are known, the molecular consequences of RPS6 phosphorylation on translation are less well understood. Here we use selective ribosome footprinting to analyze the location of ribosomes containing phosphorylated RPS6 on endogenous mRNAs in cells. We find that RPS6 becomes progressively dephosphorylated on ribosomes as they translate an mRNA. As a consequence, average RPS6 phosphorylation is higher on mRNAs with short coding sequences (CDSs) compared to mRNAs with long CDSs. We test whether RPS6 phosphorylation differentially affects mRNA translation based on CDS length by genetic removal of RPS6 phosphorylation. We find that RPS6 phosphorylation promotes translation of mRNAs with short CDSs more strongly than mRNAs with long CDSs. Interestingly, RPS6 phosphorylation does not promote translation of mRNAs with 5′ TOP motifs despite their short CDS lengths, suggesting they are translated via a different mode. In sum this provides a dynamic view of RPS6 phosphorylation on ribosomes as they translate mRNAs and the functional consequence on translation.     

A new mechanism of post-transfer editing by aminoacyl-tRNA synthetases: catalysis of hydrolytic reaction by bacterial-type prolyl-tRNA synthetase

Aminoacyl tRNA synthetases are enzymes that specifically attach amino acids to cognate tRNAs for use in the ribosomal stage of translation. For many aminoacyl tRNA synthetases, the required level of amino acid specificity is achieved either by specific hydrolysis of misactivated aminoacyl-adenylate intermediate (pre-transfer editing) or by hydrolysis of the mischarged aminoacyl-tRNA (post-transfer editing). To investigate the mechanism of post-transfer editing of alanine by prolyl-tRNA synthetase from the pathogenic bacteria Enterococcus faecalis, we used molecular modeling, molecular dynamic simulations, quantum mechanical (QM) calculations, site-directed mutagenesis of the enzyme, and tRNA modification. The results support a new tRNA-assisted mechanism of hydrolysis of misacylated Ala-tRNA^Pro. The most important functional element of this catalytic mechanism is the 2′-OH group of the terminal adenosine 76 of Ala-tRNA^Pro, which forms an intramolecular hydrogen bond with the carbonyl group of the alanine residue, strongly facilitating hydrolysis. Hydrolysis was shown by QM methods to proceed via a general acid-base catalysis mechanism involving two functionally distinct water molecules. The transition state of the reaction was identified. Amino acid residues of the editing active site participate in the coordination of substrate and both attacking and assisting water molecules, performing the proton transfer to the 3′-O atom of A76.     

Rapid high resolution single nucleotide polymorphism-comparative genome hybridization mapping in Caenorhabditis elegans

We have developed a significantly improved and simplified method for high-resolution mapping of phenotypic traits in Caenorhabditis elegans using a combination of single nucleotide polymorphisms (SNPs) and oligo array comparative genome hybridization (array CGH). We designed a custom oligonucleotide array using a subset of confirmed SNPs between the canonical wild-type Bristol strain N2 and the Hawaiian isolate CB4856, populated with densely overlapping 50-mer probes corresponding to both N2 and CB4856 SNP sequences. Using this method a mutation can be mapped to a resolution of ~200 kb in a single genetic cross. Six mutations representing each of the C. elegans chromosomes were detected unambiguously and at high resolution using genomic DNA from populations derived from as few as 100 homozygous mutant segregants of mutant N2/CB4856 heterozygotes. Our method completely dispenses with the PCR, restriction digest, and gel analysis of standard SNP mapping and should be easy to extend to any organism with interbreeding strains. This method will be particularly powerful when applied to difficult or hard-to-map low-penetrance phenotypes. It should also be possible to map polygenic traits using this method.     

Surface localization determinants of Borrelia OspC/Vsp family lipoproteins

The dimeric OspC/Vsp family surface lipoproteins of Borrelia spirochetes are crucial to the transmission and persistence of Lyme borreliosis and tick-borne relapsing fever. However, the requirements for their proper surface display remained undefined. In previous studies, we showed that localization of Borrelia burgdorferi monomeric surface lipoprotein OspA was dependent on residues in the N-terminal "tether" peptide. Here, site-directed mutagenesis of the B. burgdorferi OspC tether revealed two distinct regions affecting either release from the inner membrane or translocation through the outer membrane. Determinants of both of these steps appear consolidated within a single region of the Borrelia turicatae Vsp1 tether. Periplasmic OspC mutants still were able to form dimers. Their localization defect could be rescued by the addition of an apparently structure-destabilizing C-terminal epitope tag but not by coexpression with wild-type OspC. Furthermore, disruption of intermolecular Vsp1 salt bridges blocked dimerization but not surface localization of the resulting Vsp1 monomers. Together, these results suggest that Borrelia OspC/Vsp1 surface lipoproteins traverse the periplasm and the outer membrane as unfolded monomeric intermediates and assemble into their functional multimeric folds only upon reaching the spirochetal surface.     

Bradykinin acutely inhibits activity of the epithelial Na+ channel in mammalian aldosterone-sensitive distal nephron

Activation of the renal kallikrein-kinin system results in natriuresis and diuresis, suggesting its possible role in renal tubular sodium transport regulation. Here, we used patch-clamp electrophysiology to directly assess the effects of bradykinin (BK) on the epithelial Na(+) channel (ENaC) activity in freshly isolated split-opened murine aldosterone-sensitive distal nephrons (ASDNs). BK acutely inhibits ENaC activity by reducing channel open probability (P(o)) in a dose-dependent and reversible manner. Inhibition of B2 receptors with icatibant (HOE-140) abolished BK actions on ENaC. In contrast, activation of B1 receptors with the selective agonist Lys-des-Arg(9)-BK failed to reproduce BK actions on ENaC. This is consistent with B2 receptors playing a critical role in mediating BK signaling to ENaC. BK has little effect on ENaC P(o) when G(q/11) was inhibited with Gp antagonist 2A. Moreover, inhibition of phospholipase C (PLC) with U73122, but not saturation of cellular cAMP levels with the membrane-permeable nonhydrolysable cAMP analog 8-cpt-cAMP, prevents BK actions on ENaC activity. This argues that BK stimulates B2 receptors with subsequent activation of G(q/11)-PLC signaling cascade to acutely inhibit ENaC activity. Activation of BK signaling acutely depletes apical PI(4,5)P(2) levels. However, inhibition of Ca(2+) pump SERCA of the endoplasmic reticulum with thapsigargin does not prevent BK signaling to ENaC. Furthermore, caffeine, while producing a similar rise in [Ca(2+)](i) as in response to BK stimulation, fails to recapitulate BK actions on ENaC. Therefore, we concluded that BK acutely inhibits ENaC P(o) in mammalian ASDN via stimulation of B2 receptors and following depletion of PI(4,5)P(2), but not increases in [Ca(2+)](i).