RESUMEN
The allantoicase (allC) gene of Dictyostelium discoideum allC RNAi mutant strain was silenced using the RNA interference technique. The mutant strain is motile, aggregated, and could not undergo further morphological development. The growth rate is high and the cells show a shortened cell cycle comparing with wild-type D. discoideum. However, the mechanisms regarding these actions remain unclear. mRNA differential display was used in this study to identify genetic differences. A novel D. discoideum gene (GenBank accession number: KC759140) encoding a new zinc protease was cloned. The amino acid sequence of the novel gene exhibited a conserved zinc-binding domain (HEX2HX18E) that allowed its classification into the M1 family of metallopeptidases. The gene encoded a 345-amino acid protein with a theoretical molecular mass of 39.69 kDa and a theoretical pI of 6.05. This protein showed strong homology with leukotriene A4 (LTA4) hydrolase of Homo sapiens (41% identity and 60% similarity at the amino acid level). By analyzing quantitative reverse transcription-polymerase chain reaction data, this zinc protease gene was more highly expressed in D. discoideum allC RNAi mutant type than in wild-type KAx-3 cells during the trophophase. The novel zinc protease gene may function as an LTA4 hydrolase and contribute to the shortening of the allC RNAi mutant cell cycle.
Asunto(s)
Dictyostelium/genética , Epóxido Hidrolasas/química , Péptido Hidrolasas/química , Péptido Hidrolasas/genética , Conformación Proteica , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Dictyostelium/clasificación , Dictyostelium/metabolismo , Epóxido Hidrolasas/genética , Humanos , Datos de Secuencia Molecular , Péptido Hidrolasas/metabolismo , Filogenia , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Ureohidrolasas/química , Ureohidrolasas/genética , Ureohidrolasas/metabolismo , Zinc/metabolismoRESUMEN
Dictyostelium discoideum allC RNAi mutant cells are motile and aggregate together, but do not undergo further morphological development. The relatively quick growth rate of allC RNAi mutants compared to wild-type D. discoideum results in a shortened mutant cell cycle. However, at present, little is known about the mechanism underlying this phenomenon. Here, we used semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative RT-PCR, two-dimensional gel electrophoresis, and mass spectrometry/mass spectrometry to elucidate the phenomenon. We found significant downregulation of myosin II heavy chain, D. discoideum calcium-dependent cell adhesion molecule-1 (DdCAD-1) mRNA, DdCAD-1 protein, D. discoideum mRNA for 14-3-3 and 14-3-3 protein, and type A von Willebrand factor domain-containing protein mRNA in allC RNAi mutants. The results suggest that downregulation of the myosin II heavy chain could be one of key factors causing the developmental interruption and that downregulation of the 14-3-3 protein and the type A von Willebrand factor domain-containing protein mRNA plays an important role in shortening the cell cycle of allC RNAi mutants.
Asunto(s)
Proteínas 14-3-3/genética , Moléculas de Adhesión Celular/biosíntesis , Agregación Celular/genética , Puntos de Control del Ciclo Celular/genética , Proteínas 14-3-3/biosíntesis , Proteínas de Unión al Calcio/biosíntesis , Proteínas de Unión al Calcio/genética , Dictyostelium , Regulación de la Expresión Génica/genética , Mutación , Miosina Tipo II/biosíntesis , Estructura Terciaria de Proteína , Interferencia de ARN , ARN Mensajero/biosíntesis , Factor de von Willebrand/biosíntesisRESUMEN
The signaling molecules NH(3) (unprotonated volatile ammonia), as well as cyclic adenosine monophosphate and differentiation-inducing factor, play important roles in the multicellular development of the slime mould Dictyostelium discoideum. One of the downstream metabolic products catalyzed by allantoicase (allC) is ammonia. We observed the role of allC by RNAi-mediated manipulation of its expression. The allC gene of D. discoideum was silenced by RNAi. We found significant downregulation of allC mRNA and protein expression levels. Recombinant allC RNAi mutant cell lines had a shortened cell cycle, a reduction in cell size relative to wild-type cells and interrupted development. We conclude that the normal functions of allC include retarding cell division until a specific cell size is reached and coordinating the progression of development.
Asunto(s)
Ciclo Celular , Dictyostelium/citología , Dictyostelium/crecimiento & desarrollo , Interferencia de ARN , Ureohidrolasas/genética , Línea Celular , Tamaño de la Célula , Dictyostelium/enzimología , Dictyostelium/genética , Regulación hacia Abajo/genética , Citometría de Flujo , Regulación Enzimológica de la Expresión Génica , Mutación/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ureohidrolasas/metabolismoRESUMEN
Porcine deltacoronavirus (PDCoV) is a recently identified coronavirus in the genus Deltacoronavirus that can cause enteric disease with clinical signs including diarrhoea, vomiting, dehydration and mortality in neonatal piglets. Although evidence of the prevalence of PDCoV in China is accumulating, little published information about Chinese PDCoV isolates is available. In this study, we investigated the presence of PDCoV in 49 faecal/intestinal samples from piglets with diarrhoea on different farms in Hebei province. Five samples (10.2%) were positive for PDCoV, but no coinfection of PDCoV with other enteropathogens was observed. A PDCoV strain named HB-BD was successfully isolated from the intestinal contents of a diarrhoeic piglet and serially propagated in swine testicular (ST) cells for >40 passages. The complete genome of the HB-BD strain was sequenced and analysed. Genomic analysis showed that the HB-BD strain had a closer relationship with Chinese strains than those from other countries and was grouped within the Chinese PDCoV cluster. The results of this study will be valuable for further research of PDCoV genetic evolution and development of effective diagnostic reagents, assays and potential vaccines against newly emerged PDCoV strains.
Asunto(s)
Infecciones por Coronavirus/veterinaria , Coronavirus/genética , Coronavirus/aislamiento & purificación , Diarrea/veterinaria , Enfermedades de los Porcinos/virología , Animales , China/epidemiología , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/virología , Diarrea/epidemiología , Diarrea/virología , Evolución Molecular , Heces/virología , Genes Virales/genética , Genómica , Intestinos/virología , Filogenia , Porcinos , Enfermedades de los Porcinos/epidemiologíaRESUMEN
The opening angles of 30 canine autogenous vein grafts were measured to determine the postsurgical change of residual strain in the vein graft. Canine femoral veins were grafted to femoral arteries in the end-to-end anastomosis fashion. When harvested, the vein grafts were cut into short segments and the segments were cut open radially. The opened-up configurations were taken as the zero-stress states of the vessels. Opening angle, defined as the angle between the two lines from the middle point to the tips of the inner wall, was used to describe the zero-stress states. Results show that the opening angles (mean +/- SD) are 63.0 +/- 30.6 deg for normal femoral veins, and -0.4 +/- 4.6, 6.1 +/- 19.4, 25.4 +/- 20.1, and 47.8 +/- 11.4 deg for vein grafts at 1 day, 1 week, 4 and 12 weeks postsurgery, respectively. The postsurgical changes in opening angle reveal nonuniform transmural tissue remodeling in the vascular wall. The relations between the changes in opening angle and the changes in the morphology of the vein grafts are discussed. Intimal hyperplasia is correlated to the opening angle and is suggested to be the main factor for the postsurgical increase in opening angle. The longitudinal strain in the vein graft is found to decrease postsurgically.