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Pear (Pyrus L.) is one of the most important temperate fruit crops worldwide, with considerable economic value and significant health benefits. Red-skinned pears have an attractive appearance and relatively high anthocyanin accumulation, and are especially favored by customers. Abnormal weather conditions usually reduce the coloration of red pears. The application of exogenous sucrose obviously promotes anthocyanins accumulation in ‘Kuerle Xiangli’ (Pyrus sinkiangensis Yü); however, the underlying molecular mechanism of sucrose-mediated fruit coloration remains largely unknown. In this study, comprehensive transcriptome analysis was performed to identify the essential regulators and pathways associated with anthocyanin accumulation. The differentially expressed genes enriched in Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes items were analyzed. The transcript levels of some anthocyanin biosynthetic regulatory genes and structural genes were significantly induced by sucrose treatment. Sucrose application also stimulated the expression of some sugar transporter genes. Further RT-qPCR analysis confirmed the induction of anthocyanin biosynthetic genes. Taken together, the results revealed that sucrose promotes pear coloration most likely by regulating sugar metabolism and anthocyanin biosynthesis, and this study provides a comprehensive understanding of the complex molecular mechanisms underlying the coloration of red-skinned pear.

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Anthocyanin accumulation in vacuoles results in red coloration in pear peels. Glutathione S-transferase (GST) proteins have emerged as important regulators of anthocyanin accumulation. Here, a total of 57 PcGST genes were identified in the European pear ‘Bartlett’ (Pyrus communis) through comprehensive genomic analysis. Phylogenetic analysis showed that PcGST genes were divided into 10 subfamilies. The gene structure, chromosomal localization, collinearity relationship, cis-elements in the promoter region, and conserved motifs of PcGST genes were analyzed. Further research indicated that glutamic acid (Glu) can significantly improve anthocyanin accumulation in pear peels. RNA sequencing (RNA-seq) analysis showed that Glu induced the expression of most PcGST genes, among which PcGST57 was most significantly induced. Further phylogenetic analysis indicated that PcGST57 was closely related to GST genes identified in other species, which were involved in anthocyanin accumulation. Transcript analysis indicated that PcGST57 was expressed in various tissues, other than flesh, and associated with peel coloration at different developmental stages. Silencing of PcGST57 by virus-induced gene silencing (VIGS) inhibited the expression of PcGST57 and reduced the anthocyanin content in pear fruit. In contrast, overexpression of PcGST57 improved anthocyanin accumulation. Collectively, our results demonstrated that PcGST57 was involved in anthocyanin accumulation in pear and provided candidate genes for red pear breeding.

Figure S1. |Δ(SNP-index)| graph derived from the modified QTL-seq analysis of mapping for the red skin trait of Asian pear. The X-axis indicates the position of the 17 pseudo-chromosomes and the Y-axis indicates the |Δ(SNP-index)|. (PPTX 278 kb)

Figure S1. Percentages of different motifs among mono-(a), di- (b) and tri- (c) nucleotide repeats in the â Dangshansuliâ pear genome. (PDF 183Â kb)

KEGG annotation details of unigenes (for qPCR). (DOCX 12 kb)

The pigmentation patterns of ‘Mantianhong’ (Pyrus pyrifolia Nakai) and ‘Cascade’ (Pyrus communis L.) that develop under bagging and postharvest UV-B/visible irradiation conditions were evaluated in this study. The results showed that, unlike ‘Cascade,’ in which almost no anthocyanin was detected, ‘Mantianhong’ developed good red coloration under bagging treatment and postharvest irradiation. The activity of PAL and DFR increased during anthocyanin accumulation. The expression of PpCHS3, PpCHI3, PpUFGT1, and PyMYB10 was up-regulated in ‘Mantianhong’ but not in ‘Cascade’ during both treatments. This suggests that these four genes may be limiting factors for peel coloration in ‘Cascade.’ The expression patterns of gene family members were also different during fruit red coloration, suggesting that different gene family members may have distinct functions during pear development. The results obtained in this study are helpful in understanding the molecular mechanism of anthocyanin accumulation in red pears and can serve as the basis for the development of marker-assisted selection or for anthocyanin metabolic engineering of pears.

Orphan G-protein-coupled receptors are a large class of receptors whose cognate ligands are unknown. SP9155 (also referred to as AQ27 and GPR103) is an orphan G-protein-coupled receptor originally cloned from a human brain cDNA library. SP9155 was found to be predominantly expressed in brain, heart, kidney, retina, and testis. Phylogenetic analysis shows that SP9155 shares high homology with Orexin, NPFF, and cholecystokinin (CCK) receptors, but identification of the endogenous ligand for SP9155 has not been reported. In this study, we have used a novel method to predict peptides from genome data bases. From these predicted peptides, a novel RF-amide peptide, P52 was shown to selectively activate SP9155-transfected cells. We subsequently cloned the precursor gene of the P52 ligand and characterized the activity of other possible peptides encoded by the precursor. This revealed an extended peptide, P518, which exhibited high affinity for SP9155 (EC50 = 7 nm). mRNA expression analysis revealed that the peptide P518 precursor gene is predominantly expressed in various brain regions, coronary arteries, thyroid and parathyroid glands, large intestine, colon, bladder, testes, and prostate. These results indicate the existence of a novel RF-amide neuroendocrine peptide system, and suggest that SP9155 is likely the relevant G-protein-coupled receptor for this peptide.

The diverse physiological actions of galanin are thought to be mediated through activation of galanin receptors (GalRs). We report the genomic and cDNA cloning of a mouse GalR that possesses a genomic structure distinct from that of GalR1 and encodes a functional galanin receptor. The mouse GalR gene consists of two exons separated by a single intron within the protein-coding region. The splicing site for the intron is located at the junction between the third transmembrane domain and the second intracellular loop. The cDNA encodes a 370-amino acid putative G protein-coupled receptor that is markedly different from human GalR1 and rat GalR3 (38 and 57%) but shares high homology with rat GalR2 (94%). In binding studies utilizing membranes from COS-7 cells transfected with mouse GalR2 cDNA, the receptor displayed high affinity (K(D) = 0.47 nM) and saturable binding with 125I-galanin (Bmax = 670 fmol/mg). The radioligand binding can be displaced by galanin and its analogues in a rank order: galanin approximately = M40 approximately = M15 approximately = M35 approximately = C7 approximately = galanin(2-29) approximately = galanin(1-16) >> galanin(10-29) approximately = galanin(3-29), which resembles the pharmacological profile of the rat GalR2. Receptor activation by galanin in COS-7 cells stimulated phosphoinositide metabolism, which was not reversed by pertussis toxin. Thus, the galanin receptor encoded in the cloned mouse GalR gene is the type 2 galanin receptor and is active in both ligand binding and signaling assays.

Melanin-concentrating hormone (MCH) is a neuropeptide highly expressed in the brain that regulates several physiological functions mediated by receptors in the G protein-coupled receptor family. Recently an orphan receptor, SLC-1, has been identified as an MCH receptor (MCH-R1). Herein we identify and characterize a novel receptor for human MCH (MCH-R2). The receptor is composed of 340 amino acids encoded by a 1023-base pair cDNA and is 35% homologous to SLC-1. (125)I-MCH specifically bound to Chinese hamster ovary cells stably expressing MCH-R2. MCH stimulated dose-dependent increases in intracellular free Ca(2+) and inositol phosphate production in these cells but did not affect cAMP production. The pharmacological profile for mammalian MCH, [Phe(13),Tyr(19)]MCH, and salmon MCH at MCH-R2 differed compared with MCH-R1 as assessed by intracellular signaling and radioligand binding assays. The EC(50) in signaling assays and the IC(50) in radioligand binding assays of salmon MCH was an order of magnitude higher than mammalian MCH at MCH-R2. By comparison, the EC(50) and IC(50) values of salmon MCH and mammalian MCH at MCH-R1 were relatively similar. Blot hybridization revealed exclusive expression of MCH-R2 mRNA in several distinct brain regions, particularly in the cortical area, suggesting the involvement of MCH-R2 in the central regulation of MCH-mediated functions.

Galanin, a 29 - 30 amino acid neuropeptide, is widely distributed in both the central and peripheral nervous systems. The peptide is unique to other families of neuropeptides and is highly conserved in the N-terminal portion among several mammalian species. Cloning efforts have successfully yielded three galanin receptor (GalR) subtypes that have high affinities for galanin. A recently isolated galanin-like peptide has high affinity for at least one of the three receptors. Galanin modulates a variety of physiological processes via activation of these receptor subtypes. In the CNS, galanin alters the release of several neurotransmitters. For example, it has an inhibitory effect on the release of acetylcholine (ACh) in the hippocampus, suggesting a potential role in the modulation of memory and learning. When injected directly into the ventricles of the brain, galanin increases food intake and alters plasma levels of hormones involved in the maintenance of nutrient and body weight homeostasis. The peptide a...

Leukotriene B(4) (LTB(4)) is a product of eicosanoid metabolism and acts as an extremely potent chemotactic mediator for inflammation. LTB(4) exerts positive effects on the immigration and activation of leukocytes. These effects suggest an involvement of LTB(4) in several diseases: inflammatory bowel disease, psoriasis, arthritis, and asthma. LTB(4) elicits actions through interaction with one or more cell surface receptors that lead to chemotaxis and inflammation. One leukotriene B(4) receptor has been recently identified (LTB(4)-R1). In this report we describe cloning of a cDNA encoding a novel 358-amino acid receptor (LTB(4)-R2) that possesses seven membrane-spanning domains and is homologous (42%) and genetically linked to LTB(4)-R1. Expression of LTB(4)-R2 is broad but highest in liver, intestine, spleen, and kidney. In radioligand binding assays, membranes prepared from COS-7 cells transfected with LTB(4)-R2 cDNA displayed high affinity (K(d) = 0.17 nm) for [(3)H]LTB(4). Radioligand competition assays revealed high affinities of the receptor for LTB(4) and LTB(5), and 20-hydroxy-LTB(4), and intermediate affinities for 15(S)-HETE and 12-oxo-ETE. Three LTB(4) receptor antagonists, 14,15-dehydro-LTB(4), LTB(4)-3-aminopropylamide, and U-75302, had high affinity for LTB(4)-R1 but not for LTB(4)-R2. No apparent affinity binding for the receptors was detected for the CysLT1-selective antagonists montelukast and zafirlukast. LTB(4) functionally mobilized intracellular calcium and inhibited forskolin-stimulated cAMP production in 293 cells. The discovery of this new receptor should aid in further understanding the roles of LTB(4) in pathologies in these tissues and may provide a tool in identification of specific antagonists/agonists for potential therapeutic treatments.

Neuromedin U is a neuropeptide prominently expressed in the upper gastrointestinal tract and central nervous system. Recently, GPR66/FM-3 (NmU-R1) was identified as a specific receptor for neuromedin U. A BLAST search of the GenBankTM genomic database using the NmU-R1 cDNA sequence revealed a human genomic fragment encoding a G protein-coupled receptor that we designated NmU-R2 based on its homology to NmU-R1. The full-length NmU-R2 cDNA was subsequently cloned, stably expressed in 293 cells, and shown to mobilize intracellular calcium in response to neuromedin U. This response was dose-dependent (EC50 = 5 nm) and specific in that other neuromedins did not induce a calcium flux in receptor-transfected cells. Expression analysis of human NmU-R2 demonstrated its mRNA to be most highly expressed in central nervous system tissues. Based on these data, we conclude that NmU-R2 is a novel neuromedin U receptor subtype that is likely to mediate central nervous system-specific neuromedin U effects.

Neuromedin U (NmU) is a 25 amino acid peptide prominently expressed in the upper gastrointestinal (GI) tract and central nervous system. It is highly conserved throughout evolution and induces smooth muscle contraction in a variety of species. Our understanding of NmU biology has been limited because the identity of its receptor was unknown. Here we demonstrate that GPR66/FM-3 is specifically stimulated by NmU, causing the mobilization of intracellular calcium. This response was dose-dependent (EC(50) = 10 nM) and specific in that none of over 1000 ligands tested, including other neuromedins (NmB, C, L, K, N), induced a calcium flux in GPR66/FM-3-transfected cells. The GPR66/FM-3 mRNA is prominently expressed in the upper GI tract of humans, as is the mRNA for NmU, consistent with role for this receptor-ligand pair in regulating the function of this organ system. In addition, we show that whereas neuromedin U is expressed by monocytes and dendritic cells, GPR66/FM-3 is expressed by T cells and NK cells. These data suggest a previously unrecognized role for NmU as an immunoregulatory molecule.

The 9th International Conference on Obesity, NIDDM, Adipogenesis and Insulin Resistance was held between 3rd and 5th April, 2000 in London, UK. The three day conference was attended by more than 100 delegates from both academic and industrial institutions. Conference topics covered the use of genetics in obesity and diabetes, etiology and implications of treatment for obesity, progress in identification of new obesity and Type 2 diabetes drug targets, islet cell targets, progress in drugs for insulin enhancement and sensitisation, adipogenesis and insulin resistance. Below are meeting highlights closely related to new drug developments and drug target identification in these therapeutic areas.

The neuropeptide galanin mediates a number of diverse physiological and pathophysiological actions via interaction with membrane-bound receptors. The role that membrane cholesterol plays in modulating the interaction between galanin and one of the three cloned galanin receptor subtypes (GalR2) expressed in Chinese hamster ovary (CHO) cells was examined. Reduction of membrane cholesterol by treatment with methyl-beta-cyclodextrin (CD) or by culturing cells in lipoprotein-deficient serum markedly decreased galanin binding to the receptor. Addition of cholesterol back to CD-treated, cholesterol-depleted membranes restored galanin binding to control levels. Hill analysis suggests that the GalR2 binds multiple molecules of cholesterol (n >/= 3) in a positively cooperative manner. This interaction appears to be cholesterol-specific as only cholesterol and a limited number of cholesterol analogues were able to rescue galanin binding. The inability of some of these analogues to rescue the binding activity also suggests that binding of galanin to GalR2 is independent of membrane fluidity as, like cholesterol, cholesterol analogues generally rigidize membranes. In addition, treatment of the membranes with other modulators of membrane fluidity, e.g. ethanol, did not affect galanin binding to the GalR2. In contrast, treatment of membranes, with filipin, a molecule that clusters cholesterol within the membranes, or with cholesterol oxidase resulted in markedly reduced galanin binding. Incubation of membranes with 100 microM GTP-gamma-S did not alter the IC(50) for CD in the prebinding assay treatment suggesting that the effect of cholesterol was independent of G protein interaction. Preincubation of intact cells with CD also drastically impaired the ability of galanin to activate intracellular inositol phosphate accumulation in GalR2-transfected CHO cells. These data detail a new mechanism for the regulation of galanin receptor signaling which may link altered functions of GalRs with abnormal cholesterol metabolism.

The 8th Obesity & Non-Insulin Dependent Diabetes Mellitus and Novel Drug Developments Conference organised by International Business Communication, Inc. was held on 19th – 20th, April 1999 in London, followed by an one-day symposium on Novel Drug Developments for NIDDM & Insulin Resistance. More than 100 delegates from both academic and industrial institutes attended the two meetings. The presentations provided insights into the understanding of mechanisms and developments of novel drugs for treatments of obesity and Type 2 diabetes. This review offers a general overview of the fields in appetite suppression, thermogenesis and insulin sensitisation. Discussions focused on several emerging therapeutic areas, including novel compound developments and target identification of receptors, proteins and viruses with the use of conventional methods and recently emerged technologies, such as bioinformatics and invertebrate modelling of human systems.

The neuropeptide galanin mediates a diverse array of physiological functions through activation of specific receptors. Roles of the three recently cloned galanin receptors (GalRs) in rat intestinal contraction and food intake were examined using GalR-selective ligands and the results were compared with the pharmacological profiles of defined GalRs. The action profile of these ligands in jejunal contraction resembled only that of GalR2 and only a high level of GalR2 mRNA was detected in the tissue, supporting GalR2 as the receptor mediating jejunal contraction. The action profile for food intake in rats excluded GalR2, GalR3 and the putative pituitary galanin receptor as the `feeding receptor', suggesting that either GalR1 or an unidentified GalR is responsible for mediating this function.

The organic extract of the fermentation broth of a fungus was found to contain a depsipeptide SCH 58149 (1), containing three amino acids and a β-hydroxy acid, by spectroscopic studies. The amino acids were phenyl alanine, alanine and leucine and the β-hydroxy acid is 3-hydroxy-4-methyl octanoic acid. SCH 58149 exhibited weak activity against cholesterol ester transfer protein (CETP) with an IC50 of 50 μM.

Galanin, a 29-30 amino acid neuropeptide, is found in the central and peripheral nervous systems and displays several important physiological activities. The actions are believed to be mediated through distinct G protein-coupled receptors. To date, two galanin receptor subtypes have been cloned. In this report, we describe the cloning and expression of a cDNA encoding a novel galanin receptor (GalR3). The receptor has 370 amino acids and shares 36 and 54% homology with the rat GalR1 and GalR2 receptors. 125I-Porcine galanin binds the rat GalR3 receptor expressed in COS-7 cells with high affinity (Kd = 0.6 nM) and could be displaced by galanin and galanin fragments and galanin-chimeric peptides. The pharmacological profile of this novel receptor is distinct from those of GalR1 and GalR2, revealing different pharmacophores within galanin for the three galanin receptor subtypes. Northern blot analysis showed expression in heart, spleen, and testis. Unlike GalR1 and GalR2, no expression of GalR3 was detectable in the brain, suggesting that GalR3 may mediate some of the peripheral functions of galanin.

The synthesis and CETP inhibitory activity of a series of compounds related to wiedendiols ( 1a,1b ) are reported. It is proposed that a two point pharmacophore consisting of a catechol group and a large hydrophobic anchor is necessary for activity.

The cholesteryl ester transfer protein (CETP) binds to plasma lipoproteins and transfers neutral lipids between them. Previous studies showed that lipoprotein binding involves ionic interactions between CETP and lipoproteins, with increased binding of CETP to lipoproteins carrying increased negative charge. In order to understand the molecular determinants of lipoprotein binding in CETP, site-directed mutagenesis was carried out on positively charged amino acids within and outside regions of conserved sequence in the putative family of lipid transfer/lipopolysaccharide (LPS) binding proteins (LT/LBP). Within the conserved regions, two mutant proteins, K233A and R259D, were well secreted by the transfected cells but showed markedly reduced cholesteryl ester transfer activity. Separating the bound from free CETP by gel filtration after incubation with HDL, HDL binding by K233A was found to be impaired, suggesting that the binding deficiency of the mutant may be responsible for decreased transfer activity. Kinetic analysis showed a marked increase in the apparent Km but no change in Vmax, consistent with a lipoprotein binding defect. Thus, within CETP, K233 and R259 play an essential role in cholesteryl ester transfer activity probably by mediating binding of CETP to lipoproteins. Sequence alignment of CETP, phospholipid transfer protein, LPS binding protein, and bactericidal permeability-inducing protein showed that K223 and R259 were strictly conserved as positively charged amino acids, suggesting a common function within the LT/LBP gene family.

The plasma cholesteryl ester transfer protein (CETP, 476 amino acids) transfers cholesteryl ester (CE) from high density lipoprotein (HDL) to triglyceride-rich lipoproteins and plays a major role in HDL catabolism. Using deletional and site-directed mutagenesis, we previously showed that the carboxyl terminus of human CETP comprises the epitope of a neutralizing monoclonal antibody and is necessary for neutral lipid transfer activity. To assess the nature of the involvement of the COOH terminus in cholesteryl ester transfer activity, we characterized a deletion mutant of CETP lacking amino acid residues 470-475 in terms of CE transfer kinetics, association with HDL, and capacity to bind CE, triglyceride (TG), and phosphatidylcholine (PC). Kinetic analysis indicated a major catalytic defect of the deletion mutant, as shown by markedly decreased maximum cholesteryl ester transfer activities (apparent Vmax) for donor (HDL) and acceptor (low density lipoprotein (LDL)) lipoproteins but there were no significant changes of concentrations of the donor and acceptor at 50% Vmax (apparent Km ). The binding of CETP to HDL, as determined by native gel electrophoresis, was similar for wild-type and mutant protein. When egg PC/CE vesicles were incubated with wild type CETP and then separated by gel filtration chromatography, there was maximum binding of about 1 mol of CE/mol of CETP. Under similar conditions the mutant CETP bound 0.09-0.37 mol of CE/mol of protein. Similarly, when egg PC/TG vesicles were incubated with the CETP proteins, there was a maximum binding of 0.5 mol of triglyceride/mol of wild-type CETP, whereas there was only 0.00-0.07 mol of TG/mol of deletion mutant. The binding of phosphatidylcholine was similar for wild-type and the deletion mutant. The studies suggest that amino acids 470-475 (forming part of a COOH-terminal amphipathic helix) are involved in CE and TG binding by CETP but are not required either for the binding of PC by CETP or the association of CETP with HDL. The COOH terminus of CETP may comprise a neutral lipid binding site directly involved in the lipid transfer mechanism.

Plasma cholesteryl ester transfer protein (CETP) mediates the transfer of neutral lipids and phospholipids between the plasma lipoproteins. The deduced M(r) of the CETP polypeptide from the cDNA is 53,000, but in sodium dodecyl sulfate (SDS) gels plasma CETP appears as a broad band containing two different molecular forms of M(r) 65,000-71,000. The purpose of this study was to see if variable N-linked glycosylation could explain the microheterogeneity of CETP. Recombinant CETP (rCETP), derived from stable expression of the CETP cDNA in Chinese hamster ovary (CHO) cells, appeared as a protein doublet comparable to plasma CETP. Digestion of plasma or rCETP with N-glycosidase F (glyco F, to remove N-linked carbohydrates) resulted in the formation of a lower M(r) doublet in which the bottom band approximated the M(r) of the CETP polypeptide. Metabolic labeling of the rCETP with [3H]mannose and [3H]glucosamine, followed by digestion with glyco F, suggested that the top band of the doublet contained residual N-linked carbohydrates resistant to glyco F digestion. To explore this hypothesis further, each of the four potential N-linked glycosylation sites of CETP (at amino acid positions 88, 240, 341, and 396) was eliminated by mutagenesis of asparagine to glutamine. The wild-type (WT) and mutant CETP cDNAs were transiently expressed in COS-7 cells. Each mutant CETP showed a lower M(r) than WT, indicating that all four sites were occupied by N-linked carbohydrate.(ABSTRACT TRUNCATED AT 250 WORDS)

The cholesteryl ester transfer protein (CETP) mediates the transfer of neutral lipids between the plasma lipoproteins. A carboxyl-terminal sequence of CETP was recently shown to form the epitope of a neutralizing monoclonal antibody (TP2) and to be necessary for neutral lipid transfer activity. To determine the role of specific amino acids in the epitope and/or in lipid transfer activity, we made single amino acid substitution mutants between Pro446 and Ser476 by in vitro mutagenesis and expressed the mutants in mammalian cells. The binding of TP2 to CETP was abolished by mutations primarily of polar or charged amino acids that occurred periodically within the sequence between His466 and Leu475 and at amino acid Asp460; however, these mutants had well preserved cholesteryl ester (CE) transfer activity. By contrast, mutants of bulky hydrophobic amino acids in this region (particularly Leu475, Phe471, Leu468, Phe461, and Phe454) showed markedly decreased CE transfer specific activity, but essentially normal binding of TP2. The paradoxical effects on antibody binding and activity could be explained if amino acids determining monoclonal antibody binding and activity are disposed on opposite faces of an amphipathic helix between amino acids 465 and 476. This model was tested by substituting alanine residues for pairs of nonpolar amino acids which would be contiguous on a helix, resulting in low activity mutants equivalent to those produced by deletion of this region. We conclude that the hydrophobic face of a carboxyl-terminal helix of CETP is directly involved in the mechanism of CE transfer, and that TP2 inhibits activity by local sterical hindrance. The general hydrophobic character of this region, imparted by the bulky hydrophobic amino acids Leu and Phe, is important for normal CE transfer.

The cholesteryl ester transfer protein (CETP; 476 amino acids) mediates the transfer of neutral lipids and phospholipids between plasma lipoproteins. Previous studies showed that the epitope of a neutralizing monoclonal antibody (TP2) was located within the C-terminal 26 amino acids (aa) of CETP. To determine possible involvement of this region in lipid transfer activities, we generated six deletion mutants between Arg-451 and Leu-475 by in vitro mutagenesis and expressed mutant proteins in mammalian cells. Only deletion mutants between aa Phe-463 and Leu-475 failed to bind TP2; these mutant proteins were well secreted by cells but showed markedly reduced cholesteryl ester transfer activity. One of the deletion mutants (delta 470-475) showed similar reductions in cholesteryl ester and triglyceride transfer activities but normal or increased phospholipid transfer activity. Limited proteolysis of this mutant protein indicated a similar overall folding pattern to the wild-type protein. Thus, aa between Phe-463 and Leu-475 are necessary for binding TP2. Deletions within this sequence selectively impair neutral lipid transfer activity, suggesting a direct involvement in neutral lipid transfer.

Polarized differential phase and modulation ratios were obtained for 1,6-diphenyl-1,3,5-hexatriene (DPH) in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) vesicles by using multifrequency phase fluorometry. Data were analyzed in terms of both empirical sums-of-exponentials modeling and directly in terms of the orientational distribution functions. The orientational analysis model was used to recover the angular distribution of DPH and the rotational diffusion coefficient in the various membrane model systems throughout the phase transition. A global analysis methodology was utilized to obtain an internally consistent set of parameters that fit all of the data simultaneously. The rank order parameters (P2) and (P4) were extracted from the experimental data, and the angular distribution functions of DPH were calculated. When the time-zero anisotropy (r0) of several sets of data taken at various temperatures were linked in a single global analysis, better recovery of the fourth rank order parameter (P4), diffusion constant D, and r0 was obtained with respect to the unlinked analysis. From these recovered values, a detailed picture concerning the orientational distribution of DPH in membranes as a function of temperature was obtained. The results suggest that a single population of DPH molecules was present in the bilayers with their orientational distributions dependent upon the physical state of the membranes in the pure phases. During the phase transition, a superposition of two populations corresponding to the population of the pure phases was present. As the temperature increased in the transition region, one population was increasing at the expense of the other.

Human plasma cholesteryl ester transfer protein (CETP) enhances transfer and exchange of cholesteryl ester (CE) and triglyceride (TG) between high-density lipoprotein and other lipoproteins. To define regions responsible for the neutral lipid transfer activities at the molecular level, a total of 27 linker insertion mutants at 18 different sites along the CETP molecule were prepared and transiently expressed in a mammalian cell line (COS). The inserted linkers were small (usually 6 bp) and did not interrupt the translational reading frame of the CETP cDNA. Although secretion of each mutant protein was less than that of wild-type CETP, the majority of the mutants had normal cholesteryl ester transfer activity (transfer activity per nanogram of CETP in media). However, insertional alterations in three regions severely impaired CE transfer activity: (1) in the region of amino acids 48-53; (2) at amino acid 165; and (3) in the region of amino acids 373-379. Although the impaired activities could also be a result of globally incorrect folding of these CETP mutants, hydrophobicity analysis and secondary structure predictions tended to exclude this possibility for most of the insertion sites at which insertions resulted in inactivation. The insertion at amino acid 379 occurs immediately after a triplet of lysine residues, suggesting that this region might be involved in an essential step in the mechanism of CE and TG transfer, such as the binding of CETP to phosphatidylcholine molecules in the lipoprotein surface. Effects on TG transfer activity were generally similar to those on CE transfer activity, suggesting a similar structural requirement for both neutral lipid transfer activities.

The rat GalR1 galanin receptor was used as a prototypic G protein-coupled receptor to test the feasibility of heterologous expression in a retrovirus-based system. The system utilizes an independent retroviral vector pMX, a virus-packaging cell line BOSC23 and a pre-B cell line BA/F3 as the host for expression. A polyclonal cell population that expresses high ligand affinity (KD = 0.18 nM) and high level (7 pmol/mg) of GalR1 was generated within days with no drug sensitivity-based selection. The expression represented a 20-fold increase over the expression level of GalR1 achieved in CHO cells. The affinity of galanin for the expressed receptor was decreased by 19-fold in the presence of GTP-γ-S, suggesting that the expression system can produce active galanin receptor functionally coupled to G proteins. The fast and efficient method to generate stable cell lines and to prepared large quantities of receptors may provide a general application for expression of other G protein-coupled receptors.

The diverse physiological functions exerted by the neuropeptide galanin may be regulated by multiple G protein-coupled receptor subtypes and intracellular signaling pathways. Three galanin receptor subtypes (GalRs) have been recently cloned, but the G protein coupling profiles of these receptors are not completely understood. We have generated GalR1- and GalR2-expressing Chinese hamster ovary (CHO) cell lines and systematically examined the potential for these two receptors to couple to the Gs, Gi, Go, and Gq proteins. Galanin did not stimulate an increase in cAMP levels in GalR1/CHO or GalR2/CHO cells, suggesting an inability of either receptor to couple to Gs. Galanin inhibited forskolin-stimulated cAMP production in GalR1/CHO cells by 70% and in GalR2/CHO cells by 30%, suggesting a strong coupling of GalR1 to Gi and a more modest coupling between GalR2 and Gi. GalR1 and GalR2 both mediated pertussis toxin-sensitive MAPK activity (2-3-fold). The stimulation mediated by GalR1 was inhibited by expression of the C-terminus of beta-adrenergic receptor kinase (beta ARKct), which specifically inhibits G beta gamma signaling, but was not affected by the protein kinase C (PKC) inhibitor, bis[indolylmaleimide], or cellular depletion of PKC. In contrast, GalR2-mediated MAPK activation was not affected by beta ARKct expression but was abolished by inhibition of PKC activity. The data demonstrate that GalR1 is coupled to a Gibetagamma signaling pathway to mediate MAPK activation. In contrast, GalR2 utilizes a distinct signaling pathway to mediate MAPK activation, which is consistent with Go-mediated MAPK activation in CHO cells. Galanin was unable to stimulate inositol phosphate (IP) accumulation in CHO or COS-7 cells expressing GalR1. In contrast, galanin stimulated a 7-fold increase in IP production in CHO or COS-7 cells expressing GalR2. The GalR2-mediated IP production was not affected by pertussis toxin, suggesting a linkage of GalR2 with Gq/G11. Thus, the GalR1 receptor appears to activate only the Gi pathway. By contrast, GalR2 is capable of stimulating signaling which is consistent with activation of Go, Gq/G11, and Gi. The differential signaling profiles and the tissue distribution patterns of GalR1 and GalR2 may underlie the functional spectra of galanin action mediated by these galanin receptors and regulate the diverse physiological functions of galanin.

Galanin, a 29–30-amino acid neuropeptide, is widely distributed in central and peripheral systems and mediates a variety of physiological functions. Pharmacological studies have suggested the existence of multiple receptor subtypes but only the type I (GalR1) galanin receptor has been cloned. Now we report the cloning by a combination of sib selection and rapid amplification of cDNA ends of a cDNA encoding a new galanin receptor (GalR2) from rat hypothalamus. The receptor is 372 amino acids in length and shares only 40% homology with the rat GalR1 receptor. It contains seven putative transmembrane domains with the amino and carboxyl termini being least identical to GalR1. Northern blot analyses revealed a 2-kilobase pair mRNA species distributed in several tissues, suggesting a broader functional spectrum than GalR1. 125 I-Labeled human galanin binding to rat GalR2 receptor expressed in COS-1 cells was saturable ( K d = 0.59 nm) and could be displaced by galanin, several galanin fragments, and chimeric peptides. The pharmacological profiles of GalR1 and GalR2 receptors were distinguishable by galanin fragment(2–29), which bound the cloned GalR2 receptor with markedly higher affinity than the GalR1 receptor. Activation of the cloned receptor by galanin led to inhibition of forskolin-stimulated intracellular cAMP production. The cloning of this new receptor subtype should provide further insights into the mechanisms by which galanin mediates its diverse physiological functions. The identification of galanin(2–29) as a receptor-specific ligand should enhance the understanding of specificity of galanin-receptor interactions.

The plasma cholesteryl ester transfer protein (CETP) is known to facilitate the transfer of lipids between plasma lipoproteins. The human CETP gene is a complex locus encompassing 16 exons. The CETP mRNA is found in liver and small intestine as well as in a variety of peripheral tissues. While the CETP cDNA from human adipose tissue was being cloned, a variant CETP cDNA was discovered which excluded the complete sequence encoded by exon 9, but which was otherwise identical to the full-length CETP cDNA, suggesting modification of the CETP gene transcript by an alternative RNA splicing mechanism. RNase protection analysis of tissue RNA confirmed the presence of exon 9 deleted transcripts and showed that they represented a variable proportion of the total CETP mRNA in various human tissues including adipose tissue (25%), liver (33%), and spleen (46%). Transient expression of the exon 9 deleted cDNA in COS cells or stable expression in CHO cells showed that the protein encoded by the alternatively spliced transcript was inactive in neutral lipid transfer, smaller, and poorly secreted compared to the protein derived from the full-length cDNA. Endo H digestion suggested that the inactive, cell-associated protein was present within the endoplasmic reticulum. The experimentsmore » show that the expression of the human CETP gene is modified by alternative splicing of the ninth exon, in a tissue-specific fashion. The function of alternative splicing is unknown but could serve to produce a protein with a function other than plasma neutral lipid transfer, or as an on-off switch to regulate the local concentration of biologically active protein.« less

To investigate the effect of short-chain fatty acids (SCFAs) on production of prostaglandin E(2) (PGE(2)), cytokines and chemokines in human monocytes.Human neutrophils and monocytes were isolated from human whole blood by using 1-Step Polymorph and RosetteSep Human Monocyte Enrichment Cocktail, respectively. Human GPR41 and GPR43 mRNA expression was examined by quantitative real-time polymerase chain reaction. The calcium flux assay was used to examine the biological activities of SCFAs in human neutrophils and monocytes. The effect of SCFAs on human monocytes and peripheral blood mononuclear cells (PBMC) was studied by measuring PGE(2), cytokines and chemokines in the supernatant. The effect of SCFAs in vivo was examined by intraplantar injection into rat paws.Human GPR43 is highly expressed in human neutrophils and monocytes. SCFAs induce robust calcium flux in human neutrophils, but not in human monocytes. In this study, we show that SCFAs can induce human monocyte release of PGE(2) and that this effect can be enhanced in the presence of lipopolysaccharide (LPS). In addition, we demonstrate that PGE(2) production induced by SCFA was inhibited by pertussis toxin, suggesting the involvement of a receptor-mediated mechanism. Furthermore, SCFAs can specifically inhibit constitutive monocyte chemotactic protein-1 (MCP-1) production and LPS-induced interleukin-10 (IL-10) production in human monocytes without affecting the secretion of other cytokines and chemokines examined. Similar activities were observed in human PBMC for the release of PGE(2), MCP-1 and IL-10 after SCFA treatment. In addition, SCFAs inhibit LPS-induced production of tumor necrosis factor-alpha and interferon-gamma in human PBMC. Finally, we show that SCFAs and LPS can induce PGE(2) production in vivo by intraplantar injection into rat paws (P0.01).SCFAs can have distinct antiinflammatory activities due to their regulation of PGE(2), cytokine and chemokine release from human immune cells.

To improve the security of the power supply for local ventilator, adding an emergency power supply (EPS) to the local ventilator is proposed by the paper. The key technology and the feasibility of the proposed scheme were analyzed in detail. Also, the proposed scheme was compared with the existing scheme. This scheme was proposed can start the emergency power to provide power for the local ventilator, and minimize the gas accumulation or explosion accidents caused by the power failure, in the case of the double powers failure. Compare to the existing scheme that double powers and double local ventilator machines at the driving face required by “Coal Mine Safety Regulations”. The new scheme can improve the security of the power supply for local ventilator greatly.

A resonance energy transfer method was developed to study the distribution of phospholipids around integral membrane proteins. The method involved measuring the extent of energy transfer from tryptophan residues of the proteins to different phospholipids labeled with a dansyl moiety in the fatty acid chain. No specific interactions were observed between gramicidin and dansyl-labeled phosphatidylcholine, phosphatidylethanolamine, or phosphatidic acid. The results were consistent with a random distribution of each phospholipid in the bilayer in the presence of gramicidin. However, a redistribution of both gramicidin and dansyl-labeled phospholipids was easily observed when a phase separation was induced by adding Ca2+ to vesicles made up of phosphatidylcholine and phosphatidic acid. Polarization measurements showed that in the presence of Ca2+ a rigid phosphatidic acid rich region and a more fluid phosphatidylcholine-rich region were formed. Energy-transfer measurements from gramicidin to either dansylphosphatidylcholine or dansylphosphatidic acid showed gramicidin preferentially partitioned into the phosphatidylcholine-rich regions. Energy-transfer measurements were also carried out with D-beta-hydroxybutyrate dehydrogenase reconstituted in a vesicle composed of phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid. Although the enzyme has a specific requirement for phosphatidylcholine for activity, the extent of energy transfer decreased in the order dansylphosphatidic acid, dansylphosphatidylcholine, dansylphosphatidylethanolamine. Thus, the enzyme reorganized the phospholipids in the vesicle into a nonrandom distribution.

The fluorescence emission properties of 1,6-diphenyl-1,3,5-hexatriene (DPH) in 1,2-dipalmitoyl-3-sn-phosphatidylcholine and 1,2-dimyristoyl-3-sn-phosphatidylcholine multilamellar vesicles have been measured by using multifrequency phase fluorometry. The fluorescence decay of DPH in the phospholipid vesicles has been analyzed by assuming either that the decay is made up of a discrete sum of exponential components or that the decay is made up of one or more continuous distributions of lifetime components. The fit of the decay curve using exponentials required at least two terms, and the reduced X2 was relatively large. The fit using a continuous distribution of lifetime values used two continuous components. Several symmetric distribution functions were used: uniform, Gaussian, and Lorentzian. The distribution function that best described the decay was the Lorentzian. The full width at half-maximum of the Lorentzian distribution was about 0.6 ns at temperatures below the phase transition temperature. At the phospholipid phase transition and at higher temperatures, the distribution became quite narrow, with a width of about 0.1 ns. It is proposed that the lifetime distribution is generated by a continuum of different environments of the DPH molecule characterized by different dielectric constants. Below the transition temperature in the gel phase, the dielectric constant gradient along the membrane normal determines the distribution of decay rates. Above the transition, in the liquid-crystalline phase, the translational and rotational mobility of the DPH molecule increases, and the DPH experiences an average environment during the excited-state lifetime. Consequently, the distribution becomes narrower.(ABSTRACT TRUNCATED AT 250 WORDS)