RESUMO
In this study, the cDNA coding serum transferrin (stf) of Misgurnus anguillicaudatus (mastf) was cloned. mastf cDNA is composed of 2326 bp with a 2007 bp open reading frame encoding 668 amino acids. The deduced Mastf protein consists of a signal peptide, two lobes (N and C-lobes) and signature motifs of transferrin (Tf) family. The results of tissue distribution indicated that mastf mRNA was predominantly expressed in the liver. The results indicate that the mastf expression increased significantly in liver, blood, spleen and head kidney after the challenge with Aeromonas sobria, acting as a positive acute protein, suggesting that mastf is related to the immune response. The cloning and expression analysis of mastf further demonstrates the evolutionary conservation of Stf and immune function in vertebrates.
Assuntos
Cipriniformes/genética , Proteínas de Peixes/metabolismo , Transferrina/metabolismo , Aeromonas , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Cipriniformes/sangue , Cipriniformes/imunologia , DNA Complementar/genética , Proteínas de Peixes/genética , Rim Cefálico/metabolismo , Fígado/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Baço/metabolismo , Transferrina/genéticaRESUMO
Studies were conducted to determine the cause of high mortalities of cultured half-smooth tongue sole Cynoglossus semilaevis. Gross signs of disease included loss of appetite, erratic swimming, and haemorrhages on the head, opercula and base of fins, dorsal fin rot, swollen abdomen filled with ascitic fluid and herniation of the intestine. Histological examination of the liver showed focal areas of necrosis and extensive haemorrhages. Virtually pure, dense bacterial cultures were obtained from liver, kidney and spleen tissues, and high pathogenicity of the isolates to tongue sole was confirmed. The phenotypic characteristics of the isolates including morphological, physiological and biochemical characteristics were determined. The 16S rRNA and gyrB genes of the isolates were sequenced, and the phylogenetic trees representing genetic relatedness between the isolate and publicly available 16S rRNA and gyrB gene sequences from GenBank were constructed. The results confirmed that the diseased tongue soles were infected with Vibrio aestuarianus. The sequenced 16S rRNA gene of strain TS1 (GenBank Accession No. GQ372983) was 1446bp, the gyrB gene of strain TS1 (GenBank Accession No. GQ372984) was 1200bp and the two genes exhibited high similarity (98~99%) to those of V. aestuarianus from GenBank. In addition, the activities of extracellular enzymes and haemolysin were also studied; the results showed that the isolates produced beta haemolysis on rabbit blood agar, lecithinase, proteinase, DNase and lipase, but gelatinase was not produced.
Assuntos
DNA Girase/genética , Doenças dos Peixes/microbiologia , Linguados/microbiologia , RNA Ribossômico 16S/genética , Vibrio/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , China , DNA Bacteriano/análise , DNA Bacteriano/genética , Dados de Sequência Molecular , Filogenia , Coelhos , Vibrio/classificação , Vibrio/isolamento & purificação , Vibrio/patogenicidade , VirulênciaRESUMO
OBJECTIVE: To explore the regulatory mechanism of microRNA-328 expression level by targeting the protein ATP Binding Cassette Transporter G2 (ABCG2) in gastric cancer cells and seek for a biological marker of predicting gastric cancer. PATIENTS AND METHODS: SGC-7901 and MKN-28 human gastric cancer cell lines were cultured. Meanwhile, paired gastric cancer pathological tissues and the corresponding adjacent normal tissues were collected. Western blot analysis was used to validate the protein expression of ABCG2. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) analysis was used to detect the mRNA expression level of miR-328 and ABCG2. Cell counting kit-8 (CCK-8) and colony formation assay were performed to validate the proliferous ability of human gastric cancer cells. The transwell invasion and migration were operated to determine the migratory and invasive capacity. Dual-Luciferase reporter assay, qRT-PCR and Western blot were used to prove the target of miR-328. RESULTS: Bioinformatics analysis made a prediction that ABCG2 was a direct functional target of miR-328. Position 619-625 of ABCG2 3'-UTR had a space structure that was complementary to miR-328 by bioinformatics analysis, and there was a significant reduction in the level of miR-328 in human gastric cancer cell lines and tissues. The expression of miR-328 down-regulated proliferation, invasion and migration of human gastric cancer cells in vitro, while silencing of miR-328 accelerated proliferation, invasion and migration of human gastric cancer cells in vitro. All results displayed ABCG2 was direct target protein of miR-328 owing the binding site and they presented a negative correlation. CONCLUSIONS: ABCG2 is the target protein of miR-328. It presents a negative correlation of the expression level between miR-328 and ABCG2. Down-regulation of miR-328 inhibits the proliferation, invasion and migration of gastric cancer cell lines. MiR-328 could predict generation and development of gastric cancer as a biomarker.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , MicroRNAs/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Gástricas/genética , Regiões 3' não Traduzidas , Adulto , Idoso , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Neoplasias Gástricas/metabolismoRESUMO
This present work describes an effective new method for study traditional Chinese medicine (TCM) on meridian tropism (MT) theory, which plays an essential role in clinical selection of TCM according to syndromes and strengthens the therapeutic effects. The new thread included material basis foundation and its tissue distribution study. Xiheliu, the most popular TCM on heart tropism, was investigated by simple and accurate high performance liquid chromatography (HPLC) method. The analysis of plasma after oral administration the total flavonoid of Xiheliu (TFX) exhibited that tamarixetin and kaempferide had the highest concentration and approximately the highest level within 25 min. The mixture of them could last accelerating the urine excretion more than 7 h after a single dose and could not cause the disorder of ion in rats, which was observed in diuretic activity experiment. In view of the reported biological activities was consistent with the effects of Xiheliu, tamarixetin and kaempferide were likely to be the material basis of it. Tissue distribution study showed that the highest level of analytes was in heart, lung, kidney and liver, and most tissues reached maximum level at 30 min post-dose. Since liver was the most important blood-supply tissue, the result of this experiment was in accordance with the MT record of Xiheliu and confirmed that tamarixetin and kaempferide was the material bases of it on MT. This is the first report for the illumination of material basis and the mechanism of Xiheliu on MT by analysis the record of Xiheliu in Compendium of Materia Medica and experimental study.