Модернізація стоматологічної установки УС- 30

Background: The novel chemokine CXCL17 acts as chemoattractant for monocytes, macrophages and dendritic cells. CXCL17 also has a role in angiogenesis of importance for tumour development. Methods: Expression of CXCL17, CXCL10, CXCL9 and CCL2 was assessed in primary colon cancer tumours, colon carcinoma cell lines and normal colon tissue at mRNA and protein levels by real-time qRT-PCR, immunohistochemistry, two-colour immunofluorescence and immunomorphometry. Results: CXCL17 mRNA was expressed at 8000 times higher levels in primary tumours than in normal colon (P<0.0001). CXCL17 protein was seen in 17.2% of cells in tumours as compared with 0.07% in normal colon (P = 0.0002). CXCL10, CXCL9 and CCL2 mRNAs were elevated in tumours but did not reach the levels of CXCL17. CXCL17 and CCL2 mRNA levels were significantly correlated in tumours. Concordant with the mRNA results, CXCL10-and CXCL9-positive cells were detected in tumour tissue, but at significantly lower numbers than CXCL17. Two-colour immunofluorescence and single-colour staining of consecutive sections for CXCL17 and the epithelial cell markers carcinoembryonic antigen and BerEP4 demonstrated that colon carcinoma tumour cells indeed expressed CXCL17. Conclusions: CXCL17 is ectopically expressed in primary colon cancer tumours. As CXCL17 enhances angiogenesis and attracts immune cells, its expression could be informative for prognosis in colon cancer patients

The transcriptional response of Caenorhabditis elegans to ivermectin exposure identifies novel genes involved in the response to reduced food intake

We have examined the transcriptional response of Caenorhabditis elegans following exposure to the anthelmintic drug ivermectin (IVM) using whole genome microarrays and real-time QPCR. Our original aim was to identify candidate molecules involved in IVM metabolism and/or excretion. For this reason the IVM tolerant strain, DA1316, was used to minimise transcriptomic changes related to the phenotype of drug exposure. However, unlike equivalent work with benzimidazole drugs, very few of the induced genes were members of xenobiotic metabolising enzyme families. Instead, the transcriptional response was dominated by genes associated with fat mobilization and fatty acid metabolism including catalase, esterase, and fatty acid CoA synthetase genes. This is consistent with the reduction in pharyngeal pumping, and consequential reduction in food intake, upon exposure of DA1316 worms to IVM. Genes with the highest fold change in response to IVM exposure, cyp-37B1, mtl-1 and scl-2, were comparably up-regulated in response to short–term food withdrawal (4 hr) independent of IVM exposure, and GFP reporter constructs confirm their expression in tissues associated with fat storage (intestine and hypodermis). These experiments have serendipitously identified novel genes involved in an early response of C. elegans to reduced food intake and may provide insight into similar processes in higher organisms

Genome-wide profiling of G protein-coupled receptors in cerebellar granule neurons using high-throughput, real-time PCR

<p>Abstract</p> <p>Background</p> <p>G protein-coupled receptors (GPCRs) are major players in cell communication, regulate a whole range of physiological functions during development and throughout adult life, are affected in numerous pathological situations, and constitute so far the largest class of drugable targets for human diseases. The corresponding genes are usually expressed at low levels, making accurate, genome-wide quantification of their expression levels a challenging task using microarrays.</p> <p>Results</p> <p>We first draw an inventory of all endo-GPCRs encoded in the murine genome. To profile GPCRs genome-wide accurately, sensitively, comprehensively, and cost-effectively, we designed and validated a collection of primers that we used in quantitative RT-PCR experiments. We experimentally validated a statistical approach to analyze genome-wide, real-time PCR data. To illustrate the usefulness of this approach, we determined the repertoire of GPCRs expressed in cerebellar granule neurons and neuroblasts during postnatal development.</p> <p>Conclusions</p> <p>We identified tens of GPCRs that were not detected previously in this cell type; these GPCRs represent novel candidate players in the development and survival of cerebellar granule neurons. The sequences of primers used in this study are freely available to those interested in quantifying GPCR expression comprehensively.</p

The cys-loop ligand-gated ion channel gene superfamily of the nematode, Caenorhabditis elegans

The nematode, Caenorhabditis elegans, possesses the most extensive known superfamily of cys-loop ligand-gated ion channels (cys-loop LGICs) consisting of 102 subunit-encoding genes. Less than half of these genes have been functionally characterised which include cation-permeable channels gated by acetylcholine (ACh) and γ-aminobutyric acid (GABA) as well as anion-selective channels gated by ACh, GABA, glutamate and serotonin. Following the guidelines set for genetic nomenclature for C. elegans, we have designated unnamed subunits as lgc genes (ligand-gated ion channels of the cys-loop superfamily). Phylogenetic analysis shows that several of these lgc subunits form distinct groups which may represent novel cys-loop LGIC subtypes

Glutamate-Gated Chloride Channels of Haemonchus contortus Restore Drug Sensitivity to Ivermectin Resistant Caenorhabditis elegans

Anthelmintic resistance is a major problem in livestock farming, especially of small ruminants, but our understanding of it has been limited by the difficulty in carrying out functional genetic studies on parasitic nematodes. An important nematode infecting sheep and goats is Haemonchus contortus; in many parts of the world this species is resistant to almost all the currently available drugs, including ivermectin. It is extremely polymorphic and to date it has proved impossible to relate any sequence polymorphisms to its ivermectin resistance status. Expression of candidate drug-resistance genes in Caenorhabditis elegans could provide a convenient means to study the effects of polymorphisms found in resistant parasites, but may be complicated by differences between the gene families of target and model organisms. We tested this using the glutamate-gated chloride channel (GluCl) gene family, which forms the ivermectin drug target and are candidate resistance genes. We expressed GluCl subunits from C. elegans and H. contortus in a highly resistant triple mutant C. elegans strain (DA1316) under the control of the avr-14 promoter; expression of GFP behind this promoter recapitulated the pattern previously reported for avr-14. Expression of ivermectin-sensitive subunits from both species restored drug sensitivity to transgenic worms, though some quantitative differences were noted between lines. Expression of an ivermectin-insensitive subunit, Hco-GLC-2, had no effect on drug sensitivity. Expression of a previously uncharacterised parasite-specific subunit, Hco-GLC-6, caused the transgenic worms to become ivermectin sensitive, suggesting that this subunit also encodes a GluCl that responds to the drug. These results demonstrate that both orthologous and paralogous subunits from C. elegans and H. contortus are able to rescue the ivermectin sensitivity of mutant C. elegans, though some quantitative differences were observed between transgenic lines in some assays. C. elegans is a suitable system for studying parasitic nematode genes that may be involved in drug resistance

Engineering the Melanocortin-4 Receptor to Control Constitutive and Ligand-Mediated Gs Signaling In Vivo

The molecular and functional diversity of G protein–coupled receptors is essential to many physiological processes. However, this diversity presents a significant challenge to understanding the G protein–mediated signaling events that underlie a specific physiological response. To increase our understanding of these processes, we sought to gain control of the timing and specificity of Gs signaling in vivo. We used naturally occurring human mutations to develop two Gs-coupled engineered receptors that respond solely to a synthetic ligand (RASSLs). Our Gs-coupled RASSLs are based on the melanocortin-4 receptor, a centrally expressed receptor that plays an important role in the regulation of body weight. These RASSLs are not activated by the endogenous hormone α-melanocyte-stimulating hormone but respond potently to a selective synthetic ligand, tetrahydroisoquinoline. The RASSL variants reported here differ in their intrinsic basal activities, allowing the separation of the effects of basal signaling from ligand-mediated activation of the Gs pathway in vivo. These RASSLs can be used to activate Gs signaling in any tissue, but would be particularly useful for analyzing downstream events that mediate body weight regulation in mice. Our study also demonstrates the use of human genetic variation for protein engineering

Multidimensional Scaling Reveals the Main Evolutionary Pathways of Class A G-Protein-Coupled Receptors

Class A G-protein-coupled receptors (GPCRs) constitute the largest family of transmembrane receptors in the human genome. Understanding the mechanisms which drove the evolution of such a large family would help understand the specificity of each GPCR sub-family with applications to drug design. To gain evolutionary information on class A GPCRs, we explored their sequence space by metric multidimensional scaling analysis (MDS). Three-dimensional mapping of human sequences shows a non-uniform distribution of GPCRs, organized in clusters that lay along four privileged directions. To interpret these directions, we projected supplementary sequences from different species onto the human space used as a reference. With this technique, we can easily monitor the evolutionary drift of several GPCR sub-families from cnidarians to humans. Results support a model of radiative evolution of class A GPCRs from a central node formed by peptide receptors. The privileged directions obtained from the MDS analysis are interpretable in terms of three main evolutionary pathways related to specific sequence determinants. The first pathway was initiated by a deletion in transmembrane helix 2 (TM2) and led to three sub-families by divergent evolution. The second pathway corresponds to the differentiation of the amine receptors. The third pathway corresponds to parallel evolution of several sub-families in relation with a covarion process involving proline residues in TM2 and TM5. As exemplified with GPCRs, the MDS projection technique is an important tool to compare orthologous sequence sets and to help decipher the mutational events that drove the evolution of protein families

Origin of Secretin Receptor Precedes the Advent of Tetrapoda: Evidence on the Separated Origins of Secretin and Orexin

At present, secretin and its receptor have only been identified in mammals, and the origin of this ligand-receptor pair in early vertebrates is unclear. In addition, the elusive similarities of secretin and orexin in terms of both structures and functions suggest a common ancestral origin early in the vertebrate lineage. In this article, with the cloning and functional characterization of secretin receptors from lungfish and X. laevis as well as frog (X. laevis and Rana rugulosa) secretins, we provide evidence that the secretin ligand-receptor pair has already diverged and become highly specific by the emergence of tetrapods. The secretin receptor-like sequence cloned from lungfish indicates that the secretin receptor was descended from a VPAC-like receptor prior the advent of sarcopterygians. To clarify the controversial relationship of secretin and orexin, orexin type-2 receptor was cloned from X. laevis. We demonstrated that, in frog, secretin and orexin could activate their mutual receptors, indicating their coordinated complementary role in mediating physiological processes in non-mammalian vertebrates. However, among the peptides in the secretin/glucagon superfamily, secretin was found to be the only peptide that could activate the orexin receptor. We therefore hypothesize that secretin and orexin are of different ancestral origins early in the vertebrate lineage

GPCR Genes Are Preferentially Retained after Whole Genome Duplication

One of the most interesting questions in biology is whether certain pathways have been favored during evolution, and if so, what properties could cause such a preference. Due to the lack of experimental evidence, whether select gene families have been preferentially retained over time after duplication in metazoan organisms remains unclear. Here, by syntenic mapping of nonchemosensory G protein-coupled receptor genes (nGPCRs which represent half the receptome for transmembrane signaling) in the vertebrate genomes, we found that, as opposed to the 8–15% retention rate for whole genome duplication (WGD)-derived gene duplicates in the entire genome of pufferfish, greater than 27.8% of WGD-derived nGPCRs which interact with a nonpeptide ligand were retained after WGD in pufferfish Tetraodon nigroviridis. In addition, we show that concurrent duplication of cognate ligand genes by WGD could impose selection of nGPCRs that interact with a polypeptide ligand. Against less than 2.25% probability for parallel retention of a pair of WGD-derived ligands and a pair of cognate receptor duplicates, we found a more than 8.9% retention of WGD-derived ligand-nGPCR pairs–threefold greater than one would surmise. These results demonstrate that gene retention is not uniform after WGD in vertebrates, and suggest a Darwinian selection of GPCR-mediated intercellular communication in metazoan organisms

Neuropeptide Receptor Transcriptome Reveals Unidentified Neuroendocrine Pathways

Neuropeptides are an important class of molecules involved in diverse aspects of metazoan development and homeostasis. Insects are ideal model systems to investigate neuropeptide functions, and the major focus of insect neuropeptide research in the last decade has been on the identification of their receptors. Despite these vigorous efforts, receptors for some key neuropeptides in insect development such as prothoracicotropic hormone, eclosion hormone and allatotropin (AT), remain undefined. In this paper, we report the comprehensive cloning of neuropeptide G protein-coupled receptors from the silkworm, Bombyx mori, and systematic analyses of their expression. Based on the expression patterns of orphan receptors, we identified the long-sought receptor for AT, which is thought to stimulate juvenile hormone biosynthesis in the corpora allata (CA). Surprisingly, however, the AT receptor was not highly expressed in the CA, but instead was predominantly transcribed in the corpora cardiaca (CC), an organ adjacent to the CA. Indeed, by using a reverse-physiological approach, we purified and characterized novel allatoregulatory peptides produced in AT receptor-expressing CC cells, which may indirectly mediate AT activity on the CA. All of the above findings confirm the effectiveness of a systematic analysis of the receptor transcriptome, not only in characterizing orphan receptors, but also in identifying novel players and hidden mechanisms in important biological processes. This work illustrates how using a combinatorial approach employing bioinformatic, molecular, biochemical and physiological methods can help solve recalcitrant problems in neuropeptide research