Purification and characterization of a receptor for human parathyroid hormone and parathyroid hormone-related peptide

The human parathyroid hormone (PTH) receptor (hPTH1R), containing a 9-amino acid sequence of rhodopsin at its C terminus, was transiently expressed in COS-7 cells and solubilized with 0.25% n-dodecyl maltoside. Approximately 18 microg of hPTH1R were purified to homogeneity per mg of crude membranes by single-step affinity chromatography using 1D4, a monoclonal antibody to a rhodopsin epitope. The N terminus of the hPTH1R is Tyr(23), consistent with removal of the 22-amino acid signal peptide. Comparisons of hPTH1R by quantitative immunoblotting and Scatchard analysis revealed that 75% of the receptors in membrane preparations were functional; there was little, if any, loss of functional receptors during purification. The binding affinity of the purified hPTH1R was slightly lower than membrane-embedded hPTH1R (K(d) = 16.5 +/- 1.3 versus 11.9 +/- 1.9 nm), and the purified receptors bound rat [Nle(8,21),Tyr(34)]PTH-(1-34)-NH(2) (PTH-(1-34)), and rat [Ile(5),Trp(23),Tyr(36)]PTHrP-(5-36)-NH(2) with indistinguishable affinity. Maximal displacement of (125)I-PTH-(1-34) binding by rat [alpha-aminoisobutyric acid (Aib)(1,3),Nle(8),Gln(10),Har(11),Ala(12),Trp(14),Arg(19),Tyr(21)]PTH-(1-21)-NH(2) and rat [Aib(1,3),Gln(10),Har(11),Ala(12),Trp(14)]PTH-(1-14)-NH(2) of 80 and 10%, respectively, indicates that both N-terminal and juxtamembrane ligand binding determinants are functional in the purified hPTH1R. Finally, PTH stimulated [(35)S]GTP gamma S incorporation into G alpha(s) in a time- and dose-dependent manner, when recombinant hPTH1R, G alpha(s)-, and beta gamma-subunits were reconstituted in phospholipid vesicles. The methods described will enable structural studies of the hPTH1R, and they provide an efficient and general technique to purify proteins, particularly those of the class II G protein-coupled receptor family.     

Distinct molecular basis for differential sensitivity of the serotonin type 3A receptor to ethanol in the absence and presence of agonist

Ethanol can potentiate serotonin type 3 (5-HT(3)) receptor-mediated responses in various neurons and in cells expressing 5-HT(3A) receptors. However, the molecular basis for alcohol modulation of 5-HT(3) receptor function has not been determined. Here we report that point mutations of the arginine at amino acid 222 in the N-terminal domain of the 5-HT(3A) receptor can alter the EC(50) value of the 5-HT concentration-response curve. Some point mutations at amino acid 222 resulted in spontaneous opening of the 5-HT(3A) receptor channel and an inward current activated by ethanol in the absence of agonist. Among these mutant receptors, the amplitude of the current activated by ethanol in the absence of agonist was correlated with the amplitude of the current resulting from spontaneous channel openings, suggesting that the sensitivity of the receptor to ethanol in the absence of agonist is, at least in part, dependent on the preexisting conformational equilibrium of the receptor protein. On the other hand, point mutations that conferred greater sensitivity to ethanol potentiation of agonist-activated responses were less sensitive or insensitive to ethanol in the absence of agonist. For these receptors, the magnitude of the potentiation of agonist-activated responses by ethanol was inversely correlated with the EC(50) values of the 5-HT concentration-response curves, suggesting that these mutations may modulate ethanol sensitivity of the receptor by altering the EC(50) value of the receptor. Thus, distinct molecular processes may determine the sensitivity of 5-HT(3A) receptors to ethanol in the absence and presence of agonist.     

Association of meteorological and day-of-the-week factors with emergency hospital admissions in Fukuoka, Japan

We carried out a statistical study of the influence of meteorological and day-of-the-week factors on the intrinsic emergency patients transported to hospitals by ambulance. Multiple piecewise linear regression analysis was performed on data from 6,081 emergency admissions for 1 year between April 1997 and March 1998 in Fukuoka, Japan. The response variable was the daily number of emergency patients admitted with three types of disease: cerebrovascular, respiratory and digestive diseases. The results showed that the number of emergency patients admitted daily with cerebrovascular disease was significantly associated with temperature on the day of admission and whether the day was Sunday. As it became colder than 12 degrees C, emergency admissions of patients with cerebrovascular disease increased drastically, reaching a plateau at 4 degrees C. On the 3rd and 7th days after the temperature fell below 10 degrees C, the daily admission of patients with respiratory disease significantly increased. We also observed a weak association between emergency admissions of patients suffering from digestive disease and rising barometric pressure on the day of admission.     

Main polyol dehydrogenase of Gluconobacter suboxydans IFO 3255, membrane-bound D-sorbitol dehydrogenase,that needs product of upstream gene,sldB, for activity

The D-sorbitol dehydrogenase gene, sldA, and an upstream gene, sldB, encoding a hydrophobic polypeptide, SldB, of Gluconobacter suboxydans IFO 3255 were disrupted in a check of their biological functions. The bacterial cells with the sldA gene disrupted did not produce L-sorbose by oxidation of D-sorbitol in resting-cell reactions at pHs 4.5 and 7.0, indicating that the dehydrogenase was the main D-sorbitol-oxidizing enzyme in this bacterium. The cells did not produce D-fructose from D-mannitol or dihydroxyacetone from glycerol. The disruption of the sldB gene resulted in undetectable oxidation of D-sorbitol, D-mannitol, or glycerol, although the cells produced the dehydrogenase. The cells with the sldB gene disrupted produced more of what might be signal-unprocessed SldA than the wild-type cells did. SldB may be a chaperone-like component that assists signal processing and folding of the SldA polypeptide to form active D-sorbitol dehydrogenase.     

Heat-inducible transgenic expression in the silkmoth Bombyx mori

Germline transformation with new transposon vectors now enables causal tests of gene function via ectopic protein expression or RNA interference in non-drosophilid insects. The problem remains of how to drive the transgene expression in vivo. We employed germline transformation using the piggyBac 3xP3-EGFP vector to test whether the Drosophila heat shock hsp70 promoter will be active in the live silkworm. We modified the original vector by cloning the coding sequence for Bombyx nuclear receptor Ftz-F1 between the hsp70 promoter and the terminator. Three independent transgenic lines expressing the Pax-6-driven EGFP marker in larval and adult photoreceptors were obtained with efficiencies of up to 1.7% of fertile G0 adults that gave GFP-positive progeny. Chromosomal integration of the transposon was confirmed with inverse PCR. Heat induction of the transgenic BmFtz-F1 was proven at both the mRNA and protein levels. RT-PCR data showed that the Drosophila heat shock promoter was functional in all three transgenic lines. Although basal activity was apparent at 25 degrees C, 1 h at 42 degrees C induced BmFtz-F1 mRNA at different stages of development and in diverse tissues. The relative levels of induction differed among the transgenic lines. Northern blot hybridization detected transgenic BmFtz-F1 only after heat shock and low levels of the mRNA were still present 6 h after the heat treatment. Immunostaining of epidermis using anti-BmFtz-F1 antibody showed a clear increase of nuclear signal 90 min after a heat shock.     

Regulation of galectin-9 expression and release in Jurkat T cell line cells

Ecalectin/galectin-9 was recently described as a novel eosinophil chemoattractant highly expressed in immune tissues. We investigated the regulation of galectin-9 expression and release in Jurkat (a T cell line) cells. We demonstrated that medium and long-sized galectin-9 isoforms were constitutively expressed, and phorbol 12-myriastate 13-acetate (PMA) upregulated the level of galectin-9 mRNA in Jurkat cells. Western blotting and flow cytometry analyses revealed that PMA stimulation resulted in the upregulation of both intracellular and surface galectin-9 protein. The stimulated Jurkat cells simultaneously released evident eosinophil chemoattractant activity (ECA). Main ECA was adsorbed by both lactose and anti-galectin-9 antibody affinity column, suggesting that the ECA was ascribed to galectin-9. When Jurkat cells were stimulated with PMA in the presence of a BB94, a matrix metalloproteinase (MMP) inhibitor, but not tissue inhibitor of metalloproteinase-1 (TIMP-1), the release of galectin-9 was suppressed in a dose-dependent manner. We further found that calphostin c, a protein kinase c (PKC) inhibitor, weakly but significantly suppressed the release of galectin-9. The present data suggested that galectin-9 production in Jurkat cells is provoked by the stimulation with PMA and that some MMP and PKC is, at least, partly involved in the release of galectin-9 from Jurkat cells.     

Two flavonoid glucosyltransferases from Petunia hybrida: molecular cloning,biochemical properties and developmentally regulated expression

Two flavonoid glucosyltransferases, UDP-glucose:flavonoid 3-O-glucosyltransferase (3-GT) and UDP-glucose: anthocyanin 5-O-glucosyltransferase (5-GT), are responsible for the glucosylation of anthocyani(di)ns to produce stable molecules in the anthocyanin biosynthetic pathway. The cDNAs encoding 3-GT and 5-GT were isolated from Petunia hybrida by hybridization screening with heterologous probes. The cDNA clones of 3-GT, PGT8, and 5-GT, PH1, encode putative polypeptides of 448 and 468 amino acids, respectively. A phylogenetic tree based on amino acid sequences of the family of glycosyltransferases from various plants shows that PGT8 belongs to the 3-GT subfamily and PH1 belongs to the 5-GT subfamily. The function of isolated cDNAs was identified by the catalytic activities for 3-GT and 5-GT exhibited by the recombinant proteins produced in yeast. The recombinant PGT8 protein could convert not only anthocyanidins but also flavonols into the corresponding 3-O-glucosides. In contrast, the recombinant PH1 protein exhibited a strict substrate specificity towards anthocyanidin 3-acylrutinoside, comparing with other 5-GTs from Perilla frutescens and Verbena hybrida, which showed broad substrate specificities towards several anthocyanidin 3-glucosides. The mRNA expression of both 3-GT and 5-GT increased in the early developmental stages of P. hybrida flower, reaching the maximum at the stage before flower opening. Southern blotting analysis of genomic DNA indicates that both 3-GT and 5-GT genes exist in two copies in P. hybrida, respectively. The results are discussed in relation to the molecular evolution of flavonoid glycosyltransferases.     

Use of a biomimetic chromatographic stationary phase for study of the interactions occurring between inorganic anions and phosphatidylcholine membranes

A liquid chromatographic method for the study of ion-membrane interactions is reported. A phosphatidylcholine biomimetic stationary phase was established by loading dimyristoylphosphatidylcholine (DMPC) onto a reversed-phase octadecylsilica packed column. This column was then used to study the interaction of some inorganic anions with the stationary phase by UV and conductivity detection. Ten inorganic anions were selected as model ions and were analyzed with the proposed chromatographic system. Anion-DMPC interactions of differing magnitudes were observed for all of the model anions. Perchlorate-DMPC interactions were strongest, followed by thiocyanate-DMPC, iodide-DMPC, chlorate-DMPC, nitrate-DMPC, bromide-DMPC, chloride-DMPC, fluoride-DMPC, and then sulfate-DMPC. Cations in the eluent, especially H(+) ions and divalent cations such as Ca(2+), showed strong effects on anion-DMPC interactions. The chromatographic data suggest that DMPC interacts with both the anions and the cations. Anion-DMPC interactions were dependent on the surface potential of the stationary phase: at low surface potentials anion-DMPC interactions were predominantly solvation dependent in nature whereas at more positive surface potentials anion-DMPC interactions were predominantly electrostatic in nature. Cation-DMPC interactions served to raise the surface potential, causing the anion-DMPC interactions to vary from solvation dependent to electrostatic. The chromatographic data were used to provide quantitative estimates of the enthalpies of the anion-DMPC interactions.