RESUMEN
Most T lymphocytes express an antigen-specific receptor composed of two subunits, alpha and beta, each of which can exhibit structural variability. A complex selection process operates on T cells during development in the thymus such that cells expressing only particular alpha beta-receptors migrate to the periphery. The alpha-chain repertoire was dissected at different stages of the selection process by using the polymerase chain reaction (PCR) technique to amplify only those transcripts of a particular variable region gene (V58). Sequences from these V58 cDNAs reveal the predominant expression of four joining (J) segments by T cells in the adult thymus, suggesting that molecular or cellular processes select particular V alpha J alpha combinations during development. T cells expressing one of these V58J alpha chains appear to have been negatively selected at a later stage, since these transcripts were present in the spleen at approximately one-tenth the level in the thymus. Results also indicate that residues present at the V alpha J alpha junction may be important in an early selection process.
Asunto(s)
Complejo Mayor de Histocompatibilidad , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/fisiología , Animales , Secuencia de Bases , Genes , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Receptores de Antígenos de Linfocitos T alfa-beta , Recombinación Genética , Bazo/fisiología , Timo/fisiología , Distribución TisularRESUMEN
Cytotoxicity of peritoneal macrophages (pMs) and peritoneal natural killer (pNK) cells toward xenogenic tumor cells was studied in anemic, suckling rats. Dams were fed 6, 12, or 250 mg Fe/kg diet ad libitum throughout gestation and lactation. Pups were injected intraperitoneally with 10(5) plaque-forming units of virus. Four days later cytotoxicity of pMs and pNK cells against YAC-1 mouse lymphoma cells was measured. Body weight, hemoglobin, hematocrit, and viable cell yield of pups were significantly decreased with decreasing dietary iron. pM cytotoxicity was significantly impaired in anemic pups at pM-target-cell ratios of 10:1 and 30:1 at 4 and 16 h (P less than or equal to 0.03). pNK-cell cytotoxicity was significantly impaired in anemic pups at pNK-target-cell ratios of 10:1 and 50:1 at 16 h. Iron-deficient diet consumed by dams throughout gestation and lactation resulted in anemic offspring whose immunologic defense by pMs and pNK cells against xenogenic tumor cells was significantly reduced.
Asunto(s)
Citotoxicidad Inmunológica , Deficiencias de Hierro , Células Asesinas Naturales/inmunología , Macrófagos/inmunología , Complicaciones del Embarazo , Animales , Femenino , Lactancia , Cavidad Peritoneal/citología , Embarazo , Ratas , Ratas EndogámicasRESUMEN
The immune system is composed of two major types of lymphocytes, called B and T cells, that recognize foreign antigens. Recognition of antigens is accomplished through the generation of a large repertoire of different cell surface receptors, called immunoglobulins (Igs) on B cells and T cell receptors (TCRs) on T cells. The elucidation of Ig structure and molecular genetics preceded that of the TCR because of the greater abundance of Ig protein and mRNA. Although studies of TCRs have recently shed light on many of the issues of T cell recognition, the process of examining TCR gene structure has been tedious. Such analyses are also difficult because of the time required for the production, maintenance, and culturing of T cell clones. This report describes several strategies that use the polymerase chain reaction (PCR) to analyze very rapidly the structure of TCRs. Specific manipulations of the amplified material are discussed, as are the advantages of using the PCR to study TCR diversity.
Asunto(s)
Técnicas de Amplificación de Ácido Nucleico , Reacción en Cadena de la Polimerasa/métodos , Receptores de Antígenos de Linfocitos T/genética , Transcripción Genética , Animales , Secuencia de Bases , Biotecnología , Línea Celular , Clonación Molecular/métodos , ADN/genética , Ratones , Datos de Secuencia MolecularRESUMEN
Whole genome sequencing has revolutionized biological sciences, and is leading to a paradigm shift in microbiology. As more microbial genomes are sequenced, and more bioinformatics tools are developed, it has become possible to predict the metabolism of an organism from genomic data. In contrast, predicting the pathogenic potential of parasitic microbes and their interactions with their hosts is still a challenge, especially as the definition of pathogenesis itself is still evolving. In this review, we introduce the subsystem-based technology for genome annotation and analysis, and we discuss some subsystem-based tools available in the National Microbial Pathogen Data Resource (NMPDR, http://www.nmpdr.org) and their potential application in comparative genomics and pathogenomics.
Asunto(s)
Genoma Bacteriano , Programas Informáticos , Biología Computacional , Simulación por Computador , Bases de Datos Genéticas , Genoma , GenómicaRESUMEN
Cryptococcus neoformans is a fungus that possesses two properties unique for yeast: (1) production of a polysaccharide capsule and (2) neurotropism. The natural route of infection by C. neoformans is the respiratory tract; thus, factors that regulate the development and recruitment of memory Th1 cells and monocytes into the brain are critical for an effective response against disseminated C. neoformans infection. Production of TNFalpha prior to day 7 is required to prevent colonization of the central nervous system (CNS). Th1 type immunity is required to clear established foci. In contrast, Th2 type immunity is ineffective at eliminating the infection in the brain and results in decreased survival. C. neoformans infection of MIP-1alpha and CCR5 knockout mice has highlighted the complex role that some chemokines may play in different organs. MIP-1alpha knockout mice have decreased leukocyte recruitment and cryptococcal clearance from the brain compared to wild-type mice. Thus, the host defence mechanisms that clear C. neoformans from the CNS appear to be similar to those in the lungs: via a Th1 cell-mediated inflammatory response that requires chemokines for the recruitment of effector cells.
Asunto(s)
Enfermedades del Sistema Nervioso Central/microbiología , Quimiocinas/fisiología , Criptococosis/microbiología , Cryptococcus neoformans/fisiología , Leucocitos/fisiología , Células TH1/inmunología , Animales , Especificidad de Anticuerpos , Enfermedades del Sistema Nervioso Central/inmunología , Enfermedades del Sistema Nervioso Central/metabolismo , Quimiocina CCL3 , Quimiocina CCL4 , Quimiotaxis , Criptococosis/metabolismo , Cryptococcus neoformans/inmunología , Cryptococcus neoformans/patogenicidad , Susceptibilidad a Enfermedades , Inmunoglobulina G/farmacología , Proteínas Inflamatorias de Macrófagos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Receptores CCR5/genética , Células Th2/inmunología , Factores de Tiempo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , VirulenciaRESUMEN
IGF-I, IGF-II, and IGF binding proteins (IGFBP) were characterized in porcine serum, colostrum, and milk on d 1-28 postpartum. IGF-I and -II were measured by heterologous RIA. Serum IGFBP were characterized by Western ligand blotting and milk IGF binding activity by [125I]-IGF binding assay. IGF-II accounted for 70-85% of serum IGF and rose 2-fold between d 1 and d 28. Serum IGF-I was unaffected by duration of lactation. Milk IGF-II concentrations were higher than IGF-I concentrations on d 1-7 postpartum. After d 10, milk IGF-I and IGF-II contents were not significantly different. Serum contained IGFBP with M(r) of 43, 39, 34, 28, and 24 kD. Over the course of lactation, the 43- and 39-kD bands increased, the 24-kD band decreased, and the 34- and 28-kD bands were unchanged. Milk IGF binding activity increased between d 1 (28%) and d 3 (44%), then declined until d 28 (7%). Serum and milk were separated by isoelectric focusing into 20 fractions, across a gradient from pH 3 to 10, that were screened for IGFBP by Western ligand blotting. Milk contained six IGFBP of similar M(r) as serum IGFBP; however, the relative amounts of the IGFBP and their apparent isoelectric points differed. In conclusion, porcine milk contains both IGF-I and -II, with IGF-II predominating. Several IGFBP with similar M(r) as those found in serum are present in milk. IGF peptide concentrations were highest in prepartum secretions and colostrum, whereas IGF binding activity peaked on d 4 of lactation.
Asunto(s)
Proteínas Portadoras/sangre , Proteínas Portadoras/metabolismo , Lactancia/sangre , Lactancia/metabolismo , Leche/metabolismo , Somatomedinas/metabolismo , Animales , Western Blotting , Proteínas Portadoras/química , Calostro/metabolismo , Femenino , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor II del Crecimiento Similar a la Insulina/metabolismo , Proteínas de la Leche/metabolismo , Peso Molecular , Embarazo , Porcinos , Factores de TiempoRESUMEN
A small percentage (approximately 5%) of the cells in the adult thymus expresses a heterodimeric receptor, gamma delta, that exhibits extensive clonal diversity. The specificity and function of these cells are unclear. Furthermore, it is not known if their role in the immune system is primarily one that operates within the thymus during the selection of the T-cell repertoire or if they function primarily in an antigen-recognition capacity in the peripheral lymphoid system. To examine if gamma delta+ T cells in the periphery are as diverse as those in the thymus, we used the polymerase chain reaction to amplify delta-chain transcripts from polyclonal populations of thymic and splenic lymphocytes (the latter were derived from allogeneic mixed lymphocyte cultures). The nucleotide sequences of delta chains from the spleen, like those from the thymus, were all different. Most of the diversity was present in the region between the variable (V) and joining (J) gene segments and was generated through the use of the two known diversity (D) elements, D delta 1 and D delta 2, and by the addition or deletion of bases at the V delta D delta 1, D delta 1D delta 2, and D delta 2J delta junctions. The extensive gamma delta repertoire among peripheral cells suggests that they have the potential to recognize an array of ligands that could be as diverse as those recognized by alpha beta+ cells. The amplification strategy described here can be used to analyze rapidly the diversity exhibited by any of the members of the immunoglobulin-like gene families that undergo rearrangement.
Asunto(s)
Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/inmunología , Animales , Secuencia de Bases , Línea Celular , Células Cultivadas , Genes , Variación Genética , Sustancias Macromoleculares , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Bazo/inmunología , Timo/inmunología , Transcripción GenéticaRESUMEN
The Drosophila central nervous system (CNS) develops from a population of stem cells called neuroblasts; each neuroblast goes through an invariant cell lineage to produce a characteristic family of neurons or glia. We are interested in the molecular mechanisms controlling neuroblast cell lineage. Recently we identified the prospero (pros) gene, which is expressed in embryonic neuroblasts. Loss of pros function results in aberrant expression of the homeobox genes fushi tarazu, even-skipped and engrailed in a subset of neuroblast progeny, suggesting that pros plays an early and fundamental role in the specification of neuronal fate (Doe et al. 1991). Here we show that the pros gene encodes a highly divergent homeodomain. The homeodomain contains several of the most conserved amino acids characteristic of known homeodomains, yet it is considerably less basic than previously identified homeodomains. These data are consistent with a model in which pros controls neuroblast cell lineages by regulating gene expression.
Asunto(s)
Diferenciación Celular/genética , Sistema Nervioso Central/embriología , Drosophila/genética , Genes de Insecto/fisiología , Neuronas/fisiología , Secuencia de Aminoácidos , Animales , Sistema Nervioso Central/citología , Secuencia Conservada , Expresión Génica/genética , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
Two archaeal proteins, RadA and RadB, share similarity with the RecA/Rad51 family of recombinases, with RadA being the functional homologue. We have studied and compared the RadA and RadB proteins of mesophilic and thermophilic Archaea. In growing cells, RadA levels are similar in mesophilic Methanococcus species and the hyperthermophile Methanococcus jannaschii. Treatment of cells with mutagenic agents (methylmethane sulfonate or UV light) increased the expression of RadA (as evidenced by higher levels of both mRNA and protein) in all organisms tested, but the increase was greater in the mesophiles than in the thermophiles M. jannaschii and Sulfolobus solfataricus. Recombinantly expressed RadA proteins from the mesophile M. voltae and the thermophile M. jannaschii were similar in their ATPase- and DNA-binding activities. All the data are consistent with proposals that RadA plays the same role as eukaryotic Rad51. Surprisingly, the data also suggested that the thermophiles do not need more RadA protein or activity than the mesophiles. On the other hand, RadB is not coregulated with RadA, and its role remains unclear. Neither RadA nor RadB from a mesophile or from a thermophile rescued the UV-sensitive phenotype of an Escherichia coli recA- host.
Asunto(s)
Archaea/genética , Rec A Recombinasas/genética , Archaea/metabolismo , Daño del ADN , Regulación de la Expresión Génica Arqueal , Genes Arqueales , Rec A Recombinasas/análisis , Rec A Recombinasas/metabolismo , Recombinación GenéticaRESUMEN
Our objective was to define the role of the chemokine macrophage-inflammatory protein-1alpha (MIP-1alpha) in the efferent phase of pulmonary cell-mediated immunity (CMI) against Cryptococcus neoformans. Following intratracheal inoculation of C. neoformans (24067) into CBA/J mice, the development of CMI was required for leukocyte recruitment into the lungs at 2 wk postinfection. MIP-1alpha mRNA was expressed by day 6 postinfection, and MIP-1alpha protein in bronchoalveolar lavage fluid was detectable at day 6, but significantly elevated at days 19 and 33. Administration of neutralizing anti-MIP-1alpha Abs from days 7 to 13 blocked the increase in bronchoalveolar lavage fluid MIP-1alpha and resulted in a 37% decrease in total leukocytes in the lungs at day 16. There were 66% fewer macrophages/monocytes and 42% fewer neutrophils in the lungs of anti-MIP-1alpha-treated mice, and the pulmonary burden of C. neoformans was threefold higher. There was no significant difference in the number of eosinophils, CD4+, CD8+, or B220+ lymphocytes between the two groups of mice. Neutralization of MIP-1alpha did not significantly decrease the levels of monocyte chemotactic protein-1 (MCP-1); however, neutralization of MCP-1 significantly decreased MIP-1alpha levels, demonstrating that induction of MIP-1alpha was largely dependent on MCP-1 production. Neutralization of MIP-1alpha also blocked the cellular recruitment phase of a recall response to cryptococcal Ag in the lungs of immunized mice. Thus, in both the contexts of active cryptococcal infection or rechallenge with cryptococcal Ag, MIP-1alpha was required during the efferent phase of CMI for maximal leukocyte recruitment into the lungs, most notably the recruitment of phagocytic effector cells (neutrophils and macrophages).
Asunto(s)
Criptococosis/inmunología , Cryptococcus neoformans/inmunología , Inmunidad Celular , Pulmón/inmunología , Proteínas Inflamatorias de Macrófagos/inmunología , Macrófagos Alveolares/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/inmunología , Quimiocina CCL3 , Quimiocina CCL4 , Criptococosis/patología , Femenino , Pulmón/microbiología , Pulmón/patología , Proteínas Inflamatorias de Macrófagos/análisis , Ratones , Ratones Endogámicos CBA , ARN Mensajero/análisisRESUMEN
After intratracheal inoculation of the AIDS-associated pathogen Cryptococcus neoformans, 12-wk survival was >90% for CCR5+/+ mice but <25% for CCR5-/- mice. There were no defects in lung leukocyte recruitment (wk 5), pulmonary clearance, or delayed-type hypersensitivity in CCR5-/- mice. However, CCR5-/- mice had defects in leukocyte recruitment into the brain and, strikingly, in elimination of cryptococcal polysaccharide from the brain. In nonimmune CCR5-/- mice, there was a significant defect in macrophage recruitment after challenge with shed cryptococcal products (C. neoformans filtrate Ag) but not other nonspecific stimuli. Thus, CCR5 plays specific roles in innate immunity and organ-specific leukocyte trafficking during host defense against C. neoformans.
Asunto(s)
Criptococosis/inmunología , Cryptococcus neoformans/inmunología , Receptores CCR5/fisiología , Animales , Movimiento Celular/genética , Movimiento Celular/inmunología , Criptococosis/genética , Criptococosis/mortalidad , Criptococosis/patología , Cryptococcus neoformans/crecimiento & desarrollo , Inmunidad Innata , Enfermedades Pulmonares Fúngicas/genética , Enfermedades Pulmonares Fúngicas/inmunología , Enfermedades Pulmonares Fúngicas/mortalidad , Enfermedades Pulmonares Fúngicas/patología , Meningitis Criptocócica/genética , Meningitis Criptocócica/inmunología , Meningitis Criptocócica/mortalidad , Meningitis Criptocócica/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Especificidad de Órganos/genética , Especificidad de Órganos/inmunología , Receptores CCR5/biosíntesis , Receptores CCR5/deficiencia , Receptores CCR5/genética , Células TH1/inmunología , Células TH1/metabolismoRESUMEN
We employed a phylogenomic approach to study the evolution of alpha subunits of the proteasome gene family from early diverging eukaryotes. BLAST similarity searches of the Giardia lamblia genome identified all seven alpha proteasome genes characteristic of eukaryotes from the crown group. In addition, a PCR strategy for the amplification of multiple alpha subunit sequences generated single alpha proteasome products for representatives of the Kinetoplastida (Leishmania major), the Parabasalia (Trichomonas vaginalis), and the Microsporidia (Vairimorpha sp., Nosema sp., Endoreticulata sp., and Spraguea lophii). The kinetoplastid Trypanosoma cruzi and the eukaryote crown group Acanthamoeba castellanii yielded two distinct alpha proteasome genes each. The presence of seven distinct alpha proteasome genes in G. lamblia, one of the earliest-diverging eukaryotes, indicates that the alpha proteasome gene family evolved rapidly from a minimum of one gene in Archaea to seven or more in Eukarya. Results from the phylogenomic analysis are consistent with the idea that the Diplomonida (as represented by G. lamblia), the Kinetoplastida, the Parabasalia, and the Microsporidia diverged after the duplication events that originated the alpha proteasome gene family. A model for the early origin and evolution of the proteasome gene family is presented.
Asunto(s)
Cisteína Endopeptidasas/genética , Complejos Multienzimáticos/genética , Familia de Multigenes , Filogenia , Secuencia de Bases , Cartilla de ADN , Células Eucariotas , Reacción en Cadena de la Polimerasa , Complejo de la Endopetidasa ProteasomalRESUMEN
Archaeoglobus fulgidus is the first sulphur-metabolizing organism to have its genome sequence determined. Its genome of 2,178,400 base pairs contains 2,436 open reading frames (ORFs). The information processing systems and the biosynthetic pathways for essential components (nucleotides, amino acids and cofactors) have extensive correlation with their counterparts in the archaeon Methanococcus jannaschii. The genomes of these two Archaea indicate dramatic differences in the way these organisms sense their environment, perform regulatory and transport functions, and gain energy. In contrast to M. jannaschii, A. fulgidus has fewer restriction-modification systems, and none of its genes appears to contain inteins. A quarter (651 ORFs) of the A. fulgidus genome encodes functionally uncharacterized yet conserved proteins, two-thirds of which are shared with M. jannaschii (428 ORFs). Another quarter of the genome encodes new proteins indicating substantial archaeal gene diversity.