RESUMO
South China presents an excellent opportunity to build a phylogeographic paradigm for complex geological history, including mountain lifting, climate change, and river capture/reversal events. The phylogeography of cyprinids, particularly Opsariichthys hainanensis, an endemic species restricted to South China, was examined to explore the relationship between the populations in Red River, Hainan Island and its adjacent mainland China. A total of 37 haplotypes were genotyped for the mitochondrial cytochrome b (Cyt b) gene in 115 specimens from 11 river systems. Relatively high levels of haplotype diversity (h = 0.946) and low levels of nucleotide diversity (π = 0.014) were detected in O. hainanensis. Four major phylogenetic haplotype groups revealed a relationship between phylogeny and geography. Our results found that (i) the ancestral populations of O. hainanensis were distributed south of the Wuzhishan and Yinggeling mountains, including the Changhua River on Hainan Island, and then spread to the surrounding areas, (ii) the admixtures within lineages occurred between the Red River in North Vietnam and the Changhua River in western Hainan Island and (iii) indicated that the exposure of straits and shelves under water retreat, provides opportunities for population dispersion during glaciations.
Assuntos
Cyprinidae/genética , Citocromos b/genética , Animais , China , DNA Mitocondrial/genética , Peixes/genética , Genes Mitocondriais/genética , Estruturas Genéticas/genética , Variação Genética/genética , Genética Populacional/métodos , Genoma Mitocondrial/genética , Haplótipos/genética , Mitocôndrias/genética , Filogenia , Filogeografia , Rios , Análise de Sequência de DNA/métodosRESUMO
The baculovirus expression system has been used to express large quantities of various proteins, including membrane receptors. Here, we reveal a novel property of this expression system to be that certain membrane proteins can be displayed on the budded virus itself. We introduced the genes encoding sterol regulatory element-binding protein-2 (SREBP-2) or SREBP cleavage-activating protein (SCAP), important integral membrane proteins of the endoplasmic reticulum (ER) and/or the Golgi apparatus related to cellular cholesterol regulation, into a baculovirus vector. When insect cells were infected with SREBP-2 or SCAP recombinant viruses, it was found that these ER membrane proteins appeared on the budded baculovirus in addition to the host cell membrane fraction. Compared to proteins expressed on the cell membrane, membrane proteins displayed on virus exhibited both less aggregation and less degradation upon immunoblotting. Using this viral displayed SCAP as the screening antigen, we then generated a new monoclonal antibody specific against SCAP, which was useful for immunological localization studies. This system, which takes advantage of the viral display of membrane proteins, should prove to be a powerful additional tool for postgenomic protein analysis.
Assuntos
Baculoviridae/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas de Ligação a DNA/genética , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/genética , Fatores de Transcrição/genética , Animais , Linhagem Celular , Imuno-Histoquímica , Spodoptera , Proteína de Ligação a Elemento Regulador de Esterol 1 , Proteína de Ligação a Elemento Regulador de Esterol 2RESUMO
Apoptosis inhibitor expressed by macrophages (AIM) inhibits apoptosis of CD4(+)CD8(+) (CD4/CD8) double-positive thymocytes, and supports the viability of these cells on the thymic selection. However, pleiotropic functions of AIM have been suggested. In this study, heat-killed Corynebacterium parvum (C. parvum) was injected into mice carrying the homozygous mutation (AIM(-/-)) and wild-type (AIM(+/+)) mice, to investigate the role of AIM in the formation of hepatic granulomas. In AIM(-/-) mice, the size and the number of hepatic granulomas were larger, and the resorption of granulomas was more delayed than in AIM(+/+) mice. The production of interleukin-12 was more prominent in AIM(-/-) mice than in AIM(+/+) mice. In the liver of AIM(+/+) mice, expression of AIM messenger ribonucleic acid (mRNA) increased after C. parvum injection. In situ hybridization demonstrated that AIM mRNA was expressed in Kupffer cells and exudate macrophages in the liver, especially in granulomas. Larger numbers of T cells and natural killer T (NKT) cells underwent apoptosis in the granulomas of AIM(-/-) mice, suggesting that AIM prevents apoptosis of NKT cells and T cells in C. parvum-induced inflammation. Recombinant AIM (rAIM) protein significantly inhibited apoptosis of NKT cells and T cells obtained from C. parvum-stimulated livers in vitro. These results indicate that AIM functions to induce resistance to apoptosis within NKT cells and T cells, and supports the host defense in granulomatous inflammation.
Assuntos
Apoptose/genética , Granuloma/patologia , Propionibacterium acnes/patogenicidade , Receptores Imunológicos/fisiologia , Linfócitos T/imunologia , Animais , Proteínas Reguladoras de Apoptose , Sequência de Bases , Primers do DNA , Citometria de Fluxo , Infecções por Bactérias Gram-Positivas/patologia , Granuloma/imunologia , Granuloma/microbiologia , Hibridização In Situ , Interleucinas/genética , Células Matadoras Naturais , Hepatopatias/imunologia , Hepatopatias/microbiologia , Hepatopatias/patologia , Camundongos , Camundongos Knockout , RNA Mensageiro/genética , Receptores Imunológicos/deficiência , Receptores Imunológicos/genética , Receptores Depuradores , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Monoclonal antibodies (Mabs) are valuable reagents for the purification, characterization and immunolocalization of proteins. In this study, we raised Mabs against human peroxisome proliferator-activated receptors (PPARs) using baculovirus particles displaying surface glycoprotein gp64-fusion proteins as the immunizing agent. In this system, to display fusion proteins on the viral surface, the amino terminal sequences of human PPARd and PPARg2 are inserted in-frame between the signal sequence and the mature domain of the gp64 nucleotide sequence.Mabs were raised by immunization with whole virus without a purification of the target antigens. The Mabs generated by this novel method were shown to recognize not only the gp64-PPARs fusion protein, but also mature, expressed proteins by a wide variety of techniques, including immunohistochemistry, immunoblotting, and electrophoretic mobility shift assays (EMSAs). Transfection of the transfer vector containing a nucleotide sequence encoding less than 30 amino acids along with linearized baculovirus DNA allows for the production of a high affinity antibody against the corresponding mature form. This method is of potential utility in that it allows the production of valuable antibodies without the requirement of a protein purification step.
