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Mouse steroid receptor coactivator-1 is not essential for peroxisome proliferator-activated receptor α-regulated gene expression

Qi, Chao; Zhu, Yijun; Pan, Jie; Yeldandi, Anjana V.; Rao, M. Sambasiva; Maeda, Nobuyo; Subbarao, V.; Pulikuri, Sujata; Hashimoto, Takashi; Reddy, Janardan K.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 16/02/1999 Português
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Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors, and it is assumed that the biological effects of these receptors depend on interactions with recently identified coactivators, including steroid receptor coactivator-1 (SRC-1). We assessed the in vivo function of SRC-1 on the PPARα-regulated gene expression in liver by generating mice in which the SRC-1 gene was inactivated by gene targeting. The homozygous (SRC-1−/−) mice were viable and fertile and exhibited no detectable gross phenotypic defects. When challenged with a PPARα ligand, such as ciprofibrate or Wy-14,643, the SRC-1−/− mice displayed typical pleiotropic responses, including hepatomegaly, peroxisome proliferation in hepatocytes, and increased mRNA and protein levels of genes that are regulated by PPARα. These alterations were indistinguishable from those exhibited by SRC-1+/+ wild-type mice fed either ciprofibrate- or Wy-14,643-containing diets. These results indicate that SRC-1 is not essential for PPARα-mediated transcriptional activation in vivo and suggest redundancy in nuclear receptor coactivators.

Genomic interval engineering of mice identifies a novel modulator of triglyceride production

Zhu, Yiwen; Jong, Miek C.; Frazer, Kelly A.; Gong, Elaine; Krauss, Ronald M.; Cheng, Jan-Fang; Boffelli, Dario; Rubin, Edward M.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 01/02/2000 Português
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To accelerate the biological annotation of novel genes discovered in sequenced regions of mammalian genomes, we are creating large deletions in the mouse genome targeted to include clusters of such genes. Here we describe the targeted deletion of a 450-kb region on mouse chromosome 11, which, based on computational analysis of the deleted murine sequences and human 5q orthologous sequences, codes for nine putative genes. Mice homozygous for the deletion had a variety of abnormalities, including severe hypertriglyceridemia, hepatic and cardiac enlargement, growth retardation, and premature mortality. Analysis of triglyceride metabolism in these animals demonstrated a several-fold increase in hepatic very-low density lipoprotein triglyceride secretion, the most prevalent mechanism responsible for hypertriglyceridemia in humans. A series of mouse BAC and human YAC transgenes covering different intervals of the 450-kb deleted region were assessed for their ability to complement the deletion induced abnormalities. These studies revealed that OCTN2, a gene recently shown to play a role in carnitine transport, was able to correct the triglyceride abnormalities. The discovery of this previously unappreciated relationship between OCTN2, carnitine...

Structural requirements of six naturally occurring isoforms of the IL-18 binding protein to inhibit IL-18

Kim, Soo-Hyun; Eisenstein, Miriam; Reznikov, Leonid; Fantuzzi, Giamila; Novick, Daniela; Rubinstein, Menachem; Dinarello, Charles A.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 01/02/2000 Português
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A novel, constitutively expressed and secreted IL-18 binding protein (IL-18BP) neutralizes IL-18 and thereby suppresses the production of IFN-γ, resulting in reduced T-helper type 1 immune responses. In the present study, four human and two mouse isoforms, resulting from mRNA splicing and found in various cDNA libraries, were expressed, purified, and assessed for binding and neutralization of IL-18 biological activities. Human IL-18BP isoform a (IL-18BPa) exhibited the greatest affinity for IL-18 with a rapid on-rate, a slow off-rate, and a dissociation constant (Kd) of 399 pM. IL-18BPc shares the Ig domain of IL-18BPa except for the 29 C-terminal amino acids; the Kd of IL-18BPc is 10-fold less (2.94 nM). Nevertheless, IL-18BPa and IL-18BPc neutralize IL-18 >95% at a molar excess of two. IL-18BPb and IL-18BPd isoforms lack a complete Ig domain and lack the ability to bind or neutralize IL-18. Murine IL-18BPc and IL-18BPd isoforms, possessing the identical Ig domain, also neutralize >95% murine IL-18 at a molar excess of two. However, murine IL-18BPd, which shares a common C-terminal motif with human IL-18BPa, also neutralizes human IL-18. Molecular modeling identified a large mixed electrostatic and hydrophobic binding site in the Ig domain of IL-18BP...

Tyrosine phosphorylation of the Helicobacter pylori CagA antigen after cag-driven host cell translocation

Stein, Markus; Rappuoli, Rino; Covacci, Antonello
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 01/02/2000 Português
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Helicobacter pylori strains associated with severe tissue damage and inflammation possess a unique genetic locus, cag, containing 31 genes originating from a distant event of horizontal transfer and retained as a pathogenicity island. The cag system is an Helicobacter-specific type IV secretion engine involved in cellular responses like induction of pedestals, secretion of IL-8, and phosphorylation of proteic targets. It has previously been reported that cocultivation of epithelial cells with Helicobacter pylori triggers signal transduction and tyrosine phosphorylation of a 145-kDa putative host cell protein. Herein, we demonstrate that this protein is not derived from the host but rather is the bacterial immunodominant antigen CagA, a virulence factor commonly expressed in peptic ulcer disease and thought to be an orphan of a specific biological function. Thus, CagA is delivered into the epithelial cells by the cag type IV secretion system where it is phosphorylated on tyrosine residues by an as yet unidentified host cell kinase and wired to eukaryotic signal transduction pathways and cytoskeletal plasticity.

Isolation of peptide aptamers that inhibit intracellular processes

Blum, Jonathan H.; Dove, Simon L.; Hochschild, Ann; Mekalanos, John J.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
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We have developed a method for isolation of random peptides that inhibit intracellular processes in bacteria. A library of random peptides expressed as fusions to Escherichia coli thioredoxin (aptamers) were expressed under the tight control of the arabinose-inducible PBAD promoter. A selection was applied to the library to isolate aptamers that interfered with the activity of thymidylate synthase (ThyA) in vivo. Expression of an aptamer isolated by this method resulted in a ThyA− phenotype that was suppressed by simultaneous overexpression of ThyA. Two-hybrid analysis showed that this aptamer is likely to interact with ThyA in vivo. The library also was screened for aptamers that inhibited growth of bacteria expressing them, and five such aptamers were characterized. Four aptamers were bacteriostatic when expressed, whereas one showed a bactericidal effect. Introduction of translational stop codons into various aptamers blocked their activity, suggesting that their biological effects were likely to be due to protein aptamer rather than RNA. Combinatorial aptamers provide a new genetic and biochemical tool for identifying targets for antibacterial drug development.

Lysozyme and RNases as anti-HIV components in β-core preparations of human chorionic gonadotropin

Lee-Huang, Sylvia; Huang, Paul L.; Sun, Yongtao; Huang, Philip L.; Kung, Hsiang-fu; Blithe, Diana L.; Chen, Hao-Chia
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 16/03/1999 Português
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Human chorionic gonadotropin (hCG) preparations contain activity against HIV type 1 (HIV-1). However, there has been controversy about whether some biological activities of hCG β-subunit (hCGβ) preparations are caused by the β-subunit itself or other proteins present in the preparations. We report here the purification, characterization, and identification of three enzymes with anti-HIV activity present in the β-core fraction of hCGβ prepared from the urine of pregnant women. The N-terminal amino acid sequence of one protein is identical to human urinary lysozyme C, and those of the other two are identical to human RNase A and urinary RNase U. We thus refer to these proteins as AVL (antiviral lysozyme) and AVR (antiviral RNases). In addition to HIV-1 inhibition, AVL is capable of lysing Micrococcus lysodeikticus. AVR digests a variety of RNA substrates, including RNA from HIV-1-infected cells. We also find that lysozyme from chicken egg white, human milk, and human neutrophils and RNase A from bovine pancreas possess activity against HIV-1. These findings may offer additional strategies for the treatment of HIV-1 infection.

Yeast flavin-containing monooxygenase generates oxidizing equivalents that control protein folding in the endoplasmic reticulum

Suh, Jung-Keun; Poulsen, Lawrence L.; Ziegler, Daniel M.; Robertus, Jon D.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 16/03/1999 Português
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The flavin-containing monooxygenase from yeast (yFMO) catalyzes the O2- and NADPH-dependent oxidations of biological thiols, including oxidation of glutathione to glutathione disulfide (GSSG). Glutathione and GSSG form the principle redox buffering system in the cell, with the endoplasmic reticulum (ER) being more oxidizing than the cytoplasm. Proper folding of disulfide-bonded proteins in the ER depends on an optimum redox buffer ratio. Here we show that yFMO is localized to the cytoplasmic side of the ER membrane. We used a gene knockout strain and expression vectors to show that yFMO has a major effect on the generation of GSSG transported into the ER. The enzyme is required for the proper folding, in the ER, of test proteins with disulfide bonds, whereas those without disulfide bonds are properly folded independently of yFMO in the ER or in the cytoplasm.

Tissue-specific processing of cocaine- and amphetamine-regulated transcript peptides in the rat

Thim, Lars; Kristensen, Peter; Nielsen, Per F.; Wulff, Birgitte S.; Clausen, Jes T.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 16/03/1999 Português
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Cocaine- and amphetamine-regulated transcript (CART) is a recently discovered hypothalamic peptide regulated by leptin and with a potent appetite-suppressing activity. In the rat, the CART gene encodes a peptide of 116 amino acid residues (or a splice variant 13 residues longer). The predicted signal sequence is 27 amino acid residues, resulting in a prohormone of 89 residues. The CART prohormone contains several potential posttranslational processing sites in the form of mono- and dibasic sequences. In the present study we have purified CART peptides from extracts of adrenal gland, hypothalamus, nucleus accumbens, and pituitary gland (anterior and neurointermediate lobe) of the rat and determined the peptide structures by using microsequencing and mass spectrometry. In none of the tissues examined the long splice variant was found. From the adrenal gland, the CART(1–89) and CART(10–89) peptides were isolated, in contrast to the hypothalamus and nucleus accumbens, from which the shorter form peptides CART(42–89) and CART(49–89) were purified. From the anterior lobe of the pituitary gland, CART(42–89) was isolated, in contrast to the neurointermediate lobe, which contains only CART(49–89). This tissue-specific processing indicates that CART peptides may have different biological functions in the periphery and in the central nervous system.

Phosphorylating DNA with DNA

Li, Yingfu; Breaker, Ronald R.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 16/03/1999 Português
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Nearly 50 individual DNAs with polynucleotide kinase-like activity were isolated from a random-sequence pool by using in vitro selection. Each self-phosphorylating deoxyribozyme makes use of one or more of the eight standard NTPs or dNTPs as a source of activated phosphate. Although most prototypic deoxyribozymes poorly differentiate between the ribose and deoxyribose moieties, further optimization by in vitro selection produced variants that display up to 100-fold discrimination between related NTP and dNTP substrates. An optimized ATP-dependent deoxyribozyme uses ATP >40,000-fold more efficiently than CTP, GTP, or UTP. This enzyme operates with a rate enhancement of nearly one billion-fold over the uncatalyzed rate of ATP hydrolysis. A bimolecular version of the ATP-dependent deoxyribozyme was further engineered to phosphorylate specific target DNAs with multiple turnover. The substrate-recognition patterns and rate enhancements intrinsic to these DNAs are characteristic of naturally occurring RNA and protein enzymes, supporting the hypothesis that DNA has sufficient catalytic potential to function as an enzyme in biological systems.

Detecting patterns of protein distribution and gene expression in silico

Geraghty, Michael T.; Bassett, Doug; Morrell, James C.; Gatto, Gregory J.; Bai, Jianwu; Geisbrecht, Brian V.; Hieter, Phil; Gould, Stephen J.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 16/03/1999 Português
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Most biological information is contained within gene and genome sequences. However, current methods for analyzing these data are limited primarily to the prediction of coding regions and identification of sequence similarities. We have developed a computer algorithm, CoSMoS (for context sensitive motif searches), which adds context sensitivity to sequence motif searches. CoSMoS was challenged to identify genes encoding peroxisome-associated and oleate-induced genes in the yeast Saccharomyces cerevisiae. Specifically, we searched for genes capable of encoding proteins with a type 1 or type 2 peroxisomal targeting signal and for genes containing the oleate-response element, a cis-acting element common to fatty acid-regulated genes. CoSMoS successfully identified 7 of 8 known PTS-containing peroxisomal proteins and 13 of 14 known oleate-regulated genes. More importantly, CoSMoS identified an additional 18 candidate peroxisomal proteins and 300 candidate oleate-regulated genes. Preliminary localization studies suggest that these include at least 10 previously unknown peroxisomal proteins. Phenotypic studies of selected gene disruption mutants suggests that several of these new peroxisomal proteins play roles in growth on fatty acids, one is involved in peroxisome biogenesis and at least two are required for synthesis of lysine...

Vascular endothelial growth factor (VEGF)-like protein from orf virus NZ2 binds to VEGFR2 and neuropilin-1

Wise, Lyn M.; Veikkola, Tanja; Mercer, Andrew A.; Savory, Loreen J.; Fleming, Stephen B.; Caesar, Carol; Vitali, Angela; Makinen, Taija; Alitalo, Kari; Stacker, Steven A.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 16/03/1999 Português
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Orf virus, a member of the poxvirus family, produces a pustular dermatitis in sheep, goats, and humans. The lesions induced after infection with orf virus show extensive proliferation of vascular endothelial cells, dilation of blood vessels and dermal swelling. An explanation for the nature of these lesions may lie in the discovery that orf virus encodes an apparent homolog of the mammalian vascular endothelial growth factor (VEGF) family of molecules. These molecules mediate endothelial cell proliferation, vascular permeability, angiogenesis, and lymphangiogenesis via the endothelial cell receptors VEGFR-1 (Flt1), VEGFR-2 (KDR/Flk1), and VEGFR-3 (Flt4). The VEGF-like protein of orf virus strain NZ2 (ORFV2-VEGF) is most closely related in primary structure to VEGF. In this study we examined the biological activities and receptor specificity of the ORFV2-VEGF protein. ORFV2-VEGF was found to be a disulfide-linked homodimer with a subunit of ≈25 kDa. ORFV2-VEGF showed mitogenic activity on bovine aortic and human microvascular endothelial cells and induced vascular permeability. ORFV2-VEGF was found to bind and induce autophosphorylation of VEGFR-2 and was unable to bind or activate VEGFR-1 and VEGFR-3, but bound the newly identified VEGF165 receptor neuropilin-1. These results indicate that...

Molecular cloning and characterization of prostase, an androgen-regulated serine protease with prostate-restricted expression

Nelson, Peter S.; Gan, Lu; Ferguson, Camari; Moss, Patrick; Gelinas, Richard; Hood, Leroy; Wang, Kai
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 16/03/1999 Português
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The identification of genes with selective expression in specific organs or cell types provides an entry point for understanding biological processes that occur uniquely within a particular tissue. Using a subtraction approach designed to identify genes preferentially expressed in specific tissues, we have identified prostase, a human serine protease with prostate-restricted expression. The prostase cDNA encodes a putative 254-aa polypeptide with a conserved serine protease catalytic triad and an amino-terminal pre-propeptide sequence, indicating a potential secretory function. The genomic sequence comprises five exons and four introns and contains multiple copies of a chromosome 19q-specific minisatellite repeat. Northern analysis indicates that prostase mRNA is expressed in hormonally responsive normal and neoplastic prostate epithelial tissues, but not in prostate stromal constituents. Prostase shares 35% amino acid identity with prostate-specific antigen (PSA) and 78% identity with the porcine enamel matrix serine proteinase 1, an enzyme involved in enamel matrix degradation and with a putative role in the disruption of intercellular junctions. Radiation-hybrid-panel mapping localized prostase to chromosome 19q13, a region containing several other serine proteases...

Accurate protein crystallography at ultra-high resolution: Valence electron distribution in crambin

Jelsch, Christian; Teeter, Martha M.; Lamzin, Victor; Pichon-Pesme, Virginie; Blessing, Robert H.; Lecomte, Claude
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 28/03/2000 Português
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The charge density distribution of a protein has been refined experimentally. Diffraction data for a crambin crystal were measured to ultra-high resolution (0.54 Å) at low temperature by using short-wavelength synchrotron radiation. The crystal structure was refined with a model for charged, nonspherical, multipolar atoms to accurately describe the molecular electron density distribution. The refined parameters agree within 25% with our transferable electron density library derived from accurate single crystal diffraction analyses of several amino acids and small peptides. The resulting electron density maps of redistributed valence electrons (deformation maps) compare quantitatively well with a high-level quantum mechanical calculation performed on a monopeptide. This study provides validation for experimentally derived parameters and a window into charge density analysis of biological macromolecules.

New studies on the heat resistance of hamster-adapted scrapie agent: Threshold survival after ashing at 600°C suggests an inorganic template of replication

Brown, Paul; Rau, Edward H.; Johnson, Bruce K.; Bacote, Alfred E.; Gibbs, Clarence J.; Gajdusek, D. Carleton
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
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One-gram samples from a pool of crude brain tissue from hamsters infected with the 263K strain of hamster-adapted scrapie agent were placed in covered quartz-glass crucibles and exposed for either 5 or 15 min to dry heat at temperatures ranging from 150°C to 1,000°C. Residual infectivity in the treated samples was assayed by the intracerebral inoculation of dilution series into healthy weanling hamsters, which were observed for 10 months; disease transmissions were verified by Western blot testing for proteinase-resistant protein in brains from clinically positive hamsters. Unheated control tissue contained 9.9 log10LD50/g tissue; after exposure to 150°C, titers equaled or exceeded 6 log10LD50/g, and after exposure to 300°C, titers equaled or exceeded 4 log10LD50/g. Exposure to 600°C completely ashed the brain samples, which, when reconstituted with saline to their original weights, transmitted disease to 5 of 35 inoculated hamsters. No transmissions occurred after exposure to 1,000°C. These results suggest that an inorganic molecular template with a decomposition point near 600°C is capable of nucleating the biological replication of the scrapie agent.

Transforming growth factor-β1 hyperexpression in African-American hypertensives: A novel mediator of hypertension and/or target organ damage

Suthanthiran, Manikkam; Li, Baogui; Song, Jong O.; Ding, Ruchuang; Sharma, Vijay K.; Schwartz, Joseph E.; August, Phyllis
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
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Hypertension, a remediable risk factor for stroke, cardiovascular disease, and renal failure, affects 50 million individuals in the United States alone. African Americans (blacks) have a higher incidence and prevalence of hypertension and hypertension-associated target organ damage compared with Caucasian Americans (whites). Herein, we explored the hypotheses that transforming growth factor-β1 (TGF-β1) is hyperexpressed in hypertensives compared with normotensives and that TGF-β1 overexpression is more frequent in blacks compared with whites. These hypotheses were stimulated by our recent demonstration that TGF-β1 is hyperexpressed in blacks with end-stage renal disease compared with white end-stage renal disease patients and by the biological attributes of TGF-β1, which include induction of endothelin-1 expression, stimulation of renin release, and promotion of vascular and renal disease when TGF-β1 is produced in excess. TGF-β1 profiles were determined in black and white hypertensive subjects and normotensive controls and included circulating protein concentrations, mRNA steady-state levels, and codon 10 genotype. Our investigation demonstrated that TGF-β1 protein levels are highest in black hypertensives, and TGF-β1 protein as well as TGF-β1 mRNA levels are higher in hypertensives compared with normotensives. The proline allele at codon 10 (Pro10) was more frequent in blacks compared with whites...

AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora

Dong, Yi-Hu; Xu, Jin-Ling; Li, Xian-Zhen; Zhang, Lian-Hui
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
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N-acylhomoserine lactones, known as autoinducers (AIs), are widely conserved signal molecules present in quorum-sensing systems of many Gram-negative bacteria. AIs are involved in the regulation of diverse biological functions, including expression of pathogenic genes in the plant pathogens Pseudomonas solanacearum, several Erwinia species, and the human pathogen Pseudomonas aeruginosa. A bacterial isolate, Bacillus sp. 240B1, is capable of enzymatic inactivation of AIs. The gene (aiiA) for AI inactivation from Bacillus sp. 240B1 has been cloned and shown to encode a protein of 250 amino acids. Sequence alignment indicates that AiiA contains a “HXHXDH” zinc-binding motif that is conserved in several groups of metallohydrolases. Site-directed mutagenesis showed that conserved aspartate and most histidine residues are required for AiiA activity. Expression of aiiA in transformed Erwinia carotovora strain SCG1 significantly reduces the release of AI, decreases extracellular pectolytic enzyme activities, and attenuates pathogenicity on potato, eggplant, Chinese cabbage, carrot, celery, cauliflower, and tobacco. Our results indicate that the AI-inactivation approach represents a promising strategy for prevention of diseases in which virulence is regulated by AIs.

Detection of β2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET)

Angers, Stephane; Salahpour, Ali; Joly, Eric; Hilairet, Sandrine; Chelsky, Dan; Dennis, Michael; Bouvier, Michel
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
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Heptahelical receptors that interact with heterotrimeric G proteins represent the largest family of proteins involved in signal transduction across biological membranes. Although these receptors generally were believed to be monomeric entities, a growing body of evidence suggests that they may form functionally relevant dimers. However, a definitive demonstration of the existence of G protein-coupled receptor (GPCR) dimers at the surface of living cells is still lacking. Here, using bioluminescence resonance energy transfer (BRET), as a protein–protein interaction assay in whole cells, we unambiguously demonstrate that the human β2-adrenergic receptor (β2AR) forms constitutive homodimers when expressed in HEK-293 cells. Receptor stimulation with the hydrophilic agonist isoproterenol led to an increase in the transfer of energy between β2AR molecules genetically fused to the BRET donor (Renilla luciferase) and acceptor (green fluorescent protein), respectively, indicating that the agonist interacts with receptor dimers at the cell surface. Inhibition of receptor internalization did not prevent agonist-promoted BRET, demonstrating that it did not result from clustering of receptors within endosomes. The notion that receptor dimers exist at the cell surface was confirmed further by the observation that BS3...

Noninvasive measurement of anatomic structure and intraluminal oxygenation in the gastrointestinal tract of living mice with spatial and spectral EPR imaging

He, Guanglong; Shankar, Ravi A.; Chzhan, Michael; Samouilov, Alexandre; Kuppusamy, Periannan; Zweier, Jay L.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 13/04/1999 Português
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EPR imaging has emerged as an important tool for noninvasive three-dimensional (3D) spatial mapping of free radicals in biological tissues. Spectral–spatial EPR imaging enables mapping of the spectral information at each spatial position, and, from the observed line width, the localized tissue oxygenation can be mapped. We report the development of EPR imaging instrumentation enabling 3D spatial and spectral–spatial EPR imaging of small animals. This instrumentation, along with the use of a biocompatible charcoal oximetry-probe suspension, enabled 3D spatial imaging of the gastrointestinal (GI) tract, along with mapping of oxygenation in living mice. By using these techniques, the oxygen tension was mapped at different levels of the GI tract from the stomach to the rectum. The results clearly show the presence of a marked oxygen gradient from the proximal to the distal GI tract, which decreases after respiratory arrest. This technique for in vivo mapping of oxygenation is a promising method, enabling the noninvasive imaging of oxygen within the normal GI tract. This method should be useful in determining the alterations in oxygenation associated with disease.

Ligand specificity of a high-affinity binding site for lipo-chitooligosaccharidic Nod factors in Medicago cell suspension cultures

Gressent, Frédéric; Drouillard, Sophie; Mantegazza, Natacha; Samain, Eric; Geremia, Roberto A.; Canut, Hervé; Niebel, Andreas; Driguez, Hugues; Ranjeva, Raoul; Cullimore, Julie; Bono, Jean-Jacques
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 13/04/1999 Português
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Rhizobial lipo-chitooligosaccharides (LCOs) are signaling molecules involved in host-range recognition for the establishment of the symbiosis with leguminous plants. The major LCO of Rhizobium meliloti, the symbiont of Medicago plants contains four or five N-acetylglucosamines, O-acetylated and N-acylated with a C16:2 fatty acid on the terminal nonreducing sugar and O-sulfated on the reducing sugar. In this paper, the ligand specificity of a high-affinity binding site (Nod factor binding site 2 or NFBS2), enriched in a plasma membrane-enriched fraction of Medicago cell suspension cultures, is reported. By using chemically synthesized LCOs, the role of structural elements, important for symbiotic activities, as recognition motifs for NFBS2 was determined. The results show that the substitutions on the nonreducing sugar of the LCOs (the O-acetate group, the fatty acid, and the hydroxyl group on the C4 of the sugar) are determinants for high-affinity binding to NFBS2. In contrast, the sulfate group, which is necessary for all biological activities on Medicago, is not discriminated by NFBS2. However, the reducing sugar of the LCO seems to interact with NFBS2, because ligand binding is affected by the reduction of the free anomeric carbon and depends on the number of N-acetyl glucosamine residues. These results suggest that the recognition of the LCOs by NFBS2 is mediated by structural elements in both the lipid and oligosaccharidic moities...

Museum specimen data predict crop damage by tropical rodents

Sánchez-Cordero, Víctor; Martínez-Meyer, Enrique
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
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Museum collections constitute a massive store of information on biological diversity. We used museum specimen data to generate ecological niche models that provide predictions of geographic distributions of native rodent pest species and agricultural census data that summarize the geographic distribution of nine crops in the state of Veracruz, Mexico, as well as crop losses between planting and harvest. Herein, we show that crop damage is related significantly to the predicted presence of rodent species for seven of nine crops. Museum collections may thus provide important baseline information for designing land-use and agricultural pest-management programs.