Planar Cell Polarity Signaling: The Developing Cell��s Compass

Cells of many tissues acquire cellular asymmetry to execute their physiologic functions. The planar cell polarity system, first characterized in Drosophila, is important for many of these events. Studies in Drosophila suggest that an upstream system breaks cellular symmetry by converting tissue gradients to subcellular asymmetry, whereas a downstream system amplifies subcellular asymmetry and communicates polarity between cells. In this review, we discuss apparent similarities and differences in the mechanism that controls PCP as it has been adapted to a broad variety of morphological cellular asymmetries in various organisms.The terms tissue polarity and planar cell polarity (PCP) were coined to describe the coordinated orientation of cells and cellular structures along an axis within the plane of an epithelial surface. Polarized cellular orientation and migration controlled by PCP is critical for multiple developmental processes, and defects underlie developmental anomalies. Vertebrate PCP mutations produce problems, including neural tube, cardiac, and renal developmental defects and misorientation of hair follicles and inner ear hair cells (Wang and Nathans 2007; Simons and Mlodzik 2008). PCP may be involved in the invasive and metastatic properties of carcinomas (Jessen 2009). Recently, many PCP-related phenotypes have been observed in association with mutations affecting primary cilia, thus connecting primary cilia to the PCP process (Singla and Reiter 2006; see Hirokawa et al. 2009). Therefore, dissecting the mechanisms of PCP signaling is of considerable interest.PCP was initially characterized in Drosophila through genetic studies of PCP mutants, which led to the proposal of a PCP signaling pathway (Wong and Adler 1993; reviewed in Adler 1992). According to newer models, epithelial polarity is established by the combination of a global directional cue distributed throughout the epithelium and cellular factors that interpret this cue to align cells with each other and the axis of polarity (Tree et al. 2002a; Zallen 2007). Once molecular polarity is determined, cell-type-specific downstream proteins affect morphological polarity. PCP components are highly conserved from flies to vertebrates, and the PCP pathway is now known to be active in many processes in polarized cells and tissues not limited to epithelia. PCP components are involved in oriented cell division, acquisition of asymmetric cellular morphology, and directional cell migration, each process representing a vectorial behavior. Although the mechanism of PCP signaling in most cases is just beginning to be understood, there appear to be diverse mechanisms sharing common themes. This mechanistic diversity may be demanded by the varying PCP-dependent morphological processes, and evidently arose by divergence from a common ancestral mechanism.Here, we describe our current understanding of how the PCP pathway functions in diverse processes, highlighting both common themes and diverging mechanisms. The obvious medical importance of the PCP pathway (see, for example, Kibar et al. 2007), and the growing interest in primary cilia will surely stimulate rapid gains in our knowledge of PCP in multiple cellular contexts.     

Selective binding of coumarin enantiomers to human alpha1-acid glycoprotein genetic variants

Coumarin-type anticoagulants, warfarin, phenprocoumon and acenocoumarol, were tested for their stereoselective binding to the human orosomucoid (ORM; AGP) genetic variants ORM 1 and ORM 2. Direct binding studies with racemic ligands were carried out by the ultrafiltration method; the concentrations of free enantiomers were determined by capillary electrophoresis. The binding of pure enantiomers was investigated with quinaldine red fluorescence displacement measurements. Our results demonstrated that all investigated compounds bind stronger to ORM 1 variant than to ORM 2. ORM 1 and human native AGP preferred the binding of (S)-enantiomers of warfarin and acenocoumarol, while no enantioselectivity was observed in phenprocoumon binding. Acenocoumarol possessed the highest enantioselectivity in AGP binding due to the weak binding of its (R)-enantiomer. Furthermore, a new homology model of AGP was built and the models of ORM 1 and ORM 2 suggested that difference in binding to AGP genetic variants is caused by steric factors.     

Calculation of Fluctuating Asymmetry of the biggest Caspian whipsnake population in Hungary compared to a common snake species

The Fluctuating Asymmetry (FA) is a special type of asymmetry in the populations of bilaterally symmetrical creatures. The FA gives us numerical data on the developmental instability of the populations and refers to potential genetical and environmental stressors affecting the populations. Here we give the first data on the FA of the protected Caspian whipsnake (Hierophis caspius) from Hungary. The FA indices of the biggest population from Villány Mts were compared to the FA indices of two differently stressed Dice snake (Natrix tessellata) populations [stressed (Mád) and seminatural (Lake Balaton)]. Based on the values of the multiple and the simple indices derived from sublabial scales, we can say that the status quo of the highly protected Caspian whipsnake population does not represent significant deviation from the near-natural dice snake population from the Lake Balaton.     

Characterization of desnutrin functional domains: critical residues for triacylglycerol hydrolysis in cultured cells

Murine desnutrin/human ATGL is a triacylglycerol (TAG) hydrolase with a predicted catalytic dyad within an α-β hydrolase fold in the N-terminal region. In humans, mutations resulting in C-terminal truncation cause neutral lipid storage disease with myopathy. To identify critical functional domains, we measured TAG breakdown in cultured cells by mutated or truncated desnutrin. In vitro, C-terminally truncated desnutrin displayed an even higher apparent V_max than the full-length form without changes in K_m, which may be explained by our finding of an interaction between the C- and N-terminal domains. In live cells, however, C-terminally truncated adenoviral desnutrin had lower TAG hydrolase activity. We investigated a role for the phosphorylation of C-terminal S406 and S430 residues but found that these were not necessary for TAG breakdown or lipid droplet localization in cells. The predicted N-terminal active sites, S47 and D166, were both critical for TAG hydrolysis in live cells and in vitro. We also identified two overlapping N-terminal motifs that predict lipid substrate binding domains, a glycine-rich motif (underlined) and an amphipathic α-helix (bold) within amino acid residues 10–24 (ISFAGCGFLGVYHIG). G14, F17, L18, and V20, but not G16 and G19, were important for TAG hydrolysis, suggesting a potential role for the amphipathic α-helix in TAG binding. This study identifies for the first time critical sites in the N-terminal region of desnutrin and reveals the requirement of the C-terminal region for TAG hydrolysis in cultured cells.     

The Effect of Opioid Receptor Blockade on the Neural Processing of Thermal Stimuli

The endogenous opioid system represents one of the principal systems in the modulation of pain. This has been demonstrated in studies of placebo analgesia and stress-induced analgesia, where anti-nociceptive activity triggered by pain itself or by cognitive states is blocked by opioid antagonists. The aim of this study was to characterize the effect of opioid receptor blockade on the physiological processing of painful thermal stimulation in the absence of cognitive manipulation. We therefore measured BOLD (blood oxygen level dependent) signal responses and intensity ratings to non-painful and painful thermal stimuli in a double-blind, cross-over design using the opioid receptor antagonist naloxone. On the behavioral level, we observed an increase in intensity ratings under naloxone due mainly to a difference in the non-painful stimuli. On the neural level, painful thermal stimulation was associated with a negative BOLD signal within the pregenual anterior cingulate cortex, and this deactivation was abolished by naloxone.     

Mitochondrial NO and reactive nitrogen species production: Does mtNOS exist?

It is more than 10 years now that mitochondria are suspected to be sources of nitric oxide (NO). This hypothesis is intriguing since NO has multiple targets within the organelle and it is even suggested that mitochondria are the primary targets of NO in the cell. Most remarkably, nanomolar concentrations of NO can inhibit mitochondrial respiration, so even a small amount of NO in the mitochondrial matrix may regulate ATP synthesis. Therefore, the idea that mitochondria themselves are capable of NO production is an important concept in several physiological and pathological mechanisms. However, this field of research generates surprisingly few original papers and the published studies contain conflicting results. The reliability of the results is frequently questioned since they are seldom reproduced by independent investigators. Until 2003, all papers published in this field showed affirmative results but since then several studies directly challenged the existence of a mitochondrial nitric oxide synthase. The present review aims to summarize the most recent developments in mitochondrial NO production, highlights a few unsolved questions, and proposes new directions for future work in this research area.     

Mitochondrial nitric oxide synthase is not eNOS, nNOS or iNOS

Recent studies indicated that there is a distinct mitochondrial nitric oxide synthase (mtNOS) enzyme, which may be identical to the other known NOS isoforms. We investigated the possible involvement of the endothelial, the neuronal, and the inducible NOS isoforms (eNOS, nNOS, iNOS, respectively) in mitochondrial NO production. Mouse liver mitochondria were prepared by Percoll gradient purification from wild-type and NOS knockout animals. NOS activity was measured by the arginine conversion assay, NO production of live mitochondria was visualized by the fluorescent probe DAF-FM with confocal microscopy and measured with flow cytometry. Western blotting or immunoprecipitation was performed with 12 different anti-NOS antibodies. Mitochondrial NOS was purified by arginine, 2,5 ADP and calmodulin affinity columns. We observed NO production and NOS activity in mitochondria, which was not attenuated by classic NOS inhibitors. We also detected low amounts of eNOS protein in the mitochondria, however, NO production and NOS activity were intact in eNOS knockout animals. Neither nNOS nor iNOS were present in the mitochondria. Furthermore, we could not find mitochondrial targeting signals in the sequences of either NOS proteins. Taken together, the presented data do not support the hypothesis that any of the known NOS enzymes are present in the mitochondria in physiologically relevant levels.     

Inhibition of cytochrome P450 enzymes participating in p-nitrophenol hydroxylation by drugs known as CYP2E1 inhibitors

p-Nitrophenol hydroxylation is widely used as a probe for microsomal CYP2E1. Several drugs are known as CYP2E1 inhibitors because of their capability to inhibit p-nitrophenol hydroxylation. Our results suggest further participation of CYP2A6 and CYP2C19 enzymes in p-nitrophenol hydroxylation. Moreover, CYP2A6 and CYP2C19 may be considered as the primary catalysts, whereas CYP2E1 can also contribute to the hydroxylation of p-nitrophenol. Further aim of our study was to evaluate the selectivity of p-nitrophenol hydroxylase inhibitors towards cytochrome P450 enzymes. The effects of antifungals: bifonazole, econazole, clotrimazole, ketoconazole, miconazole; CNS-active drugs: chlorpromazine, desipramine, fluphenazine, thioridazine; and the non-steroidal anti-inflammatory drug: diclofenac were investigated on the enzyme activities selective for CYP2A6, CYP2C9, CYP2C19, CYP2E1 and CYP3A4. None of the drugs could be considered as a potent inhibitor of CYP2E1. Strong inhibition was observed for CYP3A4 by antifungals with IC(50) values in submicromolar range. However, ketoconazole was the only imidazole derivative that could be considered as a selective inhibitor of CYP3A4. The CNS-active drugs investigated were found to be weak inhibitors of CYP2A6, CYP2C9, CYP2C19, CYP2E1 and CYP3A4. Diclofenac efficiently inhibited CYP2C9 and to a less extent CYP3A4 enzyme.     

Characterization of chemotactic ability of peptides containing N-formyl-methionyl residues in Tetrahymena fMLP as a targeting ligand

The chemotactic effects of six formylated, putatively bacterial peptides (fMLP, fMLPP, fMMM, fMP, fMV, and fMS) were studied. From the set of six peptides, only fMLP (one of the most effective chemoattractant peptides in mammals) elicited a significant positive chemotactic response in the eukaryotic ciliate Tetrahymena pyriformis, while the other formylated ligands, e.g. fMMM (which is also effective in mammals), had neutral or antagonistic effects in Tetrahymena. A study of their amino acid sequences points to an, as yet obscure, interaction between C-terminal f-Met and N-terminal aromatic Phe. Some optimal physicochemical characteristics (e.g. solvent exposed area, solubility) of the molecule may be responsible for this special feature of f-MLP at such a low level of phylogeny. This means that the unicellular Tetrahymena is able to select between related molecules, giving high priority to the molecule that is the most chemoattractive in mammals. The results call attention to the possible presence of f-Met receptors at a unicellular level and to the evolutionary conservation of chemotaxis-activating processes.     

HPLC study on the carotenoid composition of Calendula products

The authors report on the HPLC investigation of the carotenoid composition of the steams, leaves, petals and pollens of Calendula officinalis L. In the petals and pollens, the main carotenoids were flavoxanthin and auroxanthin while the stem and leaves mostly contained lutein and beta-carotene.Five different herbal tea and two tinctures made from the flower of C. officinalis L. were also investigated and the carotenoid composition of the industrial products was compared to the starting material.