RESUMO
Hypoxia inducible factor-1 (HIF-1) is a key mediator during cancer cells to adapt tumor hypoxic condition. In this study, a series of adamantane-based compounds were synthesized and evaluated as potential inhibitors of HIF-1α. Examination of their structure-activity relationship (SAR) identified the adamantane-containing indole derivative 20a as a potent inhibitor of HIF-1α in Hep3B cell lines under tumor hypoxia (IC50 = 0.02 µM). The study herein may provide valuable information for the development of novel therapeutics against cancer and tumor angiogenesis.
Assuntos
Acetanilidas/farmacologia , Adamantano/análogos & derivados , Adamantano/farmacologia , Antineoplásicos/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Acetanilidas/síntese química , Acetanilidas/toxicidade , Adamantano/síntese química , Adamantano/toxicidade , Antineoplásicos/síntese química , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Relação Estrutura-Atividade , Hipóxia TumoralRESUMO
AIM: To investigate the chemical constituents of the cultures of Laetiporus sulphureus (Bull.) Murrill. METHOD: Compounds were isolated and purified by various chromatographic techniques. The structure of the new compound was determined by interpretation of MS and 1D-, 2D-NMR spectroscopic data, while the known compounds were identified by comparison of their data with those reported. RESULTS: Three mycophenolic acid derivatives, 6-((2E, 6E)-3, 7-dimethyldeca-2, 6-dienyl)-7-hydroxy-5-methoxy-4-methylphtanlan-1-one (1), 6-((2E, 6E)-3, 7, 11-trimethyldedoca-2, 6, 10-trienyl)-5, 7-dihydroxy-4-methylphtanlan-1-one (2), and 6-((2E, 6E)-3, 7, 11-trimethyldedoca-2, 6, 10-trienyl)-7-hydroxy-5-methoxy-4-methylphtanlan-1-one (3) were isolated. CONCLUSION: Among them, compound 1 was new, and compound 2 exhibited moderate cytotoxicity against HL-60, SMMC-7721, A-549, and MCF-7 cells, with IC50 values of 39.1, 31.1, 27.4, and 35.7 µmol·L(-1), respectively.
Assuntos
Produtos Biológicos/isolamento & purificação , Ácido Micofenólico/análogos & derivados , Fenóis/isolamento & purificação , Polyporales/química , Agaricales , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Produtos Biológicos/uso terapêutico , Células HL-60 , Humanos , Células MCF-7 , Estrutura Molecular , Ácido Micofenólico/química , Ácido Micofenólico/isolamento & purificação , Neoplasias/tratamento farmacológico , Fenóis/química , Fenóis/farmacologia , Fenóis/uso terapêuticoRESUMO
Two new ylangene-type sesquiterpenoids, postinins A (1) and B (2), were isolated from cultures of the fungus Postia sp. Structures 1 and 2 were elucidated on the basis of extensive spectroscopic analysis. The bioactivity evaluation showed that both compounds had significant inhibitory activities against protein tyrosine phosphatase 1B, and SH2-containing cytoplasmic tyrosine phosphatase-1 and -2 with IC50 values of 1.6-6.2 µg/ml.
Assuntos
Coriolaceae/química , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Sesquiterpenos/isolamento & purificação , Animais , Concentração Inibidora 50 , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Sesquiterpenos/química , Sesquiterpenos/farmacologiaRESUMO
To evaluate whether cerebral white matter integrity is related to cognitive function, and whether diffusion tensor imaging (DTI) could differentiate amnestic mild cognitive impairment (aMCI) from Alzheimer's disease (AD), 12 patients with AD, 12 with aMCI, and 12 controls were recruited for this study. Cognitive functions of all subjects were assessed using the Mini-Mental State Examination (MMSE) and AD Assessment Scale - Cognitive Subscale (ADAS-Cog). DTI studies were acquired, and fractional anisotropy (FA) and mean diffusivity (MD) values of normal-appearing white matter (NAWM) in multiple brain regions were obtained. Results showed that MMSE and ADAS-Cog subscores were significantly associated with white matter integrity of the temporal-parietal lobes. A decrease in FA values and an increase in MD values in multiple cortical regions were confirmed in patients with AD compared to controls. MD values in the posterior region of the corpus callosum in aMCI differed from those of early AD. Significant reductions of FA values in the NAWM of the parietal lobe was observed in aMCI compared to controls. Our data indicate that the microstructural white matter integrity in the temporal-parietal lobes is gradually impaired in the progressive process of AD, and that splenium MD values could be used as a biomarker differentiating aMCI from AD.
Assuntos
Envelhecimento/patologia , Doença de Alzheimer/patologia , Amnésia/patologia , Disfunção Cognitiva/patologia , Imagem de Tensor de Difusão/métodos , Leucoencefalopatias/patologia , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/diagnóstico , Amnésia/diagnóstico , Anisotropia , Biomarcadores , Disfunção Cognitiva/diagnóstico , Corpo Caloso/patologia , Imagem de Tensor de Difusão/instrumentação , Feminino , Humanos , Leucoencefalopatias/diagnóstico , Masculino , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: To develop a detection method for Cryptosporidium parvum oocysts from water samples, which combined immunomagnetic separation (IMS) with Taqman real-time PCR (qPCR). METHODS: Conditions of separation and enrichment of IMS method by using specific streptavidin magnetic beads coated with monoclonal antibodies Cp23 directed against C. parvum oocysts were optimized. Special primers of PCR and Taqman probes were designed referring to the 18S rDNA gene of C. parvum (GenBank Accession No. AB513881.1). The conserved genes were amplified from genomic DNA of C.parvum, and then cloned into Peasy-T1 vector. Tenfold dilutions of positive plasmids (10(4)-10(8) copy/microl) were used to construct a standard curves by Taqman qPCR. The specificity of the assay was determined using genomic DNA of C. baileyi, Toxoplasma gondii, C. canis and Escherichia coli. The sensitivity of this assay was evaluated by analyzing 10-fold serially dilutions of plasmids ranging from 10(0) to 10(8) copy/microl. Both IMS-qPCR assay and indirect immunofluorescent-antibody assay (IFA) were applied to detect 50 water samples collected from the dairy cattle farms in Hebei. RESULTS: The optimal incubation concentration and time of antibody Cp23 were 20 ng/ml and 30 min, respectively, and the catching time was 30 min, the recovery rate was more than 95%. PCR product was 272 bp, and identified by restriction enzyme digestion and nucleotide sequencing. There was a good linear relationship between the standard plasmids and Ct value (correlation r2 = 0.996 1) of the Taqman qPCR. No cross-reactivity was observed with C. baileyi, T. gondii, C. canis and E. coli. The sensitivity of C. parvum-specific assay was 10 copy/microl. Compared with IFA as golden standard method, the specificity and sensitivity of IMS-qPCR for 50 water samples was 100%(18/18) and 93.8% (30/32), respectively. CONCLUSION: The IMS-qPCR assay can be used to specifically detect C. parvum oocysts in water samples.
Assuntos
Cryptosporidium parvum/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Água/parasitologia , Cryptosporidium parvum/genética , DNA Ribossômico/isolamento & purificação , Separação Imunomagnética/métodosRESUMO
In the title compound, C(14)H(12)BrN, the fused-ring system is slightly buckled as its two benzene rings are inclined to one another by 3.41â (14)°.
RESUMO
AIM: To construct a recombinant strain BL21(DE3)(pET-28a-OmpS(2);), and obtain a genetic engineering vaccine to provide protective immunity against diseases caused by Edwardsiella tarda. METHODS: According to the GenBank sequences (GenBank Accession No. AY078509), one pair of primers was designed and the outer membrance protein gene (OmpS(2);) of Edwardsiella tarda HB01 was amplified by PCR. The OmpS(2); gene was cloned in pET-28a vector and transformed into Escherichia coli BL21(DE3). The OmpS(2); protein was highly expressed when the recombinant strain BL21 (DE3) (pET-28a-OmpS(2);) was induced by IPTG. The expressed protein was 47 kD as estimated by 150 g/L SDS-polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: The immunogenicity of the expressed OmpS(2); protein was confirmed by Western blotting. Mice and Paralichthys olivaceus were immunized with the genetic engineering vaccines of Edwardsiella tarda and Aeromonas hydrophila, showing promise that all these vaccines have a high protective ability. And the protective ability to Edwardsiella tarda and Aeromonas hydrophila in Paralichthys olivaceus respectively reached 70% and 80%. CONCLUSION: The recombinant strain BL21 (DE3)(pET-28a-OmpS(2);) could be candidate of genetic engineering vaccine to provide protective immunity against diseases caused by Edwardsiella tarda.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Edwardsiella tarda/genética , Edwardsiella tarda/imunologia , Aeromonas hydrophila/imunologia , Animais , Vacinas Bacterianas/imunologia , Clonagem Molecular , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/prevenção & controle , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Regulação Bacteriana da Expressão Gênica , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/prevenção & controle , CamundongosRESUMO
A novel phenylacetic acid (PAA)-induced CoA-ligase-encoding gene, designated as phlC, has been cloned from penicillin-producing fungus Penicillium chrysogenum. The open reading frame of phlC cDNA was 1671 bp and encoded a 556 amino acid residues protein with the consensus AMP binding site and a peroxisomal targeting signal 1 on its C terminus. The deduced amino acid sequence showed 37% and 38% identity with characterized P. chrysogenum Phl and PhlB protein, respectively. Functional recombinant PhlC protein was overexpressed in Escherichia coli. The purified recombinant enzyme was capable to convert PAA into its corresponding CoA ester with a specific activity of 129.5 ± 3.026 pmol/min per mg protein. Similar to Phl and PhlB, PhlC displayed broad substrate spectrum and showed higher activities to medium- and long-chain fatty acids. The catalytic properties of PhlC have been determined and compared to those of Phl and PhlB.
Assuntos
Acetilcoenzima A/biossíntese , Coenzima A Ligases/genética , Penicillium chrysogenum/enzimologia , Sequência de Aminoácidos , Clonagem Molecular , Coenzima A Ligases/química , Coenzima A Ligases/metabolismo , Escherichia coli/genética , Dados de Sequência Molecular , Penicillium chrysogenum/genética , Fenilacetatos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de SequênciaRESUMO
BACKGROUND/AIMS: Helicobacter pylori (H. pylori) infection is one of the leading causes of gastritis, gastric ulcer, gastric cancer and other gastrointestinal diseases. In this study we aim to evaluate those factors that involved in the prevalence of H. pylori infection, such as socioeconomic living conditions, socioeconomic level, eating habits, and H. pylori gene subtype. METHODOLOGY: Included in this study are data on the daily habits and chronic disease history obtained from personal interviews of 798 healthy adults. The presence ofH. pylori infection is identified using an ELISA kit for detection of H. pylori IgG antibodies in serum. H. pylori gene subtype is determined by polymerase chain reaction (PCR) and specific probes. RESULTS: H. pylori prevalence is 54.5% in Northern China and diabetes, hyperlipidemia and atherosclerosis, age and waist circumference played important roles in H. pylori prevalence. VacA-positive strains are the most popular genotype. The most common strain of H. pylori is vacuolating cytotoxin gene A product (VacA), VacA-sla-m2 subtype. CONCLUSIONS: These data support that personal and environmental conditions affect H. pylori infection in adults, and H. pylori gene subtype may play important role in the prevalence of its infection.
Assuntos
Infecções por Helicobacter/etiologia , Helicobacter pylori/fisiologia , Adolescente , Adulto , Idoso , China/epidemiologia , Feminino , Infecções por Helicobacter/epidemiologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/classificação , Humanos , Masculino , Pessoa de Meia-Idade , Prevalência , Fatores de RiscoRESUMO
An infectious cDNA clone of H120 vaccine strain of infectious bronchitis virus (IBV) was constructed to demonstrate its potential as a gene transfer vector. Primers were designed according to the published genome sequence of H120 strain, and ten cDNA fragments covering the entire genome of H120 strain was amplified by RT-PCR. All the PCR products were ligated into pMD19-T vector and sequenced, and the ORF5a open reading frame in the pMDTM9 plasmid was replaced by an enhanced green fluorescent protein (EGFP) gene. Recombinant plasmids were digested by the restriction enzyme Bsa I, and all the cDNA fragments were recovered. By using appropriate ligation strategy, the genomic cDNA of H120 strain were reconstituted. Then genome RNA was synthesized in vitro by T7 RNA polymerase and transfected into BHK-21 cells. Recombinant virus expressing the green fluorescent protein was rescued and identified by RT-PCR and sequencing. The characteristics of recombinant virus were evaluated by passage in embryonated chicken eggs. This study showed that the 5a ORF is a good candidate for an insertion site of recombinant genes for the development of IBV infectious cDNA clone as a gene transfer vector.
Assuntos
DNA Complementar/genética , Expressão Gênica , Técnicas de Transferência de Genes/instrumentação , Proteínas de Fluorescência Verde/genética , Vírus da Bronquite Infecciosa/genética , Animais , Linhagem Celular , Embrião de Galinha , Cricetinae , DNA Complementar/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Vírus da Bronquite Infecciosa/fisiologiaRESUMO
Mycophenolic acid (MPA) has been known for decades to be an anticancer and immunosuppressive agent and has significant anticancer properties, but its underlying molecular mechanisms are poorly characterized. Peroxisome proliferator-activated receptor gamma (PPARγ) has a central role in adipocyte differentiation, and MPA has been shown to be a potent PPARγ agonist. Whether PPARγ activation has a putative role in the anticancer efficacy of MPA via induction of adipocyte-like differentiation has not been elucidated. In the present study, MPA was demonstrated to dose-dependently activate PPARγ transcription in the GAL4-hPPARγ (LBD) chimeric receptor assay and PPRE-luc reporter gene assay with an EC(50) of 5.2-9.3 µM. Treatment of the breast cancer cell lines MDA-MB-231 and MCF-7 with MPA resulted in differentiation of adipose tissue that was characterized by accumulation of intracellular lipids, enlargement of cell volume, and permanent withdrawal from the cell cycle at the G1/G0 stage. At a molecular level, the expression of three adipocyte differentiation markers (PPARγ, adipsin D, and aP2) was remarkably induced in differentiated breast cancer cells. However, RNA interference experiments showed that PPARγ-knockdown cannot completely reverse the differentiated state of MDA-MB-231 cells after MPA treatment. These data suggest that the effects of MPA on adipocyte-like terminal differentiation of breast cancer cells are (at least in part) due to PPARγ activation, which is a novel anticancer mechanism of MPA.
Assuntos
Adipócitos/patologia , Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Diferenciação Celular/efeitos dos fármacos , Ácido Micofenólico/farmacologia , PPAR gama/metabolismo , Animais , Células CHO , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cricetinae , Cricetulus , Fase G1/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , PPAR gama/deficiência , PPAR gama/genética , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
Nuclear receptors are a superfamily of ligand-activated transcription factors that play key roles in many biological processes, and have become one class of the most important targets in drug discovery. Mammalian one-hybrid system has been used to develop a cell-based functional transactivation high-throughput screening (HTS) assay for detecting nuclear receptors ligands. In the present study, we proved that different promoters used in the reporter vector had significant different impacts on the performance of HTS assays. The assay using the SV40 promoter in the reporter vector showed the characteristics of much higher signal/noise ratios, acceptable Z' factors (>0.6), low coefficient variation (<12.5%) and higher hits rate, which could be more robust, reproducible, and sensitive. In contrast, utilizing a TATA box promoter in the assay resulted in higher variance and low sensitivity. In addition, it was found that the assay using SV40 had longer signal decay time and was easier to be miniaturized in 384-well format. It has been confirmed that the choice of a promoter is a critical factor in developing a reporter gene HTS assay. However, the SV40 promoter used in the present study has been shown to be more adaptable than the minimal promoter TATA box in the Mammalian one-hybrid HTS assays for detecting nuclear receptor agonists.
Assuntos
Receptores Ativados por Proliferador de Peroxissomo/agonistas , Regiões Promotoras Genéticas/genética , Técnicas do Sistema de Duplo-Híbrido , Animais , Bezafibrato/análise , Bezafibrato/farmacologia , Células Cultivadas , Ácido Quenodesoxicólico/análise , Ácido Quenodesoxicólico/farmacologia , Descoberta de Drogas , Vetores Genéticos/genética , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Hidrocarbonetos Fluorados/análise , Hidrocarbonetos Fluorados/farmacologia , Ligantes , Camundongos , Células NIH 3T3 , Pioglitazona , Pirimidinas/análise , Pirimidinas/farmacologia , Rosiglitazona , Sensibilidade e Especificidade , Relação Estrutura-Atividade , Sulfonamidas/análise , Sulfonamidas/farmacologia , Tiazolidinedionas/análise , Tiazolidinedionas/farmacologiaRESUMO
Smart sensors are emerging as a promising technology for a large number of application domains. This paper presents a collection of requirements and guidelines that serve as a basis for a general smart sensor architecture to monitor electricity meters. It also presents an electricity meter monitoring network, named EMMNet, comprised of data collectors, data concentrators, hand-held devices, a centralized server, and clients. EMMNet provides long-distance communication capabilities, which make it suitable suitable for complex urban environments. In addition, the operational cost of EMMNet is low, compared with other existing remote meter monitoring systems based on GPRS. A new dynamic tree protocol based on the application requirements which can significantly improve the reliability of the network is also proposed. We are currently conducting tests on five networks and investigating network problems for further improvements. Evaluation results indicate that EMMNet enhances the efficiency and accuracy in the reading, recording, and calibration of electricity meters.
Assuntos
Redes de Comunicação de Computadores , Eletricidade , Telemetria/instrumentaçãoRESUMO
OBJECTIVE: To study the chemical constituents of the stems of Clematis parviloba. METHOD: The compounds were isolated and purified by repeated column chromatography with silica gel, Sephadex LH-20 and HPLC. Their structures were identified by spectroscopic data together with physical and chemical property. RESULT: Ten compounds have been isolated from the stems of C. parviloba, and identified as: (+) pinoresionol (1), (+) pinoresionol-4'-O-p-D-glucopyranoside (2), ( +) pinoresionol4, 4'-O-bis-beta-D-glucopyranoside (3), (-) syringaresinol (4), (+) syringaresinol-4'-O-beta-D-glucopyranoside (5), (-)episyringaresinol (6), (+) medioresinol-4'-O-beta-D-glucopyranoside (7), (+) lariciresinol-4-O-beta-D-glucopyranoside (8), (+) lariciresinol-4'-O-beta-D-glucopyranoside (9), (+) lariciresinol-4, 4'-O-bis-beta-D-glucopyranoside (10), respectively. CONCLUSION: Compounds 6, 7 were isolated from this genus for the first time, and the other ones were isolated from this plant for the first time.
Assuntos
Clematis/química , Medicamentos de Ervas Chinesas/química , Lignanas/química , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Medicamentos de Ervas Chinesas/isolamento & purificação , Furanos/química , Furanos/isolamento & purificação , Glucosídeos/química , Glucosídeos/isolamento & purificação , Lignanas/isolamento & purificação , Espectroscopia de Ressonância Magnética , Caules de Planta/química , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
L-sorbose/L-sorbosone dehydrogenase from Ketogulonigenium vulgare S2 can transform L-sorbose to 2-KLG, which is widely used in production of Vitamin C. In order to obtain the engineering strain producing L-sorbose/L-sorbosone dehydrogenase and simplify the fermentation technology, firstly, this enzyme was purified by the methods of ammonium sulfate precipitation, DEAE Sepharose Fast Flow and Q Sepharose High Performance. Then, the purified L-sorbose/L-sorbosone dehydrogenase was injected to rabbit to obtain antibody. Next, the genomic library of Ketogulonigenium vulgare S2 was constructed by inserting the restriction fragments of chromatosomal DNA digested with Sau3A I into cosmid pKC505 vector digested by Hpa I and Pst I, which were packed with lamda phage package protein and transferred into E. coli DH5alpha in vitro. Finally, the positive strain K719# was selected from more than 12,000 clones via Dot-ELISA. Through the test of SDS-PAGE and thin layer chromatography, the results showed that the engineering strain K719# had the same biological activity as Ketogulonigenium vulgare S2 after adding coenzyme PQQ.
Assuntos
Desidrogenases de Carboidrato/genética , Desidrogenases de Carboidrato/isolamento & purificação , Escherichia coli/metabolismo , Biblioteca Genômica , Gluconobacter oxydans/enzimologia , Desidrogenases de Carboidrato/metabolismo , Clonagem Molecular , Escherichia coli/genética , Gluconobacter oxydans/genética , Gluconobacter oxydans/crescimento & desenvolvimento , Sorbose/metabolismo , Açúcares Ácidos/metabolismo , Transformação BacterianaRESUMO
Glutathione transferases (GSTs) are a family of multifunctional proteins that mainly catalyze the conjugation of intracellular glutathione (GSH) to a wide variety of endogenous and exogenous electrophilic compounds. GSTs play important roles in stress tolerance and in the detoxification metabolism in organisms. A novel GST gene, Pc gstB, was cloned from penicillin producing fungus Penicillium chrysogenum using RT-PCR. The open reading frame (ORF) of Pc gstB was 651 bp and encoded a peptide of 216 residues. The deduced amino acids sequence had conserved GST domain and showed 65% identity to the characterized Aspergillus fumigutus gstB. The entire ORF of Pc gstB was inserted into vector pTrc99A and transformed into Escherichia coli DH5alpha. Recombinant PcGstB was overexpressed and its GST activity toward substrate 1-chloro-2,4-dinitrobenzene (CDNB) was validated.
Assuntos
Proteínas Fúngicas/genética , Glutationa Transferase/genética , Penicillium chrysogenum/enzimologia , Penicillium chrysogenum/genética , Proteínas Recombinantes/biossíntese , Catálise , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/metabolismo , Genes Bacterianos/genética , Glutationa Transferase/metabolismo , Fases de Leitura Aberta , Proteínas Recombinantes/genética , Análise de Sequência de ProteínaRESUMO
Uvaria kweichowensis is a folk nongovernmental herb used to treat cure inflammation and tumour in the Southwest area of China. During the course of our investigation for antitumour agents from the stems of Uvaria kweichowensis, six amides were obtained by means of solvent extraction, chromatography on silica gel and Sephadex LH-20 repeatedly. And their structures were identified as uvariadiamide (1), cepharanone (2), aristololactam A II (3), enterocarpam II (4), aristololactam A Ia (5), and 4,5-dioxodehydroasimilobine (6) on the basis of chemical methods and spectral analyses (EI-MS, 1H NMR, 13C NMR). Among them, compound 1 is a new compound; the other compounds were obtained from this plant for the first time.
Assuntos
Amidas/isolamento & purificação , Ácidos Aristolóquicos/isolamento & purificação , Uvaria/química , Amidas/química , Ácidos Aristolóquicos/química , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Caules de Planta/química , Plantas Medicinais/química , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Glycoprotein Si was the major protein to determine infection and immunogenicity of Infectious bronchitis virus (IBV). The S1 glycoprotein gene of IBV isolates were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) and proved to be S1 gene by sequencing. The E. coli-mycobacterium expression shuttle plasmid pR-alpha-S1 was constructed by inserting the S1 gene to the pRR3 with human mycobacterium tuberculosis HSP70 promoter and a signal peptide. Then the plasmid pR-alpha-S1 was introduced into mycobacterium bovis BCG by electroporation to construct a recombinate strain rBCG-Sl. The S1 protein could be highly expressed in M. smegmatis mc2 155 when induced by heating and was detected by ELISA and Western blot assays using monoclonal antibody against S1 glycoprotein of IBV. 6 week-old SPF chicken were subcutaneously immunized with 10(6) cfu rBCG-S1 and each chick was immunized three times at 3 week intervals with the same antigen used for the primary immunization. The protective immunity of rBCG-S1 was identified in vaccinated chickens. Results from the protection test showed the two immunizations with rBCG-S1 could provide protection for chickens from the challenge with virulent nephropathogenic IBV strain X. Haemagglutination inhibition titers were also increased in chickens immunized with the expressed rBCG-S1, and significantly higher titers were detected after challenge. These data indicate that the rBCG-S1 could be used as candidate of a live vector vaccine for NIBV.
Assuntos
Vírus da Bronquite Infecciosa/imunologia , Glicoproteínas de Membrana/genética , Mycobacterium bovis/genética , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/genética , Vacinas Virais/imunologia , Animais , Galinhas , Testes de Inibição da Hemaglutinação , Imunização , Glicoproteínas de Membrana/imunologia , Reação em Cadeia da Polimerase , Glicoproteína da Espícula de Coronavírus , Proteínas do Envelope Viral/imunologiaRESUMO
To establish a new high-throughput screening model for the agonist of PPARdelta, PPARdelta gene was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR), and subcloned to pGEM-T Vector for sequencing, then the PPARdelta fragment was excised by restriction enzymes, and inserted into pTARGET Vector to construct expression vector pTARGET-ppARdelta. Insert three copies of PPRE into pGl3-promoter vector to construct expression vector pGl3-PPRE x 3-luc. The vector pTARGET-ppARdelta was transiently cotransfected with pGl3-PPRE x 3-luc into different cell lines to assay the expression levels of luciferase. The PPARdelta agonist screening model was established and optimized. Bezafibrate and linoleic acid can induce the expression of luciferase significantly and in a dose-dependent manner. This method can be used for high throughput screening for the agonist of PPARdelta, which might become lead compounds for new anti-atheroscleriosis or anti-adiposity drugs.
Assuntos
Luciferases/genética , PPAR delta/agonistas , PPAR delta/genética , Proteínas Recombinantes de Fusão/genética , Células 3T3 , Animais , Bezafibrato/farmacologia , Linhagem Celular , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Vetores Genéticos/química , Vetores Genéticos/genética , Células HeLa , Humanos , Ácido Linoleico/farmacologia , Lipídeos/química , Luciferases/metabolismo , Camundongos , Proteínas Recombinantes de Fusão/metabolismo , Transfecção/métodosRESUMO
OBJECTIVE: To develop a high-throughput screening assay for Farnesoid X receptor (FXR) agonists based on mammalian one-hybrid system (a chimera receptor gene system) for the purpose of identifying new lead compounds for dyslipidaemia drug from the chemical library. METHODS: cDNA encoding the human FXR ligand binding domain (LBD) was amplified by RT-PCR from a human liver total mRNA and fused to the DNA binding domain (DBD) of yeast GAL4 of pBIND to construct a GAL4-FXR (LBD) chimera expression plasmid. Five copies of the GAL4 DNA binding site were synthesized and inserted into upstream of the SV40 promoter of pGL3-promoter vector to construct a reporter plasmid pG5-SV40 Luc. The assay was developed by transient co-transfection with pG5-SV40 Luc reporter plasmid and pBIND-FXR-LBD (189-472) chimera expression plasmid. RESULTS: After optimization, CDCA, a FXR natural agonist, could induce expression of the luciferase gene in a dose-dependent manner, and had a signal/noise ratio of 10 and Z' factor value of 0.65. CONCLUSION: A stable and sensitive cell-based high-throughput screening model can be used in high-throughput screening for FXR agonists from the synthetic and natural compound library.