RESUMEN
Most rabies vaccines are based on inactivated virus, which production process demands a high level of biosafety structures. In the past decades, recombinant rabies virus glycoprotein (RVGP) produced in several expression systems has been extensively studied to be used as an alternative vaccine. The immunogenic characteristics of this protein depend on its correct conformation, which is present only after the correct post-translational modifications, typically performed by animal cells. The main challenge of using this protein as a vaccine candidate is to keep its trimeric conformation after the purification process. We describe here a new immunoaffinity chromatography method using a monoclonal antibody for RVGP Site II for purification of recombinant rabies virus glycoprotein expressed on the membrane of Drosophila melanogaster S2 cells. RVGP recovery achieved at least 93%, and characterization analysis showed that the main antigenic proprieties were preserved after purification.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Glicoproteínas/aislamiento & purificación , Virus de la Rabia/aislamiento & purificación , Proteínas Virales/aislamiento & purificación , Animales , Línea Celular , Drosophila melanogaster/citología , Glicoproteínas/biosíntesis , Glicoproteínas/genética , Humanos , Virus de la Rabia/química , Virus de la Rabia/patogenicidad , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Virales/biosíntesis , Proteínas Virales/genéticaRESUMEN
Excessive adipose tissue promotes the manifestation of endocrine disorders such as reduction of the secretion of zinc-α2-glycoprotein (ZAG), an adipokine with anti-inflammatory and lipid-mobilizing activity. The molecular structure of this adipokine includes binding sites for zinc, a trace element with important antioxidant and immunological proprieties that also participates in energy metabolism and stimulates the function of ZAG. The objective of this review is to highlight current data on the metabolism of ZAG in obesity and the role of zinc in this process. The identified studies show that subjects with obesity have low serum concentrations of zinc and ZAG, as well as low expression of the genes encoding this protein. Thus, zinc appears to be an important regulator of the homeostasis of ZAG in the body; however, alterations in the metabolism of zinc in obesity appear to compromise the functions of ZAG. Therefore, further studies are needed to clarify the relationship between zinc and ZAG metabolism and its repercussions in obesity.
Asunto(s)
Adipoquinas/biosíntesis , Proteínas Portadoras/biosíntesis , Metabolismo Energético , Glicoproteínas/biosíntesis , Metabolismo de los Lípidos , Obesidad/metabolismo , Zinc/metabolismo , Regulación de la Expresión Génica , Humanos , Obesidad/patologíaRESUMEN
The transmembrane rabies virus glycoprotein (RVGP) is the main antigen of vaccine formulations used around the world to prevent rabies, the most lethal preventable infectious disease known. The objective of this work was to evaluate the potential of a bioreactor using wave-induced agitation in the initial steps of scaling up the rRVGP production process by a Drosophila melanogaster S2 cell line to produce rRVGP in sufficient quantities for immunization and characterization studies. Taking advantage of some remarkable features recognized in Drosophila S2 cells for scaling the culture process, a robust recombinant lineage (S2MtRVGPH-His) engineered by our group for the expression of rRVGP using a copper-inducible promoter was used in the bioreactor cultures. The WAVE Bioreactor was chosen because it represents an innovative approach to the cultivation of animal cells using single-use technology. For that purpose, we firstly established a procedure for culturing the S2MtRVGPH-His lineage in 100 mL Schott flasks. Using an inoculum of 5 × 105 cells/mL in culture medium (Sf900-III) induced with solution of CuSO4 (0.7 mM) and a convenient pH range (6.2-7.0), optimal parameter values such as time of induction (72 h) and temperature (28 °C) to increase rRVGP production could be defined. This procedure was reproduced in culture experiments conducted in a WAVE Bioreactor™ 2/10 using a 2 L Cellbag. The results in Schott flasks and in WAVE Bioreactor™ were very similar, yielding a maximum titer of rRVGP above of 1 mg.L-1. The immunization study showed that the rRVGP produced in the bioreactor was of high immunogenic quality.
Asunto(s)
Reactores Biológicos , Glicoproteínas/biosíntesis , Microbiología Industrial/métodos , Proteínas Recombinantes/biosíntesis , Proteínas Virales/biosíntesis , Animales , Técnicas de Cultivo de Célula , Línea Celular , Drosophila melanogaster/citología , Virus de la RabiaRESUMEN
A strain of embryonic human kidney cells (HEK293) was transiently co-transfected with the expression vectors coding for the α- and ß-subunits of human thyroid-stimulating hormone (hTSH), and, for the first time, a human cell-derived recombinant hTSH was synthesized and extensively characterized. The purification strategy involving two steps provided an overall yield of 55% and a purity level > 90%. The purified material (hTSH-HEK) was analyzed and compared to a CHO-derived recombinant preparation (hTSH-CHO) and to a pituitary-derived (hTSH-Pit) preparation. The three preparations showed an equivalent purity (> 95%) with a hTSH-HEK molecular mass 2.1% lower than that of hTSH-CHO and 2.7% higher than that of hTSH-Pit. Remarkable differences were found in the carbohydrate moiety, the lowest sialic acid content and highest fucose content being observed in hTSH-HEK. In vivo biological activity was confirmed for the three preparations, the hTSH-HEK bioactivity being 39 and 16% lower than those of hTSH-CHO and hTSH-Pit, respectively. The hTSH-HEK circulatory half-life (t 1/2) was also shorter than those of hTSH-CHO (1.5-fold) and hTSH-Pit (1.2-fold). According to these findings, HEK-293-derived hTSH can be considered to be useful for clinical applications, in view as well of its human origin and particular carbohydrate composition.
Asunto(s)
Carbohidratos/análisis , Células Epiteliales/metabolismo , Glicoproteínas/biosíntesis , Tirotropina/biosíntesis , Animales , Células CHO , Cricetinae , Cricetulus , Fucosa/análisis , Glicosilación , Células HEK293 , Semivida , Humanos , Ácido N-Acetilneuramínico/análisis , Proteínas Recombinantes/biosíntesis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , TransfecciónRESUMEN
Glycopeptides are an important class of antibiotics used in the treatment of several infections, including those caused by methicillin resistant Staphylococcus aureus. Glycopeptides are biosynthesized by a Non Ribosomal Peptide Synthase (NRPS) and the resulting peptide precursors are decorated by several tailoring enzymes, such as halogenases and glycosyltransferases. These enzymes are important targets of protein engineering to produce new derivatives of known antibiotics. Herein we show the production of two putative halogenases, denominated StaI and StaK, involved in the biosynthesis of the glycopeptide A47,934 in Streptomyces toyocaensis NRRL 15,009. This antibiotic together with the compound UK-68,597 are the unique glycopeptides which have two putative halogenases identified in their gene clusters and three chloride substituent atoms attached to their aglycones. StaI and StaK were successfully produced in E. coli in the soluble fraction with high purity using the wild type gene for StaI and a synthetic codon optimized gene for StaK. We have purified both enzymes by two chromatographic steps and a good yield was obtained. These putative halogenases were co-purified with the co-factor FAD, which are differently reduced by the enzyme SsuE in vitro. We have further confirmed that these putative halogenases are monomeric using a calibrated gel filtration column and through circular dichroism, we confirmed that both enzymes are folded with a predominance of α-helices. Molecular models for StaI and StaK were generated and together with sequence and phylogenetic analysis, we could infer some structural insights of StaI and StaK from the biosynthesis of compound A47,934.
Asunto(s)
Proteínas Bacterianas , Clonación Molecular , Expresión Génica , Glicoproteínas , Familia de Multigenes , Streptomyces/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Glicoproteínas/biosíntesis , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/aislamiento & purificación , Estructura Secundaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Streptomyces/enzimologíaRESUMEN
Paracoccidioidomycosis is a systemic mycosis, endemic in Latin America. The etiologic agents of this mycosis are composed of 2 species: Paracoccidioides brasiliensis and P. lutzii. Murine animal models are the gold standard for in vivo studies; however, ethical, economical and logistical considerations limit their use. Galleria mellonella is a suitable model for in vivo studies of fungal infections. In this study, we compared the virulence of P. brasiliensis and P. lutzii in G. mellonella model. The deaths of larvae infected with P. brasiliensis or P. lutzii were similar, and both species were able to reduce the number of hemocytes, which were estimated by microscopy and flow cytometer. Additionally, the phagocytosis percentage was similar for both species, but when we analyze hemocyte-Paracoccidioides spp. interaction using flow cytometer, P. lutzii showed higher interactions with hemocytes. The gene expression of gp43 as well as this protein was higher for P. lutzii, and this expression may contribute to a greater adherence to hemocytes. These results helped us evaluate the behavior of Paracoccidioides spp in G. mellonella, which is a convenient model for investigating the host-Paracoccidioides spp. interaction.
Asunto(s)
Paracoccidioides/patogenicidad , Paracoccidioidomicosis/microbiología , Animales , Antígenos Fúngicos/biosíntesis , Antígenos Fúngicos/genética , Western Blotting , Adhesión Celular , Modelos Animales de Enfermedad , Citometría de Flujo , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Glicoproteínas/biosíntesis , Glicoproteínas/genética , Hemocitos/microbiología , Hemocitos/patología , Interacciones Huésped-Patógeno , Mariposas Nocturnas , Paracoccidioides/genética , Paracoccidioides/metabolismo , Paracoccidioidomicosis/patología , Fagocitosis , VirulenciaRESUMEN
All life cycle stages of the protozoan parasite Trypanosoma cruzi are enveloped by mucin-like glycoproteins which, despite major changes in their polypeptide cores, are extensively and similarly O-glycosylated. O-Glycan biosynthesis is initiated by the addition of αGlcNAc to Thr in a reaction catalyzed by Golgi UDP-GlcNAc:polypeptide O-α-N-acetyl-d-glucosaminyltransferases (ppαGlcNAcTs), which are encoded by TcOGNT1 and TcOGNT2. We now directly show that TcOGNT2 is associated with the Golgi apparatus of the epimastigote stage and is markedly downregulated in both differentiated metacyclic trypomastigotes (MCTs) and cell culture-derived trypomastigotes (TCTs). The significance of downregulation was examined by forced continued expression of TcOGNT2, which resulted in a substantial increase of TcOGNT2 protein levels but only modestly increased ppαGlcNAcT activity in extracts and altered cell surface glycosylation in TCTs. Constitutive TcOGNT2 overexpression had no discernible effect on proliferating epimastigotes but negatively affected production of both types of trypomastigotes. MCTs differentiated from epimastigotes at a low frequency, though they were apparently normal based on morphological and biochemical criteria. However, these MCTs exhibited an impaired ability to produce amastigotes and TCTs in cell culture monolayers, most likely due to a reduced infection frequency. Remarkably, inhibition of MCT production did not depend on TcOGNT2 catalytic activity, whereas TCT production was inhibited only by active TcOGNT2. These findings indicate that TcOGNT2 downregulation is important for proper differentiation of MCTs and functioning of TCTs and that TcOGNT2 regulates these functions by using both catalytic and noncatalytic mechanisms.
Asunto(s)
Glicoproteínas/genética , Mucinas/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/genética , Animales , Regulación del Desarrollo de la Expresión Génica , Glicoproteínas/biosíntesis , Aparato de Golgi/enzimología , Estadios del Ciclo de Vida/genética , Mucinas/genética , Péptidos/genética , Péptidos/metabolismo , Polisacáridos/biosíntesis , Proteínas Protozoarias/genética , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/crecimiento & desarrolloRESUMEN
The role of oestrogens in epididymal function is still unclear. Knockout of the oestrogen receptor ESR1 (Esr1(-/-) ) or treatment with the anti-oestrogen Fulvestrant affect epididymal milieu and sperm motility. We investigated the effect of in vivo treatment of rats with Fulvestrant on: (i) expression of genes that may be important for the architecture and function of the epididymal epithelium: prominins 1 and 2, metalloproteinase 7, claudin 7, beta-catenin and cadherin 13, and (ii) levels of oestradiol and testosterone, and expression of oestrogen and androgen receptors, in the initial segment (IS), caput, corpus and cauda epididymis. Fulvestrant (i) reduced gene expression of prominin 1 (variant 1) in the caput, reduced prominin 1 protein content in the caput epididymis and in the efferent ductules, and increased the localization of prominin 1 in microvilli of the caput and corpus; (ii) reduced gene expression of prominin 2 in the corpus and cauda epididymis; (iii) increased the metalloproteinase 7 content in the apical region of principal cells from IS/caput; (iv) reduced in the corpus epididymis, but increased in the efferent ductules, the cadherin 13 mRNA level; (v) reduced testosterone but increased oestradiol levels in the corpus and cauda; (vi) increased the androgen receptor protein content in all regions of the epididymis, and the oestrogen receptor GPER in the corpus and cauda epididymis. In conclusion, treatment with Fulvestrant induced regional-specific changes in hormonal and steroid receptor content, and affected expression of proteins important for epithelial organization and absorption/secretion. The mechanisms of oestrogen action may differ among epididymal regions, which may contribute to determine region-specific sperm functions.
Asunto(s)
Epidídimo/efectos de los fármacos , Estradiol/análogos & derivados , Antagonistas del Receptor de Estrógeno/farmacología , Antígeno AC133 , Animales , Antígenos CD/biosíntesis , Epidídimo/metabolismo , Estradiol/metabolismo , Estradiol/farmacología , Fulvestrant , Glicoproteínas/biosíntesis , Masculino , Metaloproteinasa 7 de la Matriz/biosíntesis , Glicoproteínas de Membrana/biosíntesis , Péptidos , Ratas Wistar , Receptores de Estrógenos/metabolismo , Testosterona/metabolismoRESUMEN
Diacylglycerol-O-acyltransferase (DGAT1) gene encodes the rate-limiting enzyme of triglyceride synthesis. A polymorphism in this gene, DGAT1 K232A, has been associated with milk production and composition in taurine breeds. However, this polymorphism is not a good tool for ascertaining the effects of this QTL in Bos indicus (Zebu), since the frequency of the DGAT1 232A allele is too low in these breeds. We sequenced the 3'-untranslated region of DGAT1 gene in a sample of bulls of the breeds Guzerá (Bos indicus) and Holstein (Bos taurus) and, using in silico analysis, we searched for genetic variation, evolutionary conservation, regulatory elements, and possible substitution effects. Six single nucleotide (SNPs) and one insertion-deletion (INDEL) polymorphisms were found in the Guzerá bulls. Additionally, we developed a preliminary association study, using this INDEL polymorphism as a genetic marker. A significant association was detected (P ≤ 0.05) between the INDEL (DGAT1 3'UTR INDEL) and the breeding values (BV) for protein, fat, and milk yields over a 305-day lactation period. The DGAT1 3' UTR INDEL genotype I/I (I, for insertion) was associated with lower BVs (-38.77 kg for milk, -1.86 kg for fat, and -1.48 kg for protein yields), when compared to the genotype I/D (D, for deletion). I/D genotype was lower D/D genotype (-34.98 kg milk, -1.73 kg fat, and -1.09 kg protein yields). This study reports the first polymorphism of DGAT1 3'UTR in the Guzerá breed, as well as its association with BV for milk protein, fat, and milk yields.
Asunto(s)
Regiones no Traducidas 3' , Diacilglicerol O-Acetiltransferasa/genética , Mutación INDEL , Lactancia/genética , Polimorfismo de Nucleótido Simple , Carácter Cuantitativo Heredable , Animales , Cruzamiento , Bovinos , Simulación por Computador , Diacilglicerol O-Acetiltransferasa/metabolismo , Femenino , Expresión Génica , Genotipo , Glucolípidos/biosíntesis , Glucolípidos/genética , Glicoproteínas/biosíntesis , Glicoproteínas/genética , Gotas Lipídicas , Masculino , Leche/química , Proteínas de la Leche/biosíntesis , Proteínas de la Leche/genética , Modelos GenéticosAsunto(s)
Enzimas/química , Glicoproteínas/química , Animales , Enzimas/biosíntesis , Enzimas/síntesis química , Enzimas/metabolismo , Enzimas/farmacología , Glicoproteínas/biosíntesis , Glicoproteínas/síntesis química , Glicoproteínas/metabolismo , Glicoproteínas/farmacología , Glicosilación , Humanos , Modelos MolecularesRESUMEN
This study analyzed the effect of muscle-fiber type composition on glycogenin-1 (GYG) gene expression and its impact on pH. The longissimus dorsi (LD) muscle contains more type IIB fibers (75.10%) than does the psoas major (PM) muscle (41.58%), while the PM has more type I (3.65 vs 0.94%), type IIA (34.15 vs 10.63%), and type IIX (20.62 vs 13.33%) fibers. Compared with PM, glycolytic potential (GP), pH45 min, and ΔpH from 45 min to 24 h post-mortem were all relatively higher in LD. Glycogen metabolites (lactate and GP) were negatively correlated with pH24 h and positively correlated with ΔpH. Expression of GYG was generally higher in LD. GYG expression was positively correlated with glycogen metabolite (lactate and GP) content and ΔpH, and was negatively correlated with pH24 h. These data confirm that the muscle-fiber type and GP have significant effects on ultimate pH and pH decline, and suggest that expression of GYG in muscles is related to the metabolism of glycogen and may impact GP, ΔpH, and ultimate pH. High expression of GYG was associated with a high glycogen content, large pH decline, and low ultimate pH in muscles post-mortem.
Asunto(s)
Glucosiltransferasas/biosíntesis , Glucógeno/metabolismo , Glicoproteínas/biosíntesis , Fibras Musculares Esqueléticas/metabolismo , Porcinos/genética , Animales , Regulación de la Expresión Génica , Glucosiltransferasas/genética , Glicoproteínas/genética , Concentración de Iones de Hidrógeno , Masculino , Carne , Fibras Musculares Esqueléticas/clasificación , Porcinos/crecimiento & desarrolloRESUMEN
Culture conditions in shake flasks affect filamentous Streptomyces lividans morphology, as well the productivity and O-mannosylation of recombinant Ala-Pro-rich O-glycoprotein (known as the 45/47 kDa or APA antigen) from Mycobacterium tuberculosis. In order to scale up from previous reported shake flasks to bioreactor, data from the literature on the effect of agitation on morphology of Streptomyces strains were used to obtain gassed volumetric power input values that can be used to obtain a morphology of S. lividans in bioreactor similar to the morphology previously reported in coiled/baffled shake flasks by our group. Morphology of S. lividans was successfully scaled-up, obtaining similar mycelial sizes in both scales with diameters of 0.21 ± 0.09 mm in baffled and coiled shake flasks, and 0.15 ± 0.01 mm in the bioreactor. Moreover, the specific growth rate was successfully scaled up (0.09 ± 0.02 and 0.12 ± 0.01 h(-1), for bioreactors and flasks, respectively), and the recombinant protein productivity measured by densitometry, as well. More interestingly, the quality of the recombinant glycoprotein measured as the amount of mannoses attached to the C-terminal of APA was also scaled- up; with up to five mannose residues in cultures carried out in shake flasks; and six in the bioreactor. However, final biomass concentration was not similar, indicating that although the process can be scaled-up using the power input, others factors like oxygen transfer rate, tip speed or energy dissipation/circulation function can be an influence on bacterial metabolism.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Reactores Biológicos/microbiología , Glicoproteínas/biosíntesis , Microbiología Industrial/métodos , Mycobacterium tuberculosis/genética , Streptomyces lividans/metabolismo , Proteínas Bacterianas/genética , Medios de Cultivo/metabolismo , Glicoproteínas/genética , Microbiología Industrial/instrumentación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Streptomyces lividans/citología , Streptomyces lividans/genéticaRESUMEN
FSH induces expansion of bovine cumulus-oocyte complexes (COCs) in cattle, which can be enhanced by oocyte-secreted factors (OSFs). In this study it was hypothesised that FSH stimulates COC expansion in part from direct stimulation of the epidermal growth factor (EGF)-like ligands amphiregulin (AREG), epiregulin (EREG) and betacellulin (BTC), but also in part through regulation of OSFs or their receptors in cumulus cells. Bovine COCs were cultured in defined medium with graded doses of FSH. In the absence of FSH, COCs did not expand. FSH caused cumulus expansion, and increased the abundance of AREG and EREG mRNA in a time- and dose-dependent manner, but decreased BTC mRNA levels. FSH had modest stimulatory effects on the levels of mRNA encoding the bone morphogenetic protein 15 (BMP15) receptor, BMPR1B, in cumulus cells, but did not alter mRNA expression of the growth and differentiation factor 9 (GDF9) receptor, TGFBR1. More interestingly, FSH dramatically stimulated levels of mRNA encoding two receptors for fibroblast growth factors (FGF), FGFR2C and FGFR3C, in cumulus cells. FSH also stimulated mRNA expression of FGFR1B, but not of FGFR2B in cumulus cells. Based on dose-response studies, FGFR3C was the receptor most sensitive to the influence of FSH. This study demonstrates that FSH stimulates the expression of EGF-like factors in bovine cumulus cells, and provides evidence that FSH differently regulates the expression of distinct receptors for OSFs in cumulus cells.
Asunto(s)
Bovinos/fisiología , Células del Cúmulo/efectos de los fármacos , Factor de Crecimiento Epidérmico/metabolismo , Fármacos para la Fertilidad Femenina/farmacología , Hormona Folículo Estimulante/farmacología , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Oocitos/efectos de los fármacos , Mataderos , Anfirregulina , Animales , Betacelulina , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/biosíntesis , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo/veterinaria , Células del Cúmulo/citología , Células del Cúmulo/metabolismo , Factor de Crecimiento Epidérmico/biosíntesis , Factor de Crecimiento Epidérmico/genética , Epirregulina , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Glicoproteínas/biosíntesis , Glicoproteínas/genética , Glicoproteínas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ligandos , Oocitos/citología , Oocitos/metabolismo , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/biosíntesis , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Sus scrofaRESUMEN
Aiming at maximizing the production of transmembrane rabies virus glycoprotein (rRVGP), the influence of hypothermic temperature on a recombinant Drosophila melanogaster S2 cell culture in Sf-900II medium was investigated. Cell growth and rRVGP production were assessed at 4 culture temperatures in Schott flasks: 16, 20, 24 and 28 °C. The maximum specific growth rates µ(max) were, respectively: 0.009, 0.019, 0.038 and 0.035 h(-1), while the maximum rRVGP levels C(max)(rRVGP) were: 0.075, 2.973, 0.480 and 1.404 mg L(-1). The best production temperature (20 °C) was then tested in a bioreactor with control of pH and dissolved oxygen in batch and fed-batch modes. In the batch culture, µ(max) and C(max)(rRVGP) were 0.060 h(-1) and 0.149 mg L(-1) at 28 °C and 0.026 h(-1) and 0.354 mg L(-1) at 20 °C, respectively. One batch-culture experiment was carried out with adaptation of the cells by the temperature falling in steps from 20 °C to 16 °C, so that µ(max) fell from 0.023 to 0.013 h(-1), while C(max)(rRVGP) was improved to 0.567 mg L(-1). In the fed-batch mode at 20 °C, µ(max) was 0.025 h(-1) and C(max)(rRVGP) was 1.155 mg L(-1). Taken together, these results indicate that the best strategy for optimized rRVGP production is the culture at hypothermic temperature of 20 °C, when µ(max) is kept low and with feeding of limitant aminoacids.
Asunto(s)
Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Glicoproteínas/biosíntesis , Virus de la Rabia/metabolismo , Recombinación Genética/genética , Temperatura , Proteínas Virales/biosíntesis , Animales , Técnicas de Cultivo Celular por Lotes , Reactores Biológicos , Proliferación Celular , Células Cultivadas , Cinética , SuspensionesRESUMEN
INTRODUCTION: Mutations in the gene ALPL in hypophosphatasia (HPP) reduce the function of tissue nonspecific alkaline phosphatase, and the resulting increase in pyrophosphate (PP(i)) contributes to bone and tooth mineralization defects by inhibiting physiologic calcium-phosphate (P(i)) precipitation. Although periodontal phenotypes are well documented, pulp/dentin abnormalities have been suggested in the clinical literature although reports are variable and underlying mechanisms remains unclear. In vitro analyses were used to identify mechanisms involved in HPP-associated pulp/dentin phenotypes. METHODS: Primary pulp cells cultured from HPP subjects were established to assay alkaline phosphatase (ALP) activity, mineralization, and gene expression compared with cells from healthy controls. Exogenous P(i) was provided to the correct P(i)/PP(i) ratio in cell culture. RESULTS: HPP cells exhibited significantly reduced ALP activity (by 50%) and mineral nodule formation (by 60%) compared with the controls. The expression of PP(i) regulatory genes was altered in HPP pulp cells, including reduction in the progressive ankylosis gene (ANKH) and increased ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). Odontoblast marker gene expression was disrupted in HPP cells, including reduced osteopontin (OPN), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), and matrix extracellular phosphoprotein (MEPE). The addition of P(i) provided a corrective measure for mineralization and partially rescued the expression of some genes although cells retained altered messenger RNA levels for PP(i)-associated genes. CONCLUSIONS: These studies suggest that under HPP conditions pulp cells have the compromised ability to mineralize and feature a disrupted odontoblast profile, providing a first step toward understanding the molecular mechanisms for dentin phenotypes observed in HPP.
Asunto(s)
Fosfatasa Alcalina/genética , Pulpa Dental/fisiopatología , Dentina/patología , Difosfatos/metabolismo , Hipofosfatasia/genética , Odontoblastos/patología , Calcificación de Dientes/genética , Adolescente , Sustitución de Aminoácidos , Análisis de Varianza , Calcio/metabolismo , Estudios de Casos y Controles , Pulpa Dental/citología , Enfermedades en Gemelos/genética , Regulación hacia Abajo , Proteínas de la Matriz Extracelular/biosíntesis , Proteínas de la Matriz Extracelular/genética , Femenino , Expresión Génica , Glicoproteínas/biosíntesis , Glicoproteínas/genética , Humanos , Hipofosfatasia/patología , Hipofosfatasia/fisiopatología , Masculino , Mutación Missense , Odontoblastos/metabolismo , Osteopontina/biosíntesis , Osteopontina/genética , Proteínas de Transporte de Fosfato/biosíntesis , Proteínas de Transporte de Fosfato/genética , Fosfoproteínas/biosíntesis , Fosfoproteínas/genética , Hidrolasas Diéster Fosfóricas/biosíntesis , Hidrolasas Diéster Fosfóricas/genética , Cultivo Primario de Células , Pirofosfatasas/biosíntesis , Pirofosfatasas/genética , Sialoglicoproteínas/biosíntesis , Sialoglicoproteínas/genética , Estadísticas no Paramétricas , Adulto JovenRESUMEN
Bovine Herpesvirus-1 (BoHV-1) is distributed worldwide and is a major pathogen in cattle, being the causal agent of a variety of clinical syndromes. The aim of this study was to isolate and to characterize (molecular and biological characterization) BoHV-1 from 29 immunosuppressed animals. It was possible to obtain 18 isolates, each from a different animal, such as from the respiratory and reproductive tracts. In some cases the cytopathic effect was visible 12 hours post-inoculation, and became characteristic after 36-48 hours. Biological characteristics were evaluated and compared with Iowa and Colorado-1 reference strains, and differences were found in plaque size, virus titer measured by TCID50 and PFU/mL, and one step virus curves. These results showed that some isolates had a highly virulent-like behavior in vitro, compared to the reference strains, with shorter eclipse periods, faster release of virus into the supernatants, and higher burst size and viral titer. There were no differences in glycoprotein expression of BoHV-1 isolates, measured by Western blot on monolayers. Moreover, using restriction endonucleases analysis, most of the viruses were confirmed as BoHV-1.1 and just one of them was confirmed as BoHV-1.2a subtype. These findings suggest that some wild-type BoHV-1 isolates could be useful as seeds to develop new monovalent vaccines.
Asunto(s)
Enfermedades de los Bovinos/virología , Infecciones por Herpesviridae/veterinaria , Herpesvirus Bovino 1/aislamiento & purificación , Animales , Antígenos Virales/biosíntesis , Western Blotting , Bovinos , Efecto Citopatogénico Viral , ADN Viral/genética , Genotipo , Glicoproteínas/biosíntesis , Infecciones por Herpesviridae/virología , Herpesvirus Bovino 1/clasificación , Herpesvirus Bovino 1/genética , Herpesvirus Bovino 1/patogenicidad , Huésped Inmunocomprometido , Tipificación Molecular , Polimorfismo de Longitud del Fragmento de Restricción , Carga Viral , Ensayo de Placa Viral , Proteínas Virales/biosíntesis , Virulencia , Cultivo de VirusRESUMEN
Aberrant expression of stem cell-related genes in tumors may confer more primitive and aggressive traits affecting clinical outcome. Here, we investigated expression and prognostic value of the neural stem cell marker CD133, as well as of the pluripotency genes LIN28 and OCT4 in 37 samples of pediatric medulloblastoma, the most common and challenging type of embryonal tumor. While most medulloblastoma samples expressed CD133 and LIN28, OCT4 expression was found to be more sporadic, with detectable levels occurring in 48% of tumors. Expression levels of OCT4, but not CD133 or LIN28, were significantly correlated with shorter survival (P ≤ 0.0001). Median survival time of patients with tumors hyperexpressing OCT4 and tumors displaying low/undetectable OCT4 expression were 6 and 153 months, respectively. More importantly, when patients were clinically stratified according to their risk of tumor recurrence, positive OCT4 expression in primary tumor specimens could discriminate patients classified as average risk but which further deceased within 5 years of diagnosis (median survival time of 28 months), a poor clinical outcome typical of high risk patients. Our findings reveal a previously unknown prognostic value for OCT4 expression status in medulloblastoma, which might be used as a further indicator of poor survival and aid postoperative treatment selection, with a particular potential benefit for clinically average risk patients.
Asunto(s)
Neoplasias Encefálicas/genética , Regulación Neoplásica de la Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/fisiología , Meduloblastoma/genética , Factor 3 de Transcripción de Unión a Octámeros/biosíntesis , Células Madre/fisiología , Antígeno AC133 , Adolescente , Antígenos CD/biosíntesis , Antígenos CD/genética , Malformación de Arnold-Chiari/genética , Biomarcadores , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Niño , Preescolar , Femenino , Glicoproteínas/biosíntesis , Glicoproteínas/genética , Humanos , Estimación de Kaplan-Meier , Masculino , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/patología , Factor 3 de Transcripción de Unión a Octámeros/genética , Péptidos/genética , Valor Predictivo de las Pruebas , Pronóstico , Proteínas de Unión al ARN/biosíntesis , Proteínas de Unión al ARN/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Medición de Riesgo , SobrevidaRESUMEN
The cancer stem cell (CSC) theory is currently a very important field in cancer research. This theory states that tumours are organised in a hierarchical manner with a subpopulation of limited number called CSCs with the ability to self-renew and undergo asymmetrical divisions, giving rise to a differentiated progeny that represents most of the tumour populations. CSCs are metastatic and chemoresistant, two features that very likely contribute to the poor response of locally advanced lung cancer. CSCs have been identified in non-small-cell lung cancer cell lines as well as those from patient primary samples. A correlation has been found in terms of chemoresistance and bad prognosis in patient-derived samples enriched with CSCs, indicating that these cells are an important target for future therapy combinations. Therefore, understanding the biology and exploring cell markers and signalling pathways specific for CSCs of lung cancer may help in achieving progress in the treatment of the disease.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Resistencia a Antineoplásicos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Células Madre Neoplásicas/citología , Antígeno AC133 , Aldehído Deshidrogenasa/metabolismo , Antígenos CD/biosíntesis , Antígenos CD34/biosíntesis , Diferenciación Celular , Glicoproteínas/biosíntesis , Humanos , Receptores de Hialuranos/biosíntesis , Oncología Médica/métodos , Metástasis de la Neoplasia , Péptidos , Transducción de Señal , Factores de Transcripción/metabolismo , Resultado del TratamientoRESUMEN
S2 cell populations (S2AcRVGP2K and S2MtRVGP-Hy) were selected after transfection of gene expression vectors carrying the cDNA encoding the rabies virus glycoprotein (RVGP) gene under the control of the constitutive (actin) or inductive (metallothionein) promoters. These cell populations were cultivated in a 1L bioreactor mimicking a large scale bioprocess. Cell cultures were carried out at 90 rpm and monitored/controlled for temperature (28 degrees C) and dissolved oxygen (10 or 50% air saturation). Cell growth attained approximately 1.5-3 x 10(7)cells/mL after 3-4 days of cultivation. The constitutive synthesis of RVGP in S2AcRVGP2K cells led to values of 0.76 microg/10(7) cells at day 4 of culture. The RVGP synthesis in S2MtRVGP-Hy cell fraction increased upon CuSO(4) induction attaining specific productivities of 1.5-2 microg/10(7) cells at days 4-5. RVGP values in supernatant as a result of cell lysis were always very low (<0.2 microg/mL) indicating good integrity of cells in culture. Overall the RVGP productivity was of 1.5-3mg/L. Our data showed an important influence of dissolved oxygen on RVGP synthesis allowing a higher and sustained productivity by S2MtRVGP-Hy cells when cultivated with a DO of 10% air saturation. The RVGP productivity in bioreactors shown here mirrors those previously observed for T-flasks and shaker bottles and allow the preparation of the large RVGP quantities required for studies of structure and function.