RESUMEN
Using differential hybridization of a guinea pig endometrial cell cDNA library, a potentially negatively estrogen-regulated gene, SOX-3, was isolated. According to the nucleotide and protein sequence similarities, SOx-3 belonged to the FAD-linked sulfhydryl oxidase family containing the egg white sulfhydryl oxidase, the rat seminal vesicle sulfhydryl oxidase-2 SOx-2, the quiescence-inducible protein hQ6. The SOX-3 transcript in the guinea pig as well as 5 different mRNAs in human tissues appeared differentially expressed in the tissues studied. In secondary endometrial cell culture, the SOX-3 mRNA level increased during a serum depletion-induced quiescence, decreased when cells enter the G1 phase after serum stimulation, and was restored during the S and G2/M phases. Thus, SOX-3 could be implicated in the negative cell cycle control. The SOx-3 protein appeared to be specific of epithelial cells in the uterus. Its expression level varied during the estrus cycle in the guinea pig, suggesting a regulation by steroid hormones.
Asunto(s)
Estro/metabolismo , Oxidorreductasas/aislamiento & purificación , Útero/enzimología , Secuencia de Aminoácidos , Animales , Ciclo Celular/fisiología , ADN Complementario/análisis , Proteínas de Unión al ADN/química , Endometrio/metabolismo , Estrógenos/farmacología , Femenino , Regulación Enzimológica de la Expresión Génica , Biblioteca de Genes , Cobayas , Proteínas HMGB , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/química , Especificidad de Órganos , Oxidorreductasas/biosíntesis , Oxidorreductasas/genética , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Factores de Transcripción SOXB1 , Homología de Secuencia de Aminoácido , Factores de Transcripción , Útero/citologíaRESUMEN
We have isolated, in guinea-pig endometrial cells, an estrogen-induced 1.8 kb RNA called gec1. Screening of a guinea-pig genomic library led to identification of gec1 gene consisting of 4 exons and 3 introns. Exon 1 contains the 5'UTR and the ATG initiation codon. A guinea-pig gec1 cDNA was obtained by 5'-RACE. The 351 bp coding sequence shares 76.8% identity with that of the human GABARAP 924 bp cDNA while UTRs of the two cDNAs differ. A gec1 probe from the 3'UTR revealed a 1.9 kb mRNA in human tissues and a human GEC1 cDNA was isolated from placenta. Its coding sequence shares 93 and 79% identity with that of guinea-pig gec1 and human GABARAP, respectively. The human and guinea-pig GEC1 proteins have 100% identity. GEC1 and GABARAP proteins have 87% identity and N terminus featuring a tubulin binding motif. Thus, estrogen-regulated gec1 is a new gene which could encode a microtubule-associated protein.
Asunto(s)
Estrógenos , Regulación de la Expresión Génica/fisiología , Proteínas Asociadas a Microtúbulos/genética , Transcripción Genética , Regiones no Traducidas 3' , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas Reguladoras de la Apoptosis , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Estrógenos/farmacología , Exones , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Biblioteca Genómica , Cobayas , Humanos , Intrones , Proteínas Asociadas a Microtúbulos/biosíntesis , Datos de Secuencia Molecular , Especificidad de Órganos , Placenta , ARN Mensajero/biosíntesis , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Transcripción Genética/efectos de los fármacosRESUMEN
Pokeweed antiviral protein (PAP), a ribosome-inactivating protein isolated from the leaves of Phytolacca americana, reveals potent antiviral activity against viruses or cytotoxic action against cells once inside the cytoplasm. Therefore PAP is a good candidate to be used as an immunotoxin. We constructed a bacterial expression plasmid encoding PAP as a fusion protein with gonadotropin-releasing hormone (GnRH), a neuropeptide with receptor sites on several gynaecologic tumors. The resulting recombinant toxin was produced in Escherichia coli and accumulated in inclusion bodies. After purification under denaturing conditions, renaturated GnRH-PAP shows an IC(50) of 3 nM on in vitro translation assays and selectively inhibits the growth of the GnRH receptor positive Ishikawa cell line (ID(50) of 15 nM); on the other hand, neither GnRH nor PAP alone had any effect.
Asunto(s)
Antineoplásicos/farmacología , Hormona Liberadora de Gonadotropina/farmacología , Inmunotoxinas/farmacología , N-Glicosil Hidrolasas , Proteínas de Plantas/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Expresión Génica , Hormona Liberadora de Gonadotropina/genética , Humanos , Inmunotoxinas/genética , Inmunotoxinas/aislamiento & purificación , Proteínas de Plantas/genética , Receptores LHRH/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/farmacología , Proteínas Inactivadoras de Ribosomas Tipo 1 , Células Tumorales CultivadasRESUMEN
Pokeweed antiviral protein (PAP) inactivates both eukaryotic and prokaryotic ribosomes via a specific depurination of rRNA. The sensitivity of pokeweed ribosomes to PAP implies the existence of a mechanism to protect the plant. Using monoclonal antibodies specific to PAP, a protein complex (PAPi) which contained PAP was identified in leaf extract. In this complex, the enzymatic activity of the toxin was strongly inhibited. This protein complex had a pI lower than that of PAP and was separated from free PAP by a preparative native gel electrophoresis. PAPi had an apparent molecular mass of 57 kDa and was dissociated by heating for 5 min at 80 degrees C or by treatment by alkaline or acidic pH or by 7 M urea. The other components involved in the complex remain unknown.
Asunto(s)
Antivirales/análisis , N-Glicosil Hidrolasas , Proteínas de Plantas/análisis , Animales , Antivirales/farmacología , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Ratones , Peso Molecular , Proteínas de Plantas/farmacología , Plantas/química , Desnaturalización Proteica , Proteínas Inactivadoras de Ribosomas Tipo 1RESUMEN
Monoclonal antibodies specific to pokeweed antiviral protein (PAP), a ribosome-inactivating protein (RIP), were obtained after six unsuccessful fusions. A special procedure including injections of low doses of purified PAP from spring leaves in a short period was adopted. Some clones highly specific to PAP react with recombinant PAP. One clone cross-reacts with PAP-S isolated from seeds but none cross-reacts with the isoform PAP II isolated from summer leaves. These antibodies represent a useful tool to investigate the mechanisms of PAP biosynthesis and plant protection involving RIPs.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/química , N-Glicosil Hidrolasas , Proteínas de Plantas/inmunología , Inhibidores de la Síntesis de la Proteína/inmunología , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Especificidad de Anticuerpos , Hibridomas/química , Hibridomas/metabolismo , Inmunización , Ratones , Ratones Endogámicos BALB C , Proteínas de Plantas/aislamiento & purificación , Inhibidores de la Síntesis de la Proteína/aislamiento & purificación , Proteínas Inactivadoras de Ribosomas Tipo 1RESUMEN
A simple method was developed to isolate dsRNA segments from disulphide crosslinked polyacrylamide gels. The dsRNA preparations from the P4 killer virus strain 77 of Ustilago maydis containing 7 genomic segments with molecular sizes ranging from 0.36 to 6.7 kbp, the 20 kbp dsRNA associated with the '447' cytoplasmic male sterility in Vicia faba and the 23 kbp genomic dsRNA of citrus tristeza virus (CTV) were separated on disulphide crosslinked polyacrylamide gels. After UV visualisation, the dsRNA bands were excised from the gel and dissolved using 2-mercaptoethanol. The dsRNA were then purified from the solubilized fractions by specific adsorption on microgranular cellulose and elution with a small volume of water. The method is rapid, simple and convenient for the isolation of all the tested dsRNAs segments.