RESUMEN
Uncoupling protein (UCP1) is a transmembrane proton transporter present in the mitochondria of brown adipose tissue (BAT), a specialized tissue which functions in temperature homeostasis and energy balance (Nicholls, D. G., and Locke, R. M. (1984) Physiol. Rev. 64, 2-40; Lowell, D. D., and Flier, J. S. (1997) Annu. Rev. Med.). UCP1 mediates the thermogenesis that is characteristic of BAT by uncoupling mitochondrial oxidation of substrates from ATP synthesis. Recently, two proteins related to UCP1 have been identified and designated UCP2 (Fleury, C., et al. (1997) Nature Genetics 15, 269-272) or UCP homolog (UCPH) (Gimeno, R. E., et al. (1997) Diabetes 46, 900-906) and UCP3 (Boss, O., et al. (1997) FEBS Lett. 408, 39-42; Vidal-Puig, A., et al. (1997) Biochem. Biophys. Res. Commun. 235, 79-82). We investigated the regulation in rats of UCP3, which is expressed primarily in skeletal muscle and BAT. Expression of rat UCP3 mRNA in BAT was upregulated by in vivo treatment with triiodothyronine (T3) and by exposure to cold, suggesting that UCP3 is active in thermogenesis and energy expenditure. In skeletal muscle, UCP3 mRNA was also upregulated by T3 but, surprisingly, not by cold exposure. A hypothesis is proposed to account for this differential regulation.
Asunto(s)
Proteínas Portadoras/efectos de los fármacos , Proteínas Portadoras/metabolismo , Frío , Triyodotironina/farmacología , Tejido Adiposo Pardo/efectos de los fármacos , Tejido Adiposo Pardo/metabolismo , Administración Oral , Animales , Clonación Molecular , Grasas de la Dieta/administración & dosificación , Humanos , Canales Iónicos , Masculino , Mitocondrias Musculares/efectos de los fármacos , Mitocondrias Musculares/metabolismo , Proteínas Mitocondriales , Músculo Esquelético/metabolismo , Especificidad de Órganos/genética , ARN Mensajero/análisis , Ratas , Ratas Endogámicas , Triyodotironina/administración & dosificación , Proteína Desacopladora 3RESUMEN
We have cloned and expressed two isoforms of the human calcitonin (hCT) receptor. Primers designed from the published sequence of a CT receptor cloned from an ovarian small cell carcinoma line were used for the polymerase chain reaction amplification of related products from human breast carcinoma MCF-7 cells. Two complementary DNAs were isolated. One clone lacks a 16-amino acid insert in the first intracellular loop and is virtually identical to the receptor recently cloned from the T47D human breast carcinoma cell line. The second clone is another splice variant lacking both the 16-amino acid insert in the first intracellular domain as well as the first 47 amino acids of the amino-terminus extracellular domain. COS-7 cells transfected with either receptor isoform bound [125I]salmon CT with high affinity and responded to hCT with increases in cAMP. Tissue distribution studies revealed the truncated extracellular domain 1 isoform transcripts in human skeletal muscle, kidney, brain, and lung. Analysis of a hCT receptor genomic clone demonstrated an exon/intron organization similar to that of the porcine CT receptor gene, except for a distinct exon coding for the alternatively spliced insert in the first intracellular domain.
Asunto(s)
Receptores de Calcitonina/genética , Secuencia de Aminoácidos , Secuencia de Bases , Células Cultivadas , Clonación Molecular , AMP Cíclico/biosíntesis , Exones , Humanos , Intrones , Datos de Secuencia Molecular , Especificidad de Órganos , Ensayo de Unión Radioligante , Receptores de Calcitonina/análisis , Receptores de Calcitonina/fisiologíaRESUMEN
Amylin is a 37-amino acid peptide first isolated, purified, and characterized from the amyloid deposits in the pancrease of type 2 diabetics. It is synthesized and secreted primarily from pancreatic beta cells along with insulin. The ability of amylin to potently reduce insulin-stimulated incorporation of glucose into glycogen in skeletal muscle requires both an intact 2Cys-7Cys disulfide bond and a COOH-terminal amide. Amylin has structural and functional relationships to two other messenger proteins, calcitonin and CGRP. Amylin has relatively potent calcitonin-like activity on bone metabolism and weaker CGRP-like activity on the vasculature. CGRP is a slightly weaker agonist than amylin for metabolic responses. Although rat calcitonins are weak, teleost fish calcitonins are very potent agonists for amylin's metabolic effects. This group of peptides appears to act on a family of related G protein-coupled receptors; several variant calcitonin receptors have recently been cloned and expressed. These receptors appear to be coupled to adenylyl cyclase in many instances; recent evidence supports the view that amylin's effects on skeletal muscle occur, at least in large part, through activation of the cAMP pathway.
Asunto(s)
Amiloide/biosíntesis , Secuencia de Aminoácidos , Amiloide/química , Amiloide/farmacología , Animales , Calcitonina/química , Calcitonina/farmacología , Péptido Relacionado con Gen de Calcitonina/farmacología , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Polipéptido Amiloide de los Islotes Pancreáticos , Islotes Pancreáticos/metabolismo , Datos de Secuencia Molecular , Salmón , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
Two receptors with high affinity for salmon calcitonin were cloned from the nucleus accumbens region of rat brain. The deduced 479 amino acid sequence of cDNA clone L2175-D20 (designated C1a receptor) is 78% and 66% identical with those reported for human and porcine calcitonin receptors, respectively. Clone U3237-A2 codes for a receptor (designated C1b) that is identical to C1a except for a 37 amino acid insert in the second extracellular domain. COS-7 cells transfected with either transcript bound [125I]salmon calcitonin with high affinity (Kd = 8 pM for C1a; Kd = 48 pM for C1b) and responded to salmon calcitonin with increases in cAMP. Tissue distribution studies revealed C1a transcript in rat brain, skeletal muscle, kidney and lung, whereas C1b was predominantly found in brain.
Asunto(s)
Encéfalo/metabolismo , Calcitonina/metabolismo , Receptores de Superficie Celular/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Línea Celular , Clonación Molecular , AMP Cíclico/metabolismo , ADN , Humanos , Datos de Secuencia Molecular , Especificidad de Órganos/genética , Ratas , Receptores de Calcitonina , Receptores de Superficie Celular/metabolismo , Salmón , Homología de Secuencia de Aminoácido , PorcinosRESUMEN
Canine amylin cDNA was cloned and sequenced from a dog pancreas library using oligonucleotide probes corresponding to portions of human amylin. The sequence reported here is significantly different from an incomplete sequence previously reported. The non-amyloidogenic canine mature peptide is 89% and 95% identical to its amyloidogenic human and cat homologues, respectively. Both the dog and cat peptides are identical over the proposed amyloidogenic region, amino acids 20-29. Hydropathy plots comparing these three peptides are nearly identical suggesting that the primary sequence of amylin is in itself insufficient to explain pancreatic amyloid formation.
Asunto(s)
Amiloide/genética , Secuencia de Aminoácidos , Amiloide/química , Animales , Secuencia de Bases , Clonación Molecular , ADN/genética , Sondas de ADN/genética , Perros , Polipéptido Amiloide de los Islotes Pancreáticos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/genética , Precursores de Proteínas/química , Precursores de Proteínas/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
IgE-binding protein (epsilon BP) refers to a protein originally identified in rat basophilic leukemia cells by virtue of its affinity for IgE. It is now known to be a beta-galactoside-binding lectin equivalent to carbohydrate-binding protein 35 (CBP 35). More recently, its identity to Mac-2, a macrophage cell-surface protein, has been established. cDNA coding for human epsilon BP has been cloned from a human HeLa cell cDNA library and contains an open reading frame of 750 base pairs encoding a 250 amino acid protein. Like the rat and murine counterparts, the human epsilon BP amino acid sequence can be divided into two domains with the amino-terminal domain consisting of a highly conserved repetitive sequence (YPGXXXPGA) and the carboxyl-terminal domain containing sequences shared by other S-type lectins. The human epsilon BP sequence exhibits extensive homology to murine and rat epsilon BP with 84% and 82% identity, respectively. The homology is particularly striking in the carboxyl-terminal domain where 95% identity is found between human and murine sequences in a stretch of over 70 amino acids. A survey of epsilon BP mRNA expression from several lymphocyte cell lines revealed that the level of epsilon BP transcription may reflect a relationship between cell differentiation and epsilon BP expression. Finally, human epsilon BP was purified from several human cell lines and shown to possess lactose-binding characteristics and cross-species reactivity to murine IgE. Surprisingly, three different human myeloma IgE proteins did not show reactivity to human epsilon BP. However, after neuraminidase treatment of each human IgE, pronounced binding to epsilon BP was observed, thereby indicating that only specific IgE glycoforms can be recognized by epsilon BP.
Asunto(s)
Antígenos de Diferenciación/genética , Proteínas Portadoras/genética , Hemaglutininas/genética , Inmunoglobulina E/metabolismo , Lectinas/genética , Secuencia de Aminoácidos , Animales , Antígenos de Diferenciación/metabolismo , Secuencia de Bases , Proteínas Portadoras/metabolismo , ADN/genética , Galactosa/metabolismo , Galectina 3 , Células HeLa/química , Hemaglutininas/metabolismo , Humanos , Lectinas/metabolismo , Ratones , Datos de Secuencia Molecular , Proteínas de Mieloma/metabolismo , Especificidad de Órganos , Ratas , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Especificidad de la EspecieRESUMEN
The high-affinity IgE receptor present on mast cells and basophils is responsible for the IgE-mediated activation of these cells. The current model for this receptor depicts a four-subunit structure, alpha beta gamma 2. A cDNA for the alpha subunit was recently cloned and predicts a structure consisting of two homologous extracellular domains, a transmembrane segment, and a cytoplasmic tail. Using a synthetic oligonucleotide corresponding to the amino-terminal sequence of the alpha subunit, we identified a number of cDNA clones from a rat basophilic leukemia cell cDNA library. Nucleotide sequencing established four different forms of cDNA: one is nearly identical to the published cDNA; the second differs from the first in the 5' untranslated sequence; the other two forms use either one or the other of the 5'-end sequences as above and lack 163 base pairs in the region coding for the second extracellular domain. RNase protection analysis with radioactive RNA probes established the heterogeneity of rat basophilic leukemia cell mRNA with regard to both the 5' and the internal sequences. Our results suggest the existence of at least four different protein forms related to the alpha subunit of the high-affinity IgE receptor.
Asunto(s)
Inmunoglobulina E/inmunología , Receptores Fc/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Basófilos/inmunología , Clonación Molecular , ADN/genética , Mastocitos/inmunología , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , ARN Mensajero/genética , Receptores Fc/inmunología , Receptores de IgERESUMEN
Proteins that bind IgE play important roles in both the synthesis and function of IgE are therefore intimately involved in IgE-mediated human allergic disorders. This report describes the structure of an IgE-binding protein, as predicted from sequencing a cDNA cloned from rat basophilic leukemia cells. This protein contains two domains: the amino-terminal domain (140 amino acids) consists of a highly conserved repetitive amino acid sequence, Tyr-Pro-Gly-Pro/Gln-Ala/Thr-Pro/Ala-Pro-Gly-Ala, whereas the carboxyl-terminal domain (122 amino acids) shares significant sequence homology with a domain of lymphocyte/macrophage receptor for the Fc portion of IgG. Other proteins with this type of structure but with affinity for other immunoglobulin isotypes may exist and may represent a heretofore unidentified component of the immune system.
Asunto(s)
Inmunoglobulina E/inmunología , Inmunoglobulina G/inmunología , Receptores Fc/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Datos de Secuencia Molecular , Receptores de IgE , Receptores de IgG , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
To understand the relative importance of germ-line genes in the generation of the functional human antibody repertoire, it is first necessary to define the number of variable region genes and to determine their fine structure. We have focused on the human VkIII variable region gene family because of its association with autoantibodies. A human genomic library was screened with a VkIII cDNA probe and subsequently with a VkIII germ-line gene probe. Seven different VkIII clones were isolated and characterized by restriction mapping and sequence analyses. Three clones have identical restriction enzyme sites over a 12-kilobase (kb) region, contain identical sequences over an 895-base pair (bp) region, and thus are likely to be different isolates of the same human VkIII gene. Another two clones have identical restriction enzyme sites over a 5-kb region, are identical over a stretch of 905 bp sequenced, and likely represent independent isolates of another human VkIII gene. The remaining two VkIII clones consist of two additional VkIII genes which are homologous to each other, but are quite different from the first two VkIII genes. Thus, four new human VkIII genes were defined. Together with four other VkIII genes previously isolated by other investigators, a total of eight human VkIII germ-like genes have now been described. A comparison of the deduced amino acid sequences of these genes with the reported amino acid sequences of all human VkIII light chains suggests that at least one additional VkIII gene exists in the germ line. Among the eight identified human germ-line VkIII genes, three are pseudogenes. Of the remaining five potential functional genes, one gene seems to encode a majority of the VkIII light chains which have been sequenced. Possible explanations for this phenomenon are discussed.
Asunto(s)
Diversidad de Anticuerpos , Cadenas Ligeras de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN/genética , HumanosRESUMEN
The role of immunoglobulin structural genes in the generation of autoantibodies in humans has not been elucidated. Human monoclonal IgM anti-IgG autoantibodies (rheumatoid factors, RFs) from unrelated people often share idiotypic antigens. Antibodies against synthetic peptides have localized two of the shared idiotypic determinants to the second and third complementarity-determining regions of the kappa light chain. The reported sequences of several human RF light chains are remarkably homologous in these regions. Animal studies have shown that some shared idiotypic antigens represent serological markers for immunoglobulin variable (V)-region genes. Therefore, we hypothesized that human RF light chains derived from a single germ-line gene, designated V kappa-(RF), or from a small family of very closely related genes. In the present experiments, we have isolated and sequenced two human V kappa germ-line genes that encode kappa light chains, which are identical or closely related to the light chains of human RF. The data indicate that the shared idiotypic antigens on RF are phenotypic markers for a kappa V-region gene that is highly conserved in the human population. The results also imply that the light chains of IgM anti-IgG autoantibodies can be encoded by germ-line genes without any somatic mutation.
Asunto(s)
Idiotipos de Inmunoglobulinas/genética , Región Variable de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genética , Factor Reumatoide/genética , Secuencia de Aminoácidos , Secuencia de Bases , Reacciones Cruzadas , ADN/aislamiento & purificación , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunologíaRESUMEN
The synthesis and function of IgE are dependent on IgE-binding proteins, which include cell surface IgE receptors and IgE-binding lymphokines. To further our understanding of the IgE system, we have engaged in the molecular cloning of genes for some of these proteins. In studying the in vitro translation products of mRNA from rat basophilic leukemia (RBL) cells, we have identified a Mr 31,000 polypeptide that binds IgE and is also reactive with antibodies to proteins affinity-purified from RBL cells with IgE immunoadsorbent. For the molecular cloning, double-stranded cDNA was synthesized from sucrose gradient-fractionated RBL mRNA, inserted into plasmid pBR322, and used to transform Escherichia coli. By screening transformants with a hybridization-selection/in vitro translation procedure, we identified one clone containing cDNA that hybridized to mRNA coding for a Mr 31,000 IgE-binding protein. The DNA sequence of this cloned cDNA (571 base pairs) was determined and the amino acid sequence corresponding to part of the protein was deduced. In RNA blot analysis, the cDNA hybridized with a mRNA of 1100 nucleotides found in RBL cells but absent in cells not expressing IgE receptors. This cloned cDNA most likely codes for the Mr 31,000 IgE-binding protein identified in RBL cells, which appears to be related to the IgE-binding phenotype of the cells and which may have a significant role in the IgE-mediated activation of basophils and mast cells.
Asunto(s)
Inmunoglobulina E/metabolismo , Linfocinas/genética , Proteínas de Secreción Prostática , Receptores Fc/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Ratones , Peso Molecular , ARN Mensajero/genética , RatasRESUMEN
cDNA corresponding to human IgE heavy (epsilon) chain mRNA was cloned from human IgE-secreting myeloma U266 cells. Partial nucleotide sequence analysis demonstrated that the cloned cDNA contained the coding region for about two-thirds of the CH2 and all of the CH3 and CH4 domains as well as the 3'-untranslated region. This epsilon cDNA was inserted into expression vector pUC7 and expression of an epsilon-chain fragment in Escherichia coli was demonstrated by protein blot analysis using 125I-labeled goat anti-human IgE as probe. The expression product was purified on a column of goat anti-human IgE-conjugated Sepharose 4B and the polypeptide was found to retain binding activity to human basophils.
Asunto(s)
Clonación Molecular , ADN/metabolismo , Escherichia coli/genética , Inmunoglobulina E/genética , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas epsilon de Inmunoglobulina/genética , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Enzimas de Restricción del ADN , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Mieloma Múltiple/inmunologíaRESUMEN
cDNA corresponding to mouse IgE heavy (epsilon) chain mRNA was cloned from mouse IgE-secreting hybridoma cells. A clone containing the epsilon cDNA insert was identified by hybridization to epsilon mRNA and subsequent translation in vitro to unprocessed epsilon chain reactive with anti-mouse IgE antibodies. This clone was used to select 20 other epsilon cDNA clones by colony hybridization. The clone containing the longest insert was selected and the epsilon cDNA insert was subjected to sequence analysis. The determined sequence is 1,279 nucleotides long and contains the coding regions for part of the constant region (C epsilon) I and all of the C epsilon 2, C epsilon 3, and C epsilon 4 domains and also the entire 3' untranslated region of epsilon mRNA. When the amino acid sequence determined from the nucleotide sequence is compared to that of human epsilon chain, significant homologies between corresponding domains of the two epsilon chains are found, including conservations in cysteine and tryptophan residues and carbohydrate attachment sites.
Asunto(s)
Cadenas Pesadas de Inmunoglobulina/genética , Cadenas epsilon de Inmunoglobulina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , ADN/genética , Ratones , ARN Mensajero/genéticaRESUMEN
Certain technical considerations which affected the status of methicillin tolerance in Staphylococcus aureus strains were studied. Methods which consistently demonstrated tolerance or intolerance of a given strain were avoidance of inoculum splashing, use of stationary-phase inoculum, 24-h tube incubation, and minimization of antibiotic carry-over. These studies suggested a need for the establishment of a standardized reference for the determination of tolerance.
Asunto(s)
Meticilina/farmacología , Staphylococcus aureus/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Resistencia a las Penicilinas , Penicilinasa/metabolismo , Factores de TiempoRESUMEN
The streptococcal long-chain reaction was adapted for the measurement of type-specific antibodies to group B beta-hemolytic streptococci (GBBHS). Rabbit antisera incubated with homologous but not heterologous GBBHS produced chains that were 18 to 33 times longer than chains produced by normal rabbit sera. The long chains were easily apparent, in most instances, by scanning the slides. Human sera with mouse protective and opsonic activity against GBBHS serotype Ia produced chains that were always significantly longer than those produced by incubation in nonimmune human sera. Absorption of rabbit or human sera with homologous but not heterologous organisms inhibited the capacity to induce the formation of long chains. The long-chain assay is a simple, rapid, and reproducible test that could constitute a valuable tool for the rapid identification of anti-GBBHS antibodies.
Asunto(s)
Anticuerpos Antibacterianos/análisis , Inmunoensayo , Streptococcus agalactiae/inmunología , Animales , Humanos , Proteínas Opsoninas , Conejos , Streptococcus agalactiae/crecimiento & desarrolloRESUMEN
Sera from 14 of 56 adult women protected mice from intraperitoneal challenge with mouse-virulent group B Streptococcus serotype Ia, and sera from seven of 25 nonparturient women in this group were bactericidal for greater than 99% of the organisms in the presence of normal polymorphonuclear leukocytes. There were no discrepancies between the in vivo and in vitro assays. Protective activity was found in the IgG class in seven sera, and in the IgM class in one. Opsonic activity was partially dependent on heat-labile serum factors. Of 31 mother-cord serum pairs studied, seven maternal sera were protective, but four of the corresponding cord sera were not. Pooled human gamma-globulin injected by either the intraperitoneal or the intramuscular route protected mice from bacterial challenge.