RESUMO
This study investigated the association of tumor necrosis factor-α (TNF-α)-308, -238, and -863 polymorphisms with osteoarticular tuberculosis (OA-TB) prognosis in a Hebei population. Genomic DNA was extracted from venous blood samples of 120 OA-TB patients and 100 healthy volunteers. TNF-α-308, -238, and -863 were analyzed by PCR-restriction fragment length polymorphism; genotype and allele frequencies were calculated. Serum TNF-α level was significantly higher in OA-TB patients (283.16 ± 51.68 ng/L) than in control (122.54 ± 54.65 ng/L; P < 0.05). Higher frequency of TNF-α-308 GG genotype in healthy volunteers (91.0%) than in OA-TB patients (79.2%) indicated that it was a protective factor against OA-TB (OR = 0.405, 95%CI = 0.147-0.657, P = 0.007). Higher frequencies of TNF-α-308 GA genotype and TNF-α-308 allele (A) in OA-TB patients (20.8 and 10.4%, respectively) than in healthy volunteers (8.0 and 5.0%, respectively) indicated an association with increased risk of OA-TB (OR = 3.112, 95%CI = 1.520-6.343, P = 0.003; OR = 3.109, 95%CI = 1.676-6.538, P = 0.006; respectively). Haplotype association analysis of TNF-α polymorphisms (-308/-238/-863) showed a higher frequency of TNF-α AGA in OA-TB patients (12.1%) than in healthy volunteers (3.5%), indicating that it was a risk factor for OA-TB (OR = 4.201, 95%CI = 1.80-9.91, P = 0.010). TNF-α-308 G/A and TNF-α AGA (-308/-238/-863) were associated with a predisposition to OA-TB, which could aid clinical detection, prevention, and prognosis of OA-TB.
Assuntos
Estudos de Associação Genética , Predisposição Genética para Doença , Tuberculose Osteoarticular/genética , Fator de Necrose Tumoral alfa/genética , Adulto , Alelos , Feminino , Genótipo , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Prognóstico , Fatores de Risco , Tuberculose Osteoarticular/patologiaRESUMO
Sucrose phosphate synthase (SPS) is an enzyme used by higher plants for sucrose synthesis. In this study, three primer sets were designed on the basis of known SPS sequences from maize (GenBank: NM_001112224.1) and sugarcane (GenBank: JN584485.1), and five novel SPS genes were identified by RT-PCR from the genomes of Pennisetum spp (the hybrid P. americanum x P. purpureum, P. purpureum Schum., P. purpureum Schum. cv. Red, P. purpureum Schum. cv. Taiwan, and P. purpureum Schum. cv. Mott). The cloned sequences showed 99.9% identity and 80-88% similarity to the SPS sequences of other plants. The SPS gene of hybrid Pennisetum had one nucleotide and four amino acid polymorphisms compared to the other four germplasms, and cluster analysis was performed to assess genetic diversity in this species. Additional characterization of the SPS gene product can potentially allow Pennisetum to be exploited as a biofuel source.
Assuntos
Variação Genética , Glucosiltransferases/genética , Pennisetum/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Análise por Conglomerados , DNA Complementar/química , DNA Complementar/genética , Genoma de Planta/genética , Dados de Sequência Molecular , Pennisetum/classificação , Pennisetum/enzimologia , Filogenia , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
Invariant chain (Ii) isoform, through its thyroglobulin-like (Tg) domain, inhibits cysteine proteases during antigen presentation in vertebrates. In birds, the Ii of Muscovy Duck (MDIi) has 2 forms: MDIi-1 and MDIi-2 (MDIi isoform). To understand the genetic information and expression characteristics of MDIi-2, polymerase chain reaction, and bioinformatic analysis were performed for MDIi-2 from healthy adult Muscovy Duck. The full-length MDIi-2 cDNA sequence was found to be 1377-base pairs, encoding a 285-amino acid protein. MDIi-2 contains 63 amino acids with an insertion sequence in the Tg domain. MDIi-2 shares high identity (72.51-94.74%) with the same protein in other birds. The Tg domain of MDIi-2 is highly conserved and showed relatively high identity (96.83%) among all tested birds. The molecular structure of the Tg domain supports this conservation. MDIi-2 expression was measured in various tissues using real-time quantitative polymerase chain reaction. Similar to MDIi-1, MDIi-2 was detected in all tissues but at different levels. Higher expression level was observed in the spleen, intestinal mucosa, and bursa stipe (bursa of Fabricius stipe) than in other tissues. This suggests that MDIi-2, like MDIi-1, plays an essential role in all tissues and that its differential expression may be related to its functions in these tissues. The coexistence of 2 MDIi isoforms indicates that their functions are correlated in Muscovy Duck. This study improves the understanding of poultry immunology and may be used to improve measures to protect Muscovy Duck from disease.
Assuntos
Antígenos de Diferenciação de Linfócitos B/genética , Patos/genética , Perfilação da Expressão Gênica , Antígenos de Histocompatibilidade Classe II/genética , Especificidade de Órgãos/genética , Sequência de Aminoácidos , Animais , Antígenos de Diferenciação de Linfócitos B/química , Antígenos de Diferenciação de Linfócitos B/metabolismo , Sequência de Bases , Sítios de Ligação/genética , DNA/química , DNA/genética , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/metabolismo , Mucosa Intestinal/metabolismo , Rim/metabolismo , Pulmão/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Baço/metabolismo , Timo/metabolismoRESUMO
Reed canary grass (RCG) is a perennial grass traditionally cultivated for forage. It is also used as fuel to produce energy in Finland and Sweden, and other countries have expressed interest in the cultivation of RCG. In China, arable land is limited. Salinity is considered to be a major factor limiting plant crop development and productivity. To boost biofuel production of RCG and extend its range in saline soil, we seek to improve its salt tolerance. Proline acts as an osmolyte that accumulates when plants are subjected to abiotic stress. P5CS plays a crucial role in proline biosynthesis. We isolated a P5CS gene from RCG, designated B231P5CS (GenBank accession No. JQ622685). B231P5CS is a fragment (971 bp) that encodes a 323-amino acid polypeptide. We also cloned an actin gene fragment from RCG as a reference gene in expression analysis of B231P5CS gene. Expression analysis revealed that B231P5CS transcripts were upregulated in leaves after treatment with salt (200 mM NaCl) and that transcript levels of B231P5CS reached a maximum 12 h after exposure, which was 14.69 times the level in control plants. The trends of expression were exactly opposite in roots; transcripts were downregulated after salt treatment. Proline concentration increased in leaves after stress. In contrast, proline content of roots decreased up to 3.6-fold relative to controls. Changes in proline concentration after stress were correlated with B231P5CS expression. Our results suggest that B231P5CS is a stress-inducible gene and plays a non-redundant role in plant development. This gene may be used to improve stress tolerance of RGC and other bioenergy feedstock.
Assuntos
Glutamato-5-Semialdeído Desidrogenase/genética , Complexos Multienzimáticos/genética , Phalaris/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glutamato-5-Semialdeído Desidrogenase/classificação , Glutamato-5-Semialdeído Desidrogenase/metabolismo , Dados de Sequência Molecular , Complexos Multienzimáticos/classificação , Complexos Multienzimáticos/metabolismo , Phalaris/enzimologia , Phalaris/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/classificação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Filogenia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/classificação , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Prolina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tolerância ao Sal/genética , Análise de Sequência de DNA , Cloreto de Sódio/farmacologia , Estresse Fisiológico/genética , Fatores de TempoRESUMO
Cross-presentation (CP) is important for priming T cell responses to many viral, bacterial, and tumor antigens. Here, we designed two Ii mutants, based on evidence that the invariant chain (Ii, also named CD74) binds newly synthesized MHC class I molecules with the class II-associated invariant chain peptide (CLIP) region of Ii, which occupies the peptide-binding groove. Specifically, we designed (1) Ii-O257, which is a CLIP-substituted Ii chimer, in which OVA257-264 (SIINFEKL) was substituted for CLIP, and (2) Ii-, also named CT257, which is a C-terminal truncated form of Ii-O257 that contains the N-terminal flanking region of Ii. We immunized C57BL/6 mice with these recombinant proteins. Real-time PCR detected that mice immunized with either Ii-O257 or Ii-CT257 recombinant proteins exhibited increased IFN-γ mRNA expression (approximately 11-fold and 13-fold, respectively) and increased IL-2 mRNA expression (approximately 9-fold and 11-fold, respectively), compared to mice immunized with the OVA257-264 peptide. In vivo cytokine analysis showed that recombinant Ii proteins were highly efficient at activating T cells. Confocal microscopy and co-immunoprecipitation showed that the 2 Ii-OVA257-264 chimers are associated intracellularly with H-2K(b) molecules. Thus, Ii-CT257 (amino acids 1-89) binds stably to MHC class I with high affinity, indicating that it is a minimal functional fragment of the Ii immune vector. In conclusion, the N-terminal functional region of the Ii fusion protein containing CTL epitopes might prove to be useful for developing peptide or DNA vaccines that use CP as the main mechanism for CD8(+) T cell stimulation.
Assuntos
Antígenos de Diferenciação de Linfócitos B/química , Apresentação Cruzada , Epitopos/imunologia , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe I/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Antígenos de Diferenciação de Linfócitos B/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Interleucina-2/imunologia , Ativação Linfocitária/imunologia , Camundongos , Peptídeos/genética , Peptídeos/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
c-Jun N-terminal kinase (JNK) is an important member of the mitogen-activated protein kinase superfamily. The allotetraploid crucian carp is a product of distant hybridization of female red crucian carp with male common carp. It is the first natural case of an allotetraploid with stable genetic characters, including fertility of both female and male animals. In this study, 2 jnk1 cDNAs (including jnk1a and jnk1b) have been cloned from the polyploid crucian carp system, consisting of the allotetraploid crucian carp, the triploid crucian carp, and their original parents (red crucian and common carp). We show that jnk1a and jnk1b represent 2 splice forms arising from the jnk1 gene. On the basis of the genetic structure of jnk1a gene in the polyploid crucian carp system, we demonstrated that the allotetraploid crucian carp is phylogenetically closer to its paternal parent (common carp) than to its maternal parent. We further show a similarity between the triploid crucian carp and its original female parent (red crucian carp). Comparisons of genetic structures indicated that the jnk1b genes of allotetraploid and triploid crucian carp are more similar to those of the original paternal parent rather than the original female parent (red crucian carp). RT-PCR analysis indicated that both the jnk1a and jnk1b genes are widely expressed in fish embryos and in the adult organs, displaying distinct features of embryonic-stage and organ specificity in the polyploid crucian carp system.
Assuntos
Carpas/genética , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Poliploidia , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Carpas/classificação , Carpas/metabolismo , Quimera/genética , Clonagem Molecular , Embrião não Mamífero/metabolismo , Evolução Molecular , Feminino , Regulação da Expressão Gênica , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Especificidade de Órgãos , FilogeniaRESUMO
The invariant chain (Ii) plays an important role as a chaperone for MHC II maturation and facilitates antigen presentation in vertebrates. We cloned, characterized and made a homology analysis of healthy adult muscovy duck Ii (MDIi), from a poultry farm in the suburban district of Hefei city in China, by rapid amplification of cDNA ends (RACE)-PCR and by measuring expression of the MDIi gene in various tissues by real-time quantitative PCR. A full-length cDNA sequence of MDIi was obtained, 1188-bp long, encoding a 222-amino acid protein. A comparison of the amino acid sequence of Ii between muscovy duck and other birds showed high similarity (66.3-95.3%). Characteristic functional domains found in Ii of other species, such as cytoplasmic domain, transmembrane domain, class II-associated Ii-derived peptide (CLIP) and trimerization domain, were identified in MDIi. Although all functional domains of Ii were found to be highly conserved, small differences in the CLIP sequence occur among the various species. Expression of MDIi was detected in all tissues at different levels. A higher expression level was found in the spleen, intestinal mucosa and the bursa stipe (bursa of Fabricius stipe) than other tissues. This tissue-specific expression suggests that MDIi plays an essential role in all tissues and differential expression may be a function of the innate structures and essential functions of these tissues.
Assuntos
Antígenos de Diferenciação de Linfócitos B/genética , Patos/genética , Antígenos de Histocompatibilidade Classe II/genética , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos B/imunologia , Proteínas Aviárias/genética , Proteínas Aviárias/imunologia , Clonagem Molecular , Sequência Conservada , DNA Complementar/biossíntese , Patos/imunologia , Patos/metabolismo , Antígenos de Histocompatibilidade Classe II/imunologia , Modelos Moleculares , Dados de Sequência Molecular , Especificidade de Órgãos , Filogenia , Reação em Cadeia da Polimerase/métodos , Estrutura Terciária de Proteína , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
A flow-injection configuration based on a dual-phase gas-permeation system from a liquid donor to a gas acceptor stream with a thermistor flow-through detector is proposed for the direct analysis of the gas in the acceptor. This system was applied for the determination of carbon dioxide (in the form of carbonate) using the following chemical reaction: CO(2)(g)+2NH(3)(g)+H(2)O(g)=(NH(4))(2)CO(3)(s), with a linear response from 1x10(-3) to 50x10(-3) mol l(-1) of CO(3)(2-). Carbon dioxide was produced in the liquid donor and permeated into the gaseous acceptor stream of air/water vapor. The detection limit is 1x10(-3) mol l(-1) of carbonate, and a sampling frequency of 60 h(-1) is achieved with a relative standard deviation of 4.1% for replicate injections. The dual-phase gas-permeation flow-injection manifold, along with the membrane and phase separations, as well as the chemical reaction, provides enhanced selectivity when compared with the system employing a liquid acceptor stream, as serious interferents in this system, for instance, acetate and formate, among others, do not interfere in the proposed system.