RESUMEN
Here, a novel cDNA encoding a serine/arginine (SR)-rich protein, designated PSR, was isolated from the true slime mold Physarum polycephalum and expressed in Escherichia coli. The deduced amino acid (aa) sequence reveals that PSR contains RS repeats at its C-terminus, similar to the conventional PSRPK substrate ASF/SF2. To study the novel protein, we generated a variety of mutant constructs by PCR and site-directed mutagenesis. Our analysis indicated that the purified recombinant PSR was phosphorylated by PSRPK in vitro and the SR-rich domain (amino acids 460-469) in the PSR protein was required for phosphorylation. In addition, removal of the docking motif (amino acids 424-450) from PSR significantly reduced the overall catalytic efficiency of the phosphorylation reaction. We also found that the conserved ATP-binding region (62)LGWGHFSTVWLAIDEKNGGREVALK(86) and the serine/threonine protein kinases active-site signature (184)IIHTDLKPENVLL(196) of PSRPK played a crucial role in substrate phosphorylation and Lys(86) and Asp(188) were crucial for PSRPK phosphorylation of PSR. These results suggest that PSR is a novel SR-related protein that is phosphorylated by PSRPK.
Asunto(s)
Mixomicetos/enzimología , Proteínas Quinasas/metabolismo , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Fosforilación , Proteínas Quinasas/química , Proteínas Quinasas/genética , Homología de Secuencia de AminoácidoRESUMEN
To study application of multi-ribozyme expression system on expression inhibition of multidrug resistance-associated protein (MRP1) in HEK293 cells, the multi-ribozyme expression system containing 20 cis-acting ribozymes for self-cleavage and 10 trans-acting ribozymes for targeting to MRP1 gene specific site were constructed. HEK293 cells cotransfected multi-ribozyme expression system with MRP1 target gene or full length of MRP1 gene respectively were analyzed by RT-PCR, Western blot analysis and MTT assay. The results showed that multi-ribozyme systems were able to dramatically decrease fluorescent fusion protein expression in HEK293 cells. RT-PCR analysis indicated that the extent of MRP1 target mRNA decrease was correlated with the number of trans-acting ribozyme contained in multi-ribozyme expression system. Similar changes have been observed from Western blot. MTT assay showed that multi-ribozyme systems were able to reverse MDR generated by MRP1 gene in HEK cells. These results suggested that inhibitory effects of multiple copies of ribozymes contained in the system were better than that of single ribozyme contained. Therefore, this strategy could be used in treatment of tumor or other diseases via gene therapy.
Asunto(s)
Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , ARN Catalítico/metabolismo , ARN Catalítico/fisiología , Western Blotting , Línea Celular , Regulación de la Expresión Génica , Humanos , ARN Mensajero , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Serine/arginine protein kinases are specific kinase family for phosphorylating SR protein regulating alternative splicing of SR protein and its distribution, localization in the nucleus. However, it is unclear how Physarum Polycephalum Serine/Arginine Protein Kinase(PSRPK) functions in the cells. In order to study its function, Oligonucleotides for transcribing siRNAs were designed and inserted into pSIREN-RetroQ vector to construct pSIREN-PSRPK-1, pSIREN-PSRPK-2, pSIREN-PSRPK-3, pSIREN-PSRPK-4, pSIREN-PSRPK-5 for expressing siRNAs targeting at PSRPK, as well as the negative control pSIREN-PSRPK-Neg. The PSRPK cDNA amplified by PCR was inserted into the pDsRed-N1 vector to construct a pPSRPK-DsRed plasmid. After the pPSRPK-DsRed was co-transfected into HEK293 cell with recombinant siRNA expression plasmids respectively, the PSRPK-DsRed fusion fluorescent protein was observed under fluorescent microscope after 72 hours co-transfection. The results indicated that pSIREN-PSRPK-2 and pSIREN-PSRPK-5 were able to inhibit the expression of PSRPK-DsRed fusion fluorescent protein efficiently. RT-PCR and Northern dot blot analysis further demonstrated that pSIREN-PSRPK-2 and pSIREN-PSRPK-5 can effectively inhibit PSRPK expression, which accorded with the results under the fluorescent microscope.
Asunto(s)
Silenciador del Gen , Physarum polycephalum/enzimología , Proteínas Quinasas/genética , Proteínas Protozoarias/genética , ARN Interferente Pequeño/genética , Animales , Línea Celular , Expresión Génica , Vectores Genéticos/genética , Humanos , Physarum polycephalum/genética , Proteínas Quinasas/metabolismo , Proteínas Protozoarias/metabolismoRESUMEN
Crude fraction of Arg/Ser-rich proteins (SR proteins) were isolated from plasmodia of Physarum polycephalum and immunoassayed by western blot with a monoclonal antibody against SC35 protein from HeLa cell. Two polypeptides were detected by the antibody, suggesting that they were SCL(SC35-like) proteins. The SCL proteins have their mass weight of 32.5 kD and 82.5 kD, respectively, and so were termed PSCL32.5 and PSCL82.5 in this paper. The PI of PSCI32.5 was ascertained as 6.19 by IEF after a further purification of the protein with SDS-PAGE. The densitometric scanning of the western blot bands of PSCI32.5 isolated at different phases of cell cycle of P. polycephalum demonstrated that the relative content of the protein varied through the cell cycle: it appeared as the lowest at early S phase, showed increases from S phase to G2 phase, and peaked at late G2 phase.
Asunto(s)
Physarum polycephalum/química , Proteínas Protozoarias/análisis , Animales , Western Blotting , Ciclo Celular , Punto Isoeléctrico , Peso MolecularRESUMEN
OBJECTIVE: To isolate and identify SARS-coronavirus in nasal and throat swabs collected from clinically diagnosed severe acute respiratory syndrome (SARS) patients. METHODS: Nasal and throat swab specimens were inoculated onto well of 24-well plate containing confluent monolayers of Vero and MRC-5 cells. Isolates were identified with serology, electron microscopy and genome sequence. RESULTS: One hundred and fifty-eight nasal and throat swabs specimens from 79 SARS patients in Peking Union Medical College Hospital between April and May, 2003 were cultured for SARS-coronavirus. Cytopathic effect (CPE) was found in three nasal swab specimens inoculated in Vero cells. Acute and convalescent phase serum specimens collected from SARS patients were found with seroconversions and/or a fourfold or greater rises in indirect fluorescence antibodies (IgG and IgM) titers when the 3 isolates (infected Vero cells) were used as antigen. Coronavirus was observed in the culture supernatant by negative-stain electron microscopy. Genome sequence confirmed the isolates were SARS-coronavirus. CONCLUSIONS: The 3 isolates from nasal and throat swabs samples collected from 79 clinically diagnosed SARS patients were SARS coronavirus.
Asunto(s)
Laringe/virología , Nasofaringe/virología , Síndrome Respiratorio Agudo Grave/virología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/aislamiento & purificación , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Antivirales/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Síndrome Respiratorio Agudo Grave/inmunología , Manejo de EspecímenesRESUMEN
AIM: To prepare mAb against bovine bFGF and identify their Ig subgroups so as to establish an ELISA for detection of bFGF level. METHODS: BALB/c mice were immunized by recombinant bovine bFGF. Hybridoma cell lines which could stably secret the monoclonal antibodies to bFGF were established by cell fusion technique, and their related characteristics were identified. In addition, polyclonal antibodies to bFGF were prepared by immunization of rabbits with bovine bFGF. The mAb and polyclonal antibodies purified through protein A affinity chromatography were used to develop a sandwich ELISA for detection of bFGF level. RESULTS: Three hybridoma cell lines which could secret the mAbs IgG 1 to bFGF were obtained. The concentration of bFGF could be detected by sandwich ELISA developed with purified mAb and polyclonal antibody at nanogram level. CONCLUSION: mAb and polyclonal antibodies against to bovine bFGF have been prepared successfully, which provide powerful tool for further clinical application and related studies.