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1.
Dev Comp Immunol ; 91: 101-107, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30385317

RESUMO

Caspase, an aspartate specific proteinase mediating apoptosis, plays a key role in immune response. In our previous study, the expression of a caspase gene was up-regulated in a transcriptome library from the haematopoietic tissue (Hpt) cells of red claw crayfish Cherax quadricarinatus post white spot syndrome virus (WSSV) infection. To further reveal the effect of caspase on WSSV infection, we cloned this caspase gene (denominated as CqCaspase) with an open reading frame of 1062 bp, which encoded 353 amino acids with a caspase domain (CASc) containing a p20 subunit and a p10 subunit. Tissue distribution analysis indicated that the mRNA transcript of CqCaspase was widely expressed in all tested tissues with the highest expression in Hpt, while the lowest expression in muscle. To further explore the effect of CqCaspase on WSSV replication, recombinant protein of CqCaspase (rCqCaspase) was delivered into Hpt cells followed by WSSV infection, which resulted in a significantly decreased expression of both an immediate early gene IE1 and a late envelope protein gene VP28 of WSSV, suggesting that CqCaspase, possibly by the enhanced apoptotic activity, had a strong negative effect on the WSSV replication. These data together indicated that CqCaspase was likely to play a vital role in immune defense against WSSV infection in a crustacean C. quadricarinatus, which shed a new light on the mechanism study of WSSV infection in crustaceans.


Assuntos
Proteínas de Artrópodes/genética , Astacoidea/imunologia , Caspases/genética , Infecções por Vírus de DNA/imunologia , Hemócitos/fisiologia , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Proteínas de Artrópodes/metabolismo , Astacoidea/virologia , Caspases/metabolismo , Clonagem Molecular , Perfilação da Expressão Gênica , Regulação Viral da Expressão Gênica , Imunidade Inata/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Replicação Viral
2.
Dev Comp Immunol ; 74: 217-226, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28479346

RESUMO

It is well known that Tolls/Toll like receptors (TLRs), a family of pattern recognition receptors, play important roles in immune responses. Previously, we found that a Toll transcript was increased in a transcriptome library of haematopoietic tissue (Hpt) cells from the red claw crayfish Cherax quadricarinatus post white spot syndrome virus infection. In the present study, a full-length cDNA sequence of Toll receptor (named as CqToll) was identified with 3482 bp which contained an open reading frame of 3021 bp encoding 1006 amino acids. The predicted structure of CqToll protein was composed of three domains, including an extracellular domain of 19 leucine-rich repeats residues, a transmembrane domain and an intracellular domain of 138 amino acids. Tissue distribution analysis revealed that CqToll was expressed widely in various tissues determined from red claw crayfish with highest expression in haemocyte but lowest expression in eyestalk. Importantly, significant lower expression of the anti-lipopolysacchride factor (CqALF), an antiviral antimicrobial peptide (AMP) in crustaceans, but not CqCrustin was observed after gene silencing of CqToll in crayfish Hpt cell cultures, indicating that the CqALF was likely to be positively regulated via Toll pathway in red claw crayfish. Furthermore, the transcription of both an immediate early gene and a late envelope protein gene VP28 of WSSV were clearly enhanced in Hpt cells if silenced with CqToll, suggesting that the increase of WSSV replication was likely to be caused by the lower expression of the CqALF resulted from the loss-of-function of CqToll. Taken together, these data implied that CqToll might play a key role in anti-WSSV response via induction of CqALF in a crustacean C. quadricarinatus.


Assuntos
Astacoidea/imunologia , Hemócitos/fisiologia , Viroses/imunologia , Vírus da Síndrome da Mancha Branca 1/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Células Cultivadas , Clonagem Molecular , Imunidade Inata , RNA Interferente Pequeno/genética , Receptores Toll-Like/metabolismo , Fatores Genéricos de Transcrição/genética , Fatores Genéricos de Transcrição/metabolismo , Transcriptoma , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
3.
Bioresour Technol ; 124: 129-36, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22989641

RESUMO

This study focused on acclimating a microbial enrichment to biodegrade benzene, toluene, ethylbenzene and xylenes (BTEX) in a wide range of salinity. The enrichment degraded 120 mg/L toluene within 5d in the presence of 2M NaCl or 150 mg/L toluene within 7d in the presence of 1-1.5M NaCl. PCR-DGGE (polymerase chain reaction-denatured gradient gel electrophoresis) profiles demonstrated the dominant species in the enrichments distributed between five main phyla: Gammaproteobacteria, Sphingobacteriia, Prolixibacter, Flavobacteriia and Firmicutes. The Marinobacter, Prolixibacter, Balneola, Zunongwangia, Halobacillus were the dominant genus. PCR detection of genotypes involved in bacterial BETX degradation revealed that the degradation pathways contained all the known initial oxidative attack of BTEX by monooxygenase and dioxygenase. And the subsequent ring fission was catalysed by catechol 1,2-dioxygenase and catechol 2,3-dioxygenase. Nuclear magnetic resonance (NMR) spectroscopy profiles showed that the bacterial consortium adjusted the osmotic pressure by ectoine and hydroxyectoine as compatible solutes to acclimate the different salinity conditions.


Assuntos
Benzeno/metabolismo , Biodegradação Ambiental , Sedimentos Geológicos/química , Poluentes Químicos da Água/metabolismo , Bactérias/classificação , Bactérias/genética , Bactérias/metabolismo , Sequência de Bases , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Filogenia , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética , Microbiologia da Água
4.
Eur J Pharmacol ; 645(1-3): 14-22, 2010 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-20655907

RESUMO

Induction of apoptosis in tumor cells has become the major focus of anti-tumor therapeutics development. Juglone, a major chemical constituent of Juglans mandshurica Maxim, possesses several bioactivities including anti-tumor. Here, for the first time, we studied the molecular mechanism of Juglone-induced apoptosis in human leukemia HL-60 cells. In the present study, HL-60 cells were incubated with Juglone at various concentrations. Occurrence of apoptosis was detected by Hoechst 33342 staining and flow cytometry. Expression of Bcl-2 and Bax mRNA was determined by quantitative polymerase chain reaction (qPCR). The results showed that Juglone inhibits the growth of human leukemia HL-60 cells in dose- and time-dependent manner. Topical morphological changes of apoptotic body formation after Juglone treatment were observed by Hoechst 33342 staining. The percentages of Annexin V-FITC-positive/PI negative cells were 7.81%, 35.46%, 49.11% and 66.02% with the concentrations of Juglone (0, 0.5, 1.0 and 1.5 microg/ml). Juglone could induce the mitochondrial membrane potential (DeltaPsim) loss, which preceded release of cytochrome c (Cyt c), Smac and apoptosis inducing factor (AIF) to cell cytoplasm. A marked increased of Bax mRNA and protein appeared with Juglone treatment, while an evidently decreased of Bcl-2 mRNA and protein appeared at the same time. These events paralleled with activation of caspase-9, -3 and PARP cleavage. And the apoptosis induced by Juglone was blocked by z-LEHD-fmk, a caspase-9 inhibitor. Those results of our studies demonstrated that Juglone-induced mitochondrial dysfunction in HL-60 cells trigger events responsible for mitochondrial-dependent apoptosis pathways and the elevated ratio of Bax/Bcl-2 was also probably involved in this effect.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Juglans , Naftoquinonas/farmacologia , Fator de Indução de Apoptose/metabolismo , Inibidores de Caspase , Caspases/metabolismo , Proliferação de Células/efeitos dos fármacos , Citocromos c/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/metabolismo , Células HL-60 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Oligopeptídeos/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/metabolismo
5.
Mol Biotechnol ; 34(1): 55-68, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16943572

RESUMO

Directed molecular evolution imitates the natural selection process in the laboratory to find mutant proteins with improved properties in the expected aspects by exploring the encoding sequence space. The success of directed molecular evolution experiment depends on the quality of artificially prepared mutant libraries and the availability of convenient high-throughput screening methods. Well-prepared libraries promise the possibility of obtaining desired mutants by screening a library containing a relatively small number of mutants. This article summarizes and reviews the currently available methodologies widely used in directed evolution practices in the hope of providing a general reference for library construction. These methods include error-prone polymerase chain reaction (epPCR), oligonucleotide-based mutagenesis, and genetic recombination exemplified by DNA shuffling and its derivatives. Another designed method is also discussed, in which B-lymphocytes are fooled to mutate nonantibody foreign proteins through somatic hypermutation (SHM).


Assuntos
Clonagem Molecular/métodos , Evolução Molecular Direcionada/métodos , Evolução Molecular , Biblioteca Gênica , Engenharia Genética/métodos , Mutagênese , Reação em Cadeia da Polimerase/métodos , Animais , Evolução Molecular Direcionada/tendências , Humanos , Reação em Cadeia da Polimerase/tendências
6.
Sheng Wu Gong Cheng Xue Bao ; 22(2): 285-92, 2006 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-16607958

RESUMO

As a novel member of the IAP (Inhibitor of apoptosis protein) family, survivin was observed to be expressed in most human cancerous cells. Fusion protein TATm-survivin (T34A) has drawn considerable attention because it is a potential anti-tumor protein that can be transduced into cancer cell with the help of HIV-TAT domain. In this study, the cDNA encoding survivin was cloned by RT-PCR from human breast cancer cell lines B-Cap-37. An expression vector of pRSET-B-HIV-tatm-survivin (T34A) was constructed by PCR after survivin (T34A) was mutated by site-directed mutagenesis. Subsequently, the resultant plasmid was transformed into E. coli BL21 (DE3). Recombinant HIV-TATm-Survivin (T34A) protein was expressed efficiently with 0.5mM IPTG as inducer, reaching a yield of 650mg/liter (as inclusion body) in fermentation culture. The inclusion bodies were solubilized, refolded and purified to a purity of 96% by ion exchange chromatograghy and size-exclusion chromatography. Remarkable effects of the purified recombinant HIV-TATm-Survivin (T34A) on the morphology of cell line SW1990 and B-Cap-37 were observed after being administrated for 4h. MTT assay showed recombinant HIV-TATm-survivin (T34A) protein could inhibit significantly cell proliferation of SW1990 and B-Cap-37 and SSMC-7721 in vitro. Apoptosis rate and cell circle of SW1990 and B-Cap-37 that had been treated with target protein (final concentration 30 microg/mL) were detected with flow cytometry. Results revealed that more than 65% cancer cells were arrested at G1 phase. The study suggested that TATm-survivin (T34A) protein was a hopeful protein drug in the treatment of cancers by facilitating apoptosis of cancer cells. Key words recombinant HIV-TATm-Survivin (T34A), expression and purification, pro-apoptosis bioactivity, SW1990 and B-Cap-37 cancer cell lines


Assuntos
Proteínas Reguladoras de Apoptose/biossíntese , Apoptose/efeitos dos fármacos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes/farmacologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/biossíntese , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/farmacologia , Sequência de Bases , Linhagem Celular Tumoral , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Survivina , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacologia
7.
Zhongguo Zhong Yao Za Zhi ; 29(1): 71-4, 2004 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-15709388

RESUMO

OBJECTIVE: To observe the protective effect of Acanthopanax senticosus saponins (ASS) on myocardial ischemia-reperfusion injury in rats. METHOD: The myocardial ischemia-reperfusion model was induced by 30 min left anterior descending coronary occlusion and 120 min reperfusion in rats. The changes of myocardial infarct size (MIS), the serum creatine phosphokinase (CK) and lactate dehydrogenase (LDH) activity, the serum lipid peroxidation (LPO) content and superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity and plasma endothelin (ET), angiotensin II (Ang II), prostacycline (PGI2) and thromboxane A2 (TXA2) levels and myocardial free fatty acid (FFA) content of infarct and noninfarct area were determined. RESULT: In rats treated by ASS (in a dosage of 25, 50 and 100 mg x kg(-1) i.v. at 30 min after coronary occulusion), the MIS was significantly reduced, the serum CK and LDH activity, the plasma ET, Ang II and TXA2 level and myocardial FFA content declined, while plasma PGI2 level and PGI2/TXA2 was increased signficantly. In addition, serum LPO content declined, SOD and GSH-Px activity were increased markedly. CONCLUSION: ASS has protective effect on myocardial ischemia-reperfusion injury, which may be due to its function of improving free radicals and myocardial metabolism, decreasing plasma ET, Ang II and TXA2 levels and increasing plasma PGI2 level and PGI2/TXA2 ratio etc.


Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Eleutherococcus , Traumatismo por Reperfusão Miocárdica/patologia , Saponinas/farmacologia , Animais , Medicamentos de Ervas Chinesas/isolamento & purificação , Eleutherococcus/química , Feminino , Masculino , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/metabolismo , Miocárdio/patologia , Folhas de Planta/química , Plantas Medicinais/química , Ratos , Ratos Wistar , Saponinas/isolamento & purificação
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