Activation of ion secretion via proteinase-activated receptor-2 in human colon

Proteinase-activated receptor (PAR) type 2 (PAR-2) has been shown to mediate ion secretion in cultured epithelial cells and rat jejunum. With the use of a microUssing chamber, we demonstrate the role of PAR-2 for ion transport in native human colonic mucosa obtained from 30 normal individuals and 11 cystic fibrosis (CF) patients. Trypsin induced Cl- secretion when added to the basolateral but not luminal side of normal epithelia. Activation of Cl- secretion by trypsin was inhibited by indomethacin and was further increased by cAMP in normal tissues but was not present in CF colon, indicating the requirement of luminal CF transmembrane conductance regulator. Effects of trypsin were largely reduced by low Cl-,by basolateral bumetanide, and in the presence of barium or clotrimazole, but not by tetrodotoxin. Furthermore, trypsin-induced secretion was inhibited by the Ca2+-ATPase inhibitor cyclopiazonic acid and in low-Ca2+ buffer. The effects of trypsin were almost abolished by trypsin inhibitor. Thrombin, an activator of PAR types 1, 3, and 4, had no effects on equivalent short-circuit currents. The presence of PAR-2 in human colon epithelium was confirmed by RT-PCR and additional experiments with PAR-2-activating peptide. PAR-2-mediated intestinal electrolyte secretion by release of mast cell tryptase and potentiation of PAR-2 expression by tumor necrosis factor-alpha may contribute to the hypersecretion observed in inflammatory processes such as chronic inflammatory bowel disease.

Effects of polyunsaturated fatty acids on voltage-gated K+ and Na+ channels in rat olfactory receptor neurons

Although the polyunsaturated fatty acids arachidonic acid (AA) and docosahexaenoic acid (DHA) are enriched in the olfactory mucosa, their possible contribution to olfactory transduction has not been investigated. This study characterized their effects on voltage-gated K+ and Na+ channels of rat olfactory receptor neurons. Physiological (3-10 mum) concentrations of AA and DHA potently and irreversibly inhibited the voltage-gated K+ current in a voltage-independent manner. In addition, both compounds significantly reduced the inhibitory potency of the odorants acetophenone and amyl acetate at these channels. By comparison, the steady-state effects of both AA and DHA on the voltage-gated Na+ channel were relatively weak, with half-maximal inhibition requiring approximate to 35 mum of either compound. However, a surprising finding was that the initial application of 3 mum AA to a naive neuron caused a strong but transient inhibition of the Na+ current. The channels became almost completely resistant to this inhibition within 1 min, and a 2-min wash in control solution was insufficient to restore the strong inhibitory effect. These observations suggest that polyunsaturated fatty acids have the potential to strongly influence the coding of odorant information by olfactory receptor neurons.

Colony-stimulating factor-1 suppresses responses to CpG DNA and expression of toll-like receptor 9 but enhances responses to lipopolysaccharide in murine macrophages

During bacterial infections, the balance between resolution of infection and development of sepsis is dependent upon the macrophage response to bacterial products. We show that priming of murine bone marrow-derived macrophages (BMMs) with CSF-1 differentially regulates the response to two such stimuli, LPS and immunostimulatory (CpG) DNA. CSF-1 pretreatment enhanced IL-6, IL-12, and TNF-alpha production in response to LPS but suppressed the same response to CpG DNA. CSF-1 also regulated cytokine gene expression in response to CpG DNA and LPS; CpG DNA-induced IL-12 p40, IL-12 p35, and TNF-alpha mRNAs were all suppressed by CSF-1 pretreatment. CSF-1 pretreatment enhanced LPS-induced IL-12 p40 mRNA but not TNF-alpha and IL-12 p35 mRNAs, suggesting that part of the priming effect is posttranscriptional. CSF-1 pretreatment also suppressed CpG DNA-induced nuclear translocation of NF-kappaB and phosphorylation of the mitogen-activated protein kinases p38 and extracellular signal-related kinases-1/2 in BMMs, indicating that early events in CpG DNA signaling were regulated by CSF-1. Expression of Toll-like receptor (TLR)9, which is necessary for responses to CpG DNA, was markedly suppressed by CSF-1 in both BMMs and thioglycolate-elicited peritoneal macrophages. CSF-1 also down-regulated expression of TLR1, TLR2, and TLR6, but not the LPS receptor, TLR4, or TLR5. Hence, CSF-1 may regulate host responses to pathogens through modulation of TLR expression. Furthermore, these results suggest that CSF-1 and CSF-1R antagonists may enhance the efficacy of CpG DNA in vivo.

Association of the SULT1A1 R213H polymorphism with colorectal cancer

1. Sulphotransferases are a superfamily of enzymes involved in both detoxification and bioactivation of endogenous and exogenous compounds. The arylsulphotransferase SULT1A1 has been implicated in a decreased activity and thermostability when the wild-type arginine at position 213 of the coding sequence is substituted by a histidine. SULT1A1 is the isoform primarily associated with the conversion of dietary N -OH arylamines to DNA binding adducts and is therefore of interest to determine whether this polymorphism is linked to colorectal cancer. 2. Genotyping, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, was performed using DNA samples of healthy control subjects (n = 402) and patients with histologically proven colorectal cancer (n = 383). Both control and test populations possessed similar frequencies for the mutant allele (32.1 and 31%, respectively; P = 0.935). Results were not altered when age and gender were considered as potential confounders in a logistic regression analysis. 3. Examination of the sulphonating ability of the two allozymes with respect to the substrates p -nitrophenol and paracetamol showed that the affinity and rate of sulphonation was unaffected by substitution of arginine to histidine at position 213 of the amino acid sequence. 4. From this study, we conclude that the SULT1A1 R213H polymorphism is not linked with colorectal cancer in this elderly Australian population.

Temporal and spatial expression of fibroblast growth factor receptor 4 isoforms in murine tissues

Fibroblast growth factor receptors (FGFRs) undergo highly regulated spatial and temporal changes of expression during development. This study describes the use of quantitative reverse transcriptase-polymerase chain reaction and immunochemistry to assess the changes in expression of FGFR4 as compared to its FGFR4-17a and -17b isoforms in mouse tissues, from early embryogenesis through to adulthood. Compared to FGFR4, the expression of the isoforms is more restricted at all developmental stages tested. The reverse transcriptase-polymerase chain reaction demonstrated that FGFR4 is expressed in more tissue types than either of its isoforms: it was found predominantly in lung, liver, brain, skeletal muscle and kidney, whereas the FGFR4-17a form was detected in lung and skeletal muscle, and the FGFR4-17b form only in lung, liver, skeletal muscle and kidney. Immunohistochemistry confirmed strong FGFR4-17b expression in the postnatal lung. When combined, the results suggest that FGFR4 variants play important roles particularly in lung and skeletal muscle development.

Exploring complex fitness surfaces: Multiple ornamentation and polymorphism in male guppies

The sexual ornamentation used by male guppies to attract females comprises many components, each of which varies considerably among males. Although natural and sexual selection have been shown to contribute to divergence among populations in male sexual ornaments, the role of sexual selection in maintaining polymorphism within populations is less clear. We used both parametric quadratic regression and nonparametric projection pursuit regression techniques to reveal the major axes of non-linear sexual selection on male ornaments. We visualized the fitness surfaces defined by these axes using thin-plate splines to allow a direct comparison of the two methodologies. Identification of the major axes of selection and their visualization was critical in determining the form and strength of nonlinear selection. Both types of analysis revealed fitness surfaces comprising three peaks, suggesting that there is more than one way to make an attractive guppy. Disruptive selection may be an important process underlying the presence of multiple sexual ornaments and may contribute to the maintenance of the high levels of polymorphism in male sexual ornaments found in guppy populations.

Single-nucleotide polymorphism in the CD14 promoter and periodontal disease expression in a Japanese population

It has been reported that there is a relationship between a single-nucleotide polymorphism (SNP) in the promoter region of the CD 14 gene at position -159 (C-->T) and infectious diseases. The aim of the present study was to test the hypthesis that expression of this SNP correlates with periodontal disease in a Japanese population. The CD14 genotype was determined in 163 subjects with periodontitis and in 104 age- and gender-matched control subjects without periodontitis. The genotype distribution and allele frequency within the periodontitis patients were not significantly different from those of control subjects. There was, however, a significant difference in the genotype distribution between young patients (< 35 yrs) and older patients (greater than or equal to 35 yrs). These findings suggest that CD14-159C/T polymorphism is not related to the development of periodontitis in a Japanese population, but that, within the periodontitis subjects, expression of the SNP may be related to early disease activity.

A genomewide survey of developmentally relevant genes in Ciona intestinalis - V. Genes for receptor tyrosine kinase pathway and Notch signaling pathway

In the present survey, we identified most of the genes involved in the receptor tyrosine kinase (RTK), mitogen activated protein kinase (MAPK) and Notch signaling pathways in the draft genome sequence of Ciona intestinalis, a basal chordate. Compared to vertebrates, most of the genes found in the Ciona genome had fewer paralogues, although several genes including ephrin, Eph and fringe appeared to have multiplied or duplicated independently in the ascidian genome. In contrast, some genes including kit/flt, PDGF and Trk receptor tyrosine kinases were not found in the present survey, suggesting that these genes are innovations in the vertebrate lineage or lost in the ascidian lineage. The gene set identified in the present analysis provides an insight into genes for the RTK, MAPK and Notch signaling pathways in the ancient chordate genome and thereby how chordates evolved these signaling pathway.

Caveolin interacts with the angiotensin II type 1 receptor during exocytic transport but not at the plasma membrane

The mechanisms involved in angiotensin II type 1 receptor (AT(1)-R) trafficking and membrane localization are largely unknown. In this study, we examined the role of caveolin in these processes. Electron microscopy of plasma membrane sheets shows that the AT(1)-R is not concentrated in caveolae but is clustered in cholesterol-independent microdomains; upon activation, it partially redistributes to lipid rafts. Despite the lack of AT(1)-R in caveolae, AT(1)-R. caveolin complexes are readily detectable in cells co-expressing both proteins. This interaction requires an intact caveolin scaffolding domain because mutant caveolins that lack a functional caveolin scaffolding domain do not interact with AT(1)-R. Expression of an N-terminally truncated caveolin-3, CavDGV, that localizes to lipid bodies, or a point mutant, Cav3-P104L, that accumulates in the Golgi mislocalizes AT(1)-R to lipid bodies and Golgi, respectively. Mislocalization results in aberrant maturation and surface expression of AT(1)-R, effects that are not reversed by supplementing cells with cholesterol. Similarly mutation of aromatic residues in the caveolin-binding site abrogates AT(1)-R cell surface expression. In cells lacking caveolin-1 or caveolin-3, AT(1)-R does not traffic to the cell surface unless caveolin is ectopically expressed. This observation is recapitulated in caveolin-1 null mice that have a 55% reduction in renal AT(1)-R levels compared with controls. Taken together our results indicate that a direct interaction with caveolin is required to traffic the AT(1)-R through the exocytic pathway, but this does not result in AT(1)-R sequestration in caveolae. Caveolin therefore acts as a molecular chaperone rather than a plasma membrane scaffold for AT(1)-R.

Osteoarthritis of the hands, hips and knees in an Australian twin sample - Evidence of association with the aggrecan VNTR polymorphism

Age-related changes in the composition of the cartilage matrix may be associated with the development of osteoarthritis, a relatively late-onset disease characterised by the destruction of joint cartilage. In order to investigate whether differences in the VNTR polymorphic region of aggrecan affect cartilage functionality and therefore the development of osteoarthritis, we examined the aggrecan polymorphic genotypes of a sample of 134 Australian twins aged over 50 (including 34 monozygotic and 27 dizygotic twin pairs). Clinical measures of hand, hip and knee osteoarthritis, as well as self-reported bone and joint pain, were tested for association with the aggrecan polymorphism. The results were consistent with either a deleterious effect of allele 27, or a protective effect of alleles 25 and 28, providing some additional evidence for an association between the aggrecan VNTR polymorphism and osteoarthritis of the hands, hips and knees.

Quantitation of alternatively spliced NMDA receptor NR1 isoform mRNA transcripts in human brain by competitive RT-PCR

The N-methyl-D-aspartate (NMDA)-selective subtype of ionotropic glutamate receptor is of importance in neuronal differentiation and synapse consolidation, activity-dependent forms of synaptic plasticity, and excitatory amino acid-mediated neuronal toxicity [Neurosci. Res. Program, Bull. 19 (1981) 1; Lab. Invest. 68 (1993) 372]. NMDA receptors exist in vivo as tetrameric or pentameric complexes comprising proteins from two families of homologous subunits, designated NR1 and NR2(A-D) [Biochem. Biophys. Res. Commun. 185 (1992) 826]. The gene coding for the human NR1 subunit (hNR1) is composed of 21 exons, three of which (4, 20 and 21) can be differentially spliced to generate a total of eight distinct subunit variants. We detail here a competitive RT-PCR (cRT-PCR) protocol to quantify endogenous levels of hNR1 splice variants in autopsied human brain. Quantitation of each hNR1 splice variant is performed using standard curve methodology in which a known amount of synthetic ribonucleic acid competitor (internal standard) is co-amplified against total RNA. This method can be used for the quantitation of hNR1 mRNA levels in response to acute or chronic disease states, in particular in the glutamatergic-associated neuronal loss observed in Alzheimer's disease [J. Neurochem. 78 (2001) 175]. Furthermore, alterations in hNR1 mRNA expression may be reflected at the translational level, resulting in functional changes in the NMDA receptor. (C) 2003 Elsevier Science B.V. All rights reserved.

Quantitation of human brain GABAA receptor isoforms by competitive RT-PCR

We have developed a competitive RT-PCR assay, adapted from Lewohl et al. [Brain Res. Brain Res. Protoc. 1 (1997) 347]. for the quantitation of GABA, receptor beta isoforms in human brain using an internal standard that shares high sequence homology to the targets. The internal standard is identical to the beta(1) sequence except for a 61 bp deletion and the incorporation of a Hind III restriction enzyme site. Unlike traditional competitive RT-PCR, which requires a range of internal standard concentrations to be titrated against a constant amount of unknown, this method relies on a standard curve for quantitation of each sample and thus permits increased sample throughput. This method is suitable for the quantitation of beta(1), beta(2) and beta(3) isoforms of the GABA(A) receptor in human alcoholic and control brain. (C) 2003 Elsevier Science B.V. All rights reserved.