RESUMO
The early localization of mRNA transcripts is critical in sorting cell fate determinants in the developing embryo. In the glossiphoniid leech, Helobdella robusta, maternal mRNAs, such as Hro-twist, localize to the zygotic teloplasm. Ten seven nucleotide repeat elements (AAUAAUA) called ARE2 and a predicted secondary structural motif, called teloplasm localization motif (TLM), are present in the 3'UTR of Hro-twist mRNA. We used site-directed mutagenesis, deletions, and microinjection of labeled, exogenous transcripts to determine if ARE2 elements, and the TLM, play a role in Hro-twist mRNA localization. Deleting the poly-A tail and the cytoplasmic polyadenylation element (CPE) had no effect on Hro-twist mRNA localization. Site-directed mutagenesis of nucleotides that altered ARE2 element sequences or the TLM suggest that the ARE2 elements and the TLM are important for Hro-twist mRNA localization to the teloplasm of pre-cleavage zygotes. Hro-Twist protein expression data suggest that the localization of Hro-twist transcripts in zygotes and stage two embryos is not involved in ensuring mesoderm specification, as Hro-Twist protein is expressed uniformly in most cells before gastrulation. Our data may support a shared molecular mechanism for leech transcripts that localize to the teloplasm.
Assuntos
Regiões 3' não Traduzidas , Regulação da Expressão Gênica no Desenvolvimento , Sanguessugas/genética , Transporte de RNA , RNA Mensageiro/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Zigoto/metabolismo , Animais , Padronização Corporal , Clonagem Molecular , Biologia Computacional , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Desenvolvimento Embrionário , Sanguessugas/embriologia , Mesoderma/citologia , Mesoderma/embriologia , Mutagênese Sítio-Dirigida , Motivos de Nucleotídeos , Mutação Puntual , Estrutura Secundária de Proteína , Dobramento de RNA , RNA Mensageiro/genética , Sequências Repetitivas de Ácido Nucleico , Proteína 1 Relacionada a Twist/genética , Zigoto/citologiaRESUMO
Disintegrins and disintegrin-like peptides interact with integrins and interfere with cell-cell and cell-matrix interactions. A disintegrin-like snake venom gene, Acocostatin was cloned from the venom gland mRNA of Agkistrodon contortrix contortrix. Acocostatin belongs to the PIII-SVMP subfamily of disintegrin-like peptides. The recombinant acocostatin peptide was produced and purified as GST-fusion. The GST-acocostatin peptide, at 44 µg/mL, inhibited platelet aggregation by 30% in PRP and 18% in whole blood. In addition GST-acocostatin, at 220 µg/mL, inhibited SK-Mel-28 cell migration by 48%, but did not inhibit T24 cell migration. The GST-acocostatin peptide ability to induce apoptosis on HUVEC, HeLa, and SK-Mel-28 cells was determined using Annexin V-FITC and chromatin fragmentation assays after 24 h of treatment. At 5 µM GST-acocostatin peptide, 19.68%+/- 3.09 of treated HUVEC, and 35.86% +/- 2.05 of treated HeLa cells were in early apoptosis. The GST-acocostatin peptide also caused chromatin fragmentation of HUVEC and HeLa cells as determined by fluorescent microscopy and Hoechst staining. The GST-acocostatin peptide failed to induce apoptosis of SK-Mel-28 cells. We characterized the HUVEC, HeLa, and T24 integrin expression by flow cytometry, as the first step in determining GST-acocostatin binding specificity. Our results indicate that HUVEC express αv, αvß3, αvß5, α6, ß1, and ß3 integrin receptors. HeLa cells express α1, α2, α6, αv, αvß5, and ß1 integrin receptors. T24 cells express α1, α3, α6, αv, αvß3, αvß5, ß1, ß3, and ß6 integrin receptors.
Assuntos
Agkistrodon , Desintegrinas/genética , Integrinas/metabolismo , Peptídeos/genética , Proteínas Recombinantes/genética , Venenos de Serpentes/genética , Animais , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Cromatina/metabolismo , Citometria de Fluxo , Fluoresceína-5-Isotiocianato , Glutationa Transferase/metabolismo , Células HeLa , Humanos , Microscopia de Fluorescência , Peptídeos/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Veias Umbilicais/citologiaRESUMO
In this study, three recombinant mojastin peptides (Moj-WN, Moj-NN, and Moj-DM) were produced and compared functionally. Recombinant Moj peptides were purified as GST-fusions. GST-Moj-WN and GST-Moj-NN inhibited ADP-induced platelet aggregation in platelet rich plasma. The GST-Moj-WN had an IC(50) of 160nM, while the GST-Moj-NN had an IC(50) of 493nM. The GST-Moj-DM did not inhibit platelet aggregation. All three GST-Moj peptides inhibited SK-Mel-28 cell adhesion to fibronectin. The GST-Moj-WN inhibited the binding of SK-Mel-28 cells to fibronectin with an IC(50) of 11nM, followed by the GST-Moj-NN (IC(50) of 28nM), and the GST-Moj-DM (IC(50) of 46nM). The GST-Moj peptides' ability to induce apoptosis on SK-Mel-28 cells was determined using Annexin-V-FITC and nuclear fragmentation assays. Cells were incubated with 5muM GST-Moj peptides for 24h. At 5microM GST-Moj-DM peptide, 13.56%+/-2.08 of treated SK-Mel-28 cells were in early apoptosis. The GST-Moj-DM peptide also caused nuclear fragmentation as determined by fluorescent microscopy and Hoechst staining. The GST-Moj-WN and GST-Moj-NN peptides failed to induce apoptosis. We characterized the SK-Mel-28 integrin expression, as the first step in determining r-Moj binding specificity. Our results indicate that SK-Mel-28 cells express alphavbeta3, alphav, alpha6, beta1, and beta3 integrin receptors.