Re-evaluation of the cytokinin receptor role of the Arabidopsis gene GCR1

GCR1 has been tentatively identified in Arabidopsis thaliana as the first plant G-protein coupled receptor (GPCR) (Josefsson and Rask 1997) implicated in the cytokinin sensory pathway (Plakidou-Dymock et al. 1998). A protein fusion of GCR1 and green fluorescent protein has been expressed in Arabidopsis and shown GCR1 to be located on the plasma membrane. Studies of plants with altered GCR1 expression have led us to question GCR1's involvement in cytokinin signaling. Transgenic Arabidopsis plants containing sense and antisense constructs for GCR1 have been produced and over- and under-expression confirmed. The analysis of 12 antisense and 17 sense lines has failed to reveal the previously reported Dainty phenotype or altered cytokinin sensitivity. We have used the Gauntlet approach to test the plants' response to various plant hormones although this has not yet identified a mutant phenotype. The yeast-two hybrid system has been used and so far there is no evidence to suggest GCR1 interacts with heterotrimeric G proteins. Before GCR1 can be identified as genuine G-protein coupled receptor, the identification of a ligand and a proof of association with heterotrimeric G-proteins should be obtained.

Ionic selectivity of native ATP-activated (P2X) receptor channels in dissociated neurones from rat parasympathetic ganglia

1. The relative permeability of the native P2X receptor channel to monovalent and divalent inorganic and organic cations was determined from reversal potential measurements of ATP-evoked currents in parasympathetic neurones dissociated from rat submandibular ganglia using the dialysed whole-cell patch clamp technique. 2. The P2X receptor-channel exhibited weak selectivity among the alkali metals with a selectivity sequence of Na+ > Li+ > Cs+ > Rb+ > K+, and permeability ratios relative to Cs+ (P-X/P-Cs) ranging from 1. 11 to 0.86. 3. The selectivity for the divalent alkaline earth cations was also weak with the sequence Ca2+ > Sr2+ > Ba2+ > Mn2+ > Mg2+. ATP-evoked currents were strongly inhibited when the extracellular divalent cation concentration was increased. 4, The calculated permeability ratios of different ammonium cations are higher than those of the alkali metal cations. The permeability sequence obtained for the saturated organic cations is inversely correlated with the size of the cation. The unsaturated organic cations have a higher permeability than that predicted by molecular size. 5. Acidification to pH 6.2 increased the ATP-induced current amplitude twofold, whereas alkalization to 8.2 and 9.2 markedly reduced current amplitude. Cell dialysis with either anti-P2X(2) and/or anti-P2X(4) but not anti-P2X(1) antibodies attenuated the ATP-evoked current amplitude. Taken together, these data are consistent with homomeric and/or heteromeric P2X(2) and P2X(4) receptor subtypes expressed in rat submandibular neurones. 6. The permeability ratios for the series of monovalent organic cations, with the exception of unsaturated cations, were approximately related to the ionic size. The relative permeabilities of the monovalent inoganic and organic cations tested are similar to those reported previously for cloned rat P2X2 receptors expressed in mammalian cells.

The gene encoding the immunoregulatory signaling molecule CMRF-35A localized to human chromosome 17 in close proximity to other members of the CMRF-35 family

The immunoregulatory signaling (IRS) family includes several molecules, which play major roles in the regulation of the immune response. The CMRF-35A and CMRF-35H molecules are two new members of the IRS family of molecules, that are found on a wide variety of haemopoietic lineages. The extracellular functional interactions of these molecules is presently unknown, although CMRF-35H on initiate an inhibitory signal and is internalized when cross-linked. In this paper, we described the gene structure for the CMRF-35A gene and its localization to human chromosome 17. The gene consists of four exons spanning approximately 4.5 kb. Exon 1 encodes the 5' untranslated region and leader sequence, exon 2 encodes the immunoglobulin (Ig)-like domain, exon 3 encodes the membrane proximal region and exon 4 encodes the transmembrane region, the cytoplasmic tail and the 3' untranslated region. A region in the 5' flanking sequence of the CMRF-35A gene, that promoted expression of a reporter gene was identified. The genes for the CMRF-35A and CMRF-35H molecules are closely linked on chromosome 17. Similarity between the Ig-like exons and the preceding intron of the two genes suggests exon duplication was involved in their evolution. We also identified a further member of the CMRF-35 family, the CMRF-35J pseudogene. This gene appears to have arisen by gene duplication of the CMRF-35A gene. These three loci-the CMRF-35A, CMRF-35J and CMRF-35H genes-form a new complex of IRS genes on chromosome 17.

Characterization of the EphA1 receptor tyrosine kinase: Expression in epithelial tissues

The Eph family (of receptor tyrosine kinases plays a crucial role during development and is implicated in oncogenesis. Using a partial cDNA clone of an Eph-related kinase (Esk) we isolated the complete coding region of a gene which we show to be murine EphA1 by both structural and functional criteria. The chromosomal localization is shown to be syntenic to hEphA1 and the genomic organization also shows distinct features found in the hEphA1 gene. Functionally, in keeping with findings for the human homologue, both soluble recombinant and native mEphA1 show preferential binding to ephrin A1. However, we also observed significant binding to other A-type ligands as has been observed for other Eph receptors. We analysed the expression of mEphA1 mRNA by in situ hybridization on tissue sections. mEphA1 was expressed in epithelial elements of skin, adult thymus, kidney and adrenal cortex. Taken together with previous Northern blotting data these results suggest that mEphA1 is expressed widely in differentiated epithelial cells.

Premature calvarial synostosis and epidermal hyperplasia (Beare-Stevenson syndrome-like anomalies) resulting from a P250R missense mutation in the gene encoding fibroblast growth factor receptor 3

We report on a patient with a severe premature calvarial synostosis and epidermal hyperplasia. The phenotype was consistent with that of a mild presentation of Beare-Stevenson syndrome but molecular analysis of the IgIII-transmembrane linker region and the transmembrane domain of the gene encoding the FGFR2 receptor, revealed wild-type sequence only. Subsequently, molecular analysis of the FGFR3 receptor gene identified a heterozygous P250R missense mutation in both the proposita and her mildly affected father. This communication extends the clinical spectrum of the FGFR3 P250R mutation to encompass epidermal hyperplasia and documents the phenomenon of activated FGFR receptors stimulating common downstream developmental pathways, resulting in overlapping clinical outcomes. (C) 2001 Wiley-Liss, Inc.

Transcriptional modulation of mouse mu-opioid receptor distal promoter activity by Sox18

Previously, we reported the presence of dual promoters, referred to as distal (DP) and proximal, with a negative regulatory element between them in the mouse mu -opioid receptor (mor) gene. Here we have identified a positive regulatory element influencing mor DP transcription, which contains multiple consensus binding motifs for Sox factors (sex-determining Sry-like high mobility group box-containing genes). In gel supershift assays, the Sox family member Sox18 bound directly to the multiple Sox consensus binding motifs of the mor DP enhancer. Overexpression of Sox18 cDNA increased luciferase activity regulated by the mor DP, and did so in a Sox18 concentration-dependent manner. In contrast, overexpression of another Sox member, Sox5, triggered no such trans-activation of mor DP-driven luciferase activity or DNA-protein binding activity. These results suggest that Sox18 directly and specifically stimulates mor gene expression, by trans-activating the mor DP enhancer.

The progesterone receptor exon 4 Va1660Leu G/T polymorphism and risk of breast cancer in Australian women

An Alu insertion polymorphism of the progesterone receptor (PR) was reported recently to be associated with a reduced risk of breast cancer, with risks of 0.8- and 0.3-fold associated with the heterozygote and homozygote genotypes, respectively. This intronic variant is considered to be in linkage disequilibrium with an exon 4 hinge region G to T Val660Leu polymorphism. We investigated whether the exon 4 PR polymorphism was associated with breast cancer in Australian women, using a population-based study of 1452 cases and 793 controls, half of whom were

Evolution and developmental expression of nuclear receptor genes in the ascidian Herdmania

Nuclear receptors are a superfamily of metazoan transcription factors that have been shown to be involved in a wide range of developmental and physiological processes. A PCR-based survey of genomic DNA and developmental cDNAs from the ascidian Herdmania identifies eight members of this multigene family. Sequence comparisons and phylogenetic analyses reveal that these ascidian nuclear receptors are representative of five of the six previously defined nuclear receptor subfamilies and are apparent homologues of retinoic acid [NR1B], retinoid X [NR2B], peroxisome proliferator-activated [NR1C], estrogen related [NR3B], neuron-derived orphan (NOR) [NR4A3], nuclear orphan [NR4A], TR2 orphan [NR2C1] and COUP orphan [NR2F3] receptors. Phylogenetic analyses that include the ascidian genes produce topologically distinct trees that suggest a redefinition of some nuclear receptor subfamilies. These trees also suggest that extensive gene duplication occurred after the vertebrates split from invertebrate chordates. These ascidian nuclear receptor genes are expressed differentially during embryogenesis and metamorphosis.

Selective loss of expression of glutamate GluR2/R3 receptor subunits in cerebellar tissue from a patient with olivopontocerebellar atrophy

Expression of the mRNAs encoding the astrocytic (EAAT1, EAAT2) and neuronal (EAAT3, EAAT4) excitatory amino acid transporters and the AMPA-type glutamate receptor subunits GluR2 and GluR3 was investigated in postmortem cerebellar extracts from a patient with olivopontocerebellar atrophy (OPCA) and in material from three age-matched controls. Decreased expression in the steady state level of EAAT4 mRNA in the OPCA sample was correlated with the selective loss of Purkinje cells. Neuropathological evaluation revealed reactive gliosis and concomitantly increased expression of the mRNA encoding astrocytic glial fibrillary acidic protein (GFAP). Expression of the mRNAs encoding the AMPA receptor subunits GluR2 and GluR3 subunits was found to be decreased in OPCA suggesting that excitotoxic mechanism could play a role in the pathogenesis of the selective neuronal cell death in this disorder.

H-1 NMR study of the states of water in equilibrium Poly(HEMA-co-THFMA) hydrogels

The spin-spin relaxation times, T-2, of hydrated samples of poly(hydroxymethyl methacrylate), PHEMA, poly(tetrahydrofurfuryl methacrylate),PTHFMA, and the,corresponding HEMA-THFMA copolymers have been examined to probe the states of,the imbibed water in these polymers. The decay in the transverse magnetization of water. in fully hydrated samples of PHEMA, PTHFMA, and copolymers of HEMA and THFMA was described by a multiexponential function. The short component of T-2 was interpreted as water molecules that were strongly interacting with the polymer chains. The intermediate component of T-2 was assigned to water residing in the porous structure of the samples. The long component of T-2 was believed to arise from water residing in the remnants of cracks formed in the polymer network during water sorption.

Copolymers obtained by the radiation-induced grafting of styrene onto poly(tetrafluoroethylene-co-perfluoropropylvinyl ether) substrates. 1. Preparation and structural investigation

A study has been made to investigate the radiation grafting of styrene onto poly(tetrafluoroethylene-co-perfluoropropylvinyl ether) (PFA) substrates, using the simultaneous irradiation method. Two PFA polymers of different comonomer perfluoropropyl vinyl ether (PPVE) content and degree of crystallinity were used. Effects of grafting conditions such as monomer concentrations, type of solvent, dose rate, and irradiation dose on the grafting yield were investigated. Of the six different solvents used, the most efficient in terms of increasing grafting yield were dichloromethane, benzene, and methanol. The degree of grafting increased with increasing radiation dose up to 500 kGy, stabilizing above this dose. However, the grafting yield decreased with an increase in the dose rate. The grafting of styrene onto the PFA substrates was confirmed by FTIR-ATR and micro-Raman spectroscopy, The increase in the overall grafting yield was accompanied by a proportional increase in the penetration depth of the grafts into the substrate.

PFG-NMR measurements of the self-diffusion coefficients of water in equilibrium poly(HEMA-co-THFMA) hydrogels

The self-diffusion coefficients for water in a series of copolymers of 2-hydroxyethyl methacrylate, HEMA, and tetrahydrofurfuryl methacrylate, THFMA, swollen with water to their equilibrium states have been studied at 310 K using PFG-NMR. The self-diffusion coefficients calculated from the Stejskal-Tanner equation, D-obs, for all of the hydrated polymers were found to be dependent on the NMR storage time, as a result of spin exchange between the proton reservoirs of the water and the polymers, reaching an equilibrium plateau value at long storage times. The true values of the diffusion coefficients were calculated from the values of D-obs, in the plateau regions by applying a correction for the fraction of water protons present, obtained from the equilibrium water contents of the gels. The true self-diffusion coefficient for water in polyHEMA obtained at 310 K by this method was 5.5 x 10(-10) m(2) s(-1). For the copolymers containing 20% HEMA or more a single value of the self-diffusion coefficient was found, which was somewhat larger than the corresponding values obtained for the macroscopic diffusion coefficient from sorption measurements. For polyTHFMA and copolymers containing less than 20% HEMA, the PFG-NMR stimulated echo attenuation decay curves and the log-attenuation plots were characteristic of the presence of two diffusing water species. The self-diffusion coefficients of water in the equilibrium-hydrated copolymers were found to be dependent on the copolymer composition, decreasing with increasing THFMA content.

Spectroscopic study of the penetration depth of grafted polystyrene onto poly (tetrafluoroethylene-co-perfluoropropylvinylether) substrates. 1. Effect of grafting conditions

This study concerns the radiation grafting of styrene onto poly(tetrafluoroethylene-co-perfluoropropylvinylether) (PFA) substrates and the penetration depth of the graft. Grafting was obtained by the simultaneous irradiation method, and the spectroscopic analysis was made with the micro-Raman technique. Effects of grafting conditions such as the type of solvent, dose rate, and irradiation dose on the grafting yield were investigated. Of the different solvents used, the most efficient in terms of increasing grafting yield were dichloromethane, benzene, and methanol, respectively. A mixture of methanol and dichloromethane used as a solvent for styrene achieved a higher degree of grafting and concentration of grafted polystyrene onto the surface of PFA substrates than solutions of the monomer in the separate solvents. The degree of grafting increased with increasing radiation dose up to 500 kGy, stabilizing above this dose. However, the grafting yield decreased with an increase in the dose rate. The increase in the overall grafting yield was accompanied by a proportional increase in the penetration depth of the grafts into the substrate. (C) 2002 Wiley Periodicals, Inc.