RESUMEN

To date, limited information is available on cytomegalovirus (CMV) and lymphocryptovirus (LCV) from Chlorocebus monkeys. We report here high detection rates of herpesviruses in free-roaming African green monkeys (AGMs, Chlorocebus sabaeus) (26.4%, 23/87) and in captive AGMs (75%, 3/4) with respiratory disease on the Caribbean Island of St. Kitts. LCV (81.25%) was more prevalent than CMV (18.75%) in the AGMs. Applying a bigenic PCR approach (targeting DNA polymerase (DPOL) and glycoprotein B (gB) genes), long sequences were obtained from representative AGM CMV (KNA-SD6) and LCV (KNA-E4, -N6 and -R15) samples, and mixed LCV infections were identified in KNA-N6 and -R15. The nucleotide (nt) sequence (partial DPOL-intergenic region-partial gB) and partial DPOL- and gB-amino acid (aa) sequences of AGM CMV KNA-SD6 were closely related to Cytomegalovirus cercopithecinebeta5 isolates from grivet monkeys, whilst those of AGM LCV KNA-E4 and -N6 (and E4-like gB of KNA-R15) were more closely related to cognate sequences of erythrocebus patas LCV1 from patas monkey than other LCVs, corroborating the concept of cospeciation in the evolution of CMV/LCV. On the other hand, the partial DPOL aa sequence of KNA-R15, and additional gB sequences (N6-gB-2 and R15-gB-2) from samples KNA-N6 and -R15 (respectively) appeared to be distinct from those of Old World monkey LCVs, indicating LCV evolutionary patterns that were not synchronous with those of host species. The present study is the first to report the molecular prevalence and genetic diversity of CMV/LCV from free-roaming/wild and captive AGMs, and is the first report on analysis of CMV nt/deduced aa sequences from AGMs and LCV gB sequences from Chlorocebus monkeys.

Asunto(s)

Infecciones por Citomegalovirus , Lymphocryptovirus , Animales , Chlorocebus aethiops , Lymphocryptovirus/genética , Citomegalovirus/genética , Filogenia , Herpesvirus Humano 4 , Glicoproteínas/genética , Variación Genética

RESUMEN

The ORF 70 gene of equid alphaherpesvirus type 3 (EHV-3) encodes glycoprotein G (gG), which is conserved in the majority of alphaherpesviruses. This glycoprotein is located in the viral envelope and has the characteristic of being secreted into the culture medium after proteolytic processing. It modulates the antiviral immune response of the host by interacting with chemokines. The aim of this study was to identify and characterize EHV-3 gG. By constructing viruses with HA-tagged gG, it was possible to detect gG in lysates of infected cells, their supernatants, and purified virions. A 100-, 60-, and 17-kDa form of the protein were detected in viral particles, while a 60-kDa form was identified in supernatants of infected cells. The role of EHV-3 gG in the viral infection cycle was assessed by the construction of a gG-minus EHV-3 mutant and its gG-positive revertant. When growth characteristics in an equine dermal fibroblast cell line were compared, the plaque size and the growth kinetics of the gG-minus mutant were similar to those of the revertant virus, suggesting that EHV-3 gG does not play a role in direct cell-to-cell transmission or virus proliferation of EHV-3 in tissue culture. The identification and characterization of EHV-3 gG described here provide a solid background for further studies to assess whether this glycoprotein has a function in modulating the host immune response.

Asunto(s)

Infecciones por Herpesviridae , Herpesvirus Équido 1 , Herpesvirus Équido 3 , Animales , Caballos , Proteínas del Envoltorio Viral/metabolismo , Herpesvirus Équido 1/genética , Herpesvirus Équido 3/metabolismo , Línea Celular , Glicoproteínas/genética

RESUMEN

Germin-like proteins (GLPs) play an important role against various stresses. Vitis vinifera L. genome contains 7 GLPs; many of them are functionally unexplored. However, the computational analysis may provide important new insight into their function. Currently, physicochemical properties, subcellular localization, domain architectures, 3D structures, N-glycosylation & phosphorylation sites, and phylogeney of the VvGLPs were investigated using the latest computational tools. Their functions were predicted using the Search tool for the retrieval of interacting genes/proteins (STRING) and Blast2Go servers. Most of the VvGLPs were extracellular (43%) in nature but also showed periplasmic (29%), plasma membrane (14%), and mitochondrial- or chloroplast-specific (14%) expression. The functional analysis predicted unique enzymatic activities for these proteins including terpene synthase, isoprenoid synthase, lipoxygenase, phosphate permease, receptor kinase, and hydrolases generally mediated by Mn+ cation. VvGLPs showed similarity in the overall structure, shape, and position of the cupin domain. Functionally, VvGLPs control and regulate the production of secondary metabolites to cope with various stresses. Phylogenetically VvGLP1, -3, -4, -5, and VvGLP7 showed greater similarity due to duplication while VvGLP2 and VvGLP6 revealed a distant relationship. Promoter analysis revealed the presence of diverse cis-regulatory elements among which CAAT box, MYB, MYC, unnamed-4 were common to all of them. The analysis will help to utilize VvGLPs and their promoters in future food programs by developing resistant cultivars against various biotic (Erysiphe necator and in Powdery Mildew etc.) and abiotic (Salt, drought, heat, dehydration, etc.) stresses.

Asunto(s)

Vitis , Glicoproteínas/genética , Glicoproteínas/metabolismo , Proteínas de Plantas/metabolismo , Vitis/genética

RESUMEN

Chimeric virus-like particles are self-assembling structures composed of viral proteins that had been modified to incorporate sequences from different organisms, being able to trigger immune responses against the heterologous sequence. However, the identification of suitable sites for that purpose in the carrier protein is not an easy task. In this work, we describe the generation of rabies chimeric VLPs that expose a major antigenic site of foot-and-mouth disease virus (FMDV) by identifying suitable regions in rabies glycoprotein (RVG), as a proof of concept of a novel heterologous display platform for vaccine applications. To identify adequate sites for insertion of heterologous sequences without altering the correct folding of RVG, we identified regions that were evolutionally non-conserved in Lyssavirus glycoproteins and performed a structural analysis of those regions using a 3D model of RVG trimer that we generated. The heterologous sequence was inserted in three different sites within RVG sequence. In every case, it did not affect the correct folding of the protein and was surface exposed, being recognized by anti-FMDV antibodies in expressing cells as well as in the surface of VLPs. This work sets the base for the development of a heterologous antigen display platform based on rabies VLPs. KEY POINTS: • Adequate regions for foreign epitope display in RVG were found. • G-H loop of FMDV was inserted in three regions of RVG. • The foreign epitope was detected by specific antibodies on fusion proteins. • G-H loop was detected on the surface of chimeric VLPs.

Asunto(s)

Virus de la Fiebre Aftosa , Fiebre Aftosa , Rabia , Vacunas , Animales , Anticuerpos Antivirales , Virus de la Fiebre Aftosa/genética , Glicoproteínas/genética

RESUMEN

This study investigated the occurrence of Giardia duodenalis and Cryptosporidium spp. in rodents and marsupials from the Atlantic Forest in southern Bahia, northeastern Brazil. Two hundred and four fecal samples were collected from different forest areas in the municipalities of Ilhéus, Una, Belmonte, and Mascote. Identifications were performed using PCR and nested PCR followed by sequencing of the gdh and tpi genes for G. duodenalis, and the gp60 and Hsp-70 genes for Cryptosporidium. The total frequency of positive PCR samples for both G. duodenalis and Cryptosporidium spp. was 5.4% (11/204). Giardia duodenalis occurred in 2.94% (4/136) of rodents and 2.94% (2/68) of marsupials. The prevalence of Cryptosporidium in rodents and marsupials was 1.47% (2/136) and 4.41% (3/68), respectively. In the areas sampled, the frequency of parasitism was 50% (7/14), while the Mascote region alone had no parasitized animals. The G. duodenalis subgenotype AI was identified in the rodent species Hylaeamys laticeps, Oecomys catherinae, Oligoryzomys nigripes and Akodon cursor, and in the marsupials Gracilinanus agilis and Monodelphis americana. In the rodents Rhipidomys mastacalis, H. laticeps and in the marsupial Marmosa murina the protozoa Cryptosporidium fayeri, Cryptosporidium parvum and Cryptosporidium ubiquitum with subtypes IIa and IVg by the gp60 gene were found. In conclusion, this study provides the genetic characterization of Giardia and Cryptosporidium species and genotypes in rodents and marsupials. And, these findings reinforce that the rodent and marsupial species mentioned above play a role as new hosts for Giardia and Cryptosporidium.

Asunto(s)

Criptosporidiosis/epidemiología , Cryptosporidium/genética , Giardia lamblia/genética , Giardiasis/epidemiología , Giardiasis/veterinaria , Zoonosis/epidemiología , Animales , Brasil/epidemiología , Criptosporidiosis/parasitología , Cryptosporidium/aislamiento & purificación , ADN-Topoisomerasas/genética , ADN Protozoario/genética , Heces/parasitología , Giardia lamblia/aislamiento & purificación , Giardiasis/parasitología , Glicoproteínas/genética , Proteínas HSP70 de Choque Térmico/genética , Marsupiales/parasitología , Reacción en Cadena de la Polimerasa , Prevalencia , Proteínas Protozoarias/genética , Roedores/parasitología , Análisis de Secuencia de ADN , Deshidrogenasas del Alcohol de Azúcar/genética , Zoonosis/parasitología

RESUMEN

Plant long noncoding RNAs (lncRNAs) are key chromatin dynamics regulators, directing the transcriptional programs driving a wide variety of developmental outputs. Recently, we uncovered how the lncRNA AUXIN REGULATED PROMOTER LOOP (APOLO) directly recognizes the locus encoding the root hair (RH) master regulator ROOT HAIR DEFECTIVE 6 (RHD6) modulating its transcriptional activation and leading to low temperature-induced RH elongation. We further demonstrated that APOLO interacts with the transcription factor WRKY42 in a novel ribonucleoprotein complex shaping RHD6 epigenetic environment and integrating signals governing RH growth and development. In this work, we expand this model showing that APOLO is able to bind and positively control the expression of several cell wall EXTENSIN (EXT) encoding genes, including EXT3, a key regulator for RH growth. Interestingly, EXT3 emerged as a novel common target of APOLO and WRKY42. Furthermore, we showed that the ROS homeostasis-related gene NADPH OXIDASE C (NOXC) is deregulated upon APOLO overexpression, likely through the RHD6-RSL4 pathway, and that NOXC is required for low temperature-dependent enhancement of RH growth. Collectively, our results uncover an intricate regulatory network involving the APOLO/WRKY42 hub in the control of master and effector genes during RH development.

Asunto(s)

Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Pared Celular , Ácidos Indolacéticos/metabolismo , Raíces de Plantas/crecimiento & desarrollo , ARN Largo no Codificante/metabolismo , Factores de Transcripción/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular , Proliferación Celular , Cromatina/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Glicoproteínas/genética , Glicoproteínas/metabolismo , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Desarrollo de la Planta/genética , Raíces de Plantas/metabolismo , Regiones Promotoras Genéticas

RESUMEN

Geoffroea decorticans (chañar) is commonly used for culinary and medicinal purposes in rural communities. The aim of this work was to chemically characterize three Geoffroea decorticans extracts and determine their capacity to modulate the wnt/ß-catenin pathway. This signaling pathway plays a key role in embryonic development but its overactivation leads to cancer cell growth. Phytochemical analysis of extracts showed presence of major classes of phytochemicals. Gas chromatography-mass spectrometry results revealed the presence of acids, esters and furanic compounds. Using Xenopus embryos as in vivo model organisms, we found that the extracts modulated dorso-ventral axis formation and rescued hyperdorsalized phenotypes produced by LiCl treatment. In agreement with these findings, Geoffroea decorticans extracts decreased ß-catenin levels and suppressed the expression of wnt target genes such as xnr3 and chordin, thus demonstrating an inhibitory regulation of the wnt/ß-catenin signaling pathway. All these results support a new role for Geoffroea decorticans fruit derivatives with possible anti-carcinogenic actions.

Asunto(s)

Fabaceae/química , Frutas/química , Terapia Molecular Dirigida , Neoplasias/metabolismo , Extractos Vegetales/farmacología , Vía de Señalización Wnt/efectos de los fármacos , Xenopus laevis , beta Catenina/antagonistas & inhibidores , Animales , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Glicoproteínas/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Cloruro de Litio/farmacología , Masculino , Neoplasias/tratamiento farmacológico , Extractos Vegetales/química , Factor de Crecimiento Transformador beta/genética , Vía de Señalización Wnt/genética , Proteínas de Xenopus/genética , Xenopus laevis/embriología , Xenopus laevis/genética , beta Catenina/genética , beta Catenina/metabolismo

RESUMEN

In the present work, we established and characterized a 3D functional polarized primary bovine oviduct epithelial cells (BOECs) culture on free-floating type I collagen hydrogels (rafts) at an air-liquid interface (ALI). Intercellular junctions, ultrastructural cellular morphology and the expression of the OVGP1 closely recapitulated those of the in vivo epithelium lining. These morphological and physiological epithelial cell features were maintained under standard DMEM/F12 with 10% foetal bovine serum culture medium for at least 28 days of ALI culture. The versatility of the BOECs raft cultures should allow testing of toxicity compounds, in vitro evaluation of physiological or pathological oviductal states, and the study of epithelial-mesenchymal interactions that are critical for the maintenance of oviductal homeostasis.

Asunto(s)

Técnicas de Cultivo de Célula/veterinaria , Células Epiteliales/metabolismo , Oviductos/citología , Animales , Bovinos , Polaridad Celular , Células Cultivadas , Colágeno , Medios de Cultivo , Células Epiteliales/ultraestructura , Femenino , Glicoproteínas/genética , Glicoproteínas/metabolismo , Hidrogeles

RESUMEN

Sea urchins live in a challenging environment that requires rapid and efficient responses against pathogens and invaders. This response may be also important in reproductive processes once males and females release their gametes into water. In addition, the gonads are organs with dual function: reproductive organ and nutrient reserve, therefore it needs efficient protective mechanisms to preserve the nutrients as well as the reproductive cells. The aim of this study was to evaluate the presence and characterize antimicrobial molecules in the male and female gonads of the sea urchin Lytechinus variegatus. Through HPLC purification, antimicrobial activity test and mass spectrometry several antimicrobial molecules were found in the gonads of both gender. Computational in silico analyses showed that they are fragments of a glycoprotein called toposome, also known as major yolk protein (MYP) which is one of the major proteins found in the gonads. Although different functions have been reported for this protein, this is the first description of a direct antimicrobial activity in Lytechinus variegatus. The results indicate that when undergoing proteolysis the toposome generates different fragments with antimicrobial activity which may indicate the importance of a rapid defense response strategy against invading microorganisms in the gonads used by both males and females sea urchins.

Asunto(s)

Antiinfecciosos/inmunología , Glicoproteínas/genética , Glicoproteínas/inmunología , Inmunidad Innata/genética , Lytechinus/genética , Lytechinus/inmunología , Secuencia de Aminoácidos , Animales , Femenino , Perfilación de la Expresión Génica , Glicoproteínas/química , Masculino , Ovario/inmunología , Ovario/metabolismo , Alineación de Secuencia , Testículo/inmunología , Testículo/metabolismo

RESUMEN

Acid-labile subunit (ALS) deficiency (ACLSD) constitutes the first monogenic defect involving a member of the Insulin-like Growth Factor (IGF) binding protein system. The lack of ALS completely disrupts the circulating IGF system. Autocrine/paracrine action of local produced IGF-I could explain the mild effect on growth. In the present work we have revised the more relevant clinical and biochemical consequences of complete ACLSD in 61 reported subjects from 31 families. Low birth weight and/or length, reduced head circumference, height between -2 and -3 SD, pubertal delay and insulin resistance are commonly observed. Partial ACLSD could be present in children initially labeled as idiopathic short stature, presenting low IGF-I levels, suggesting that one functional IGFALS allele is insufficient to stabilize ternary complexes. Dysfunction of the GH-IGF axis observed in ACLSD may eventually result in increased risk for type-2 diabetes and tumor progression. Consequently, long term surveillance is recommended in these patients.

Asunto(s)

Proteínas Portadoras/fisiología , Glicoproteínas/fisiología , Hormona de Crecimiento Humana/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Estatura/efectos de los fármacos , Estatura/genética , Proteínas Portadoras/genética , Proteínas Portadoras/farmacología , Niño , Femenino , Glicoproteínas/deficiencia , Glicoproteínas/genética , Glicoproteínas/farmacología , Trastornos del Crecimiento/genética , Trastornos del Crecimiento/metabolismo , Humanos , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Masculino , Pubertad Tardía/genética , Pubertad Tardía/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología

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/genética

RESUMEN

In the past decade, a large number of studies have detected herpesvirus sequences from many bat species around the world. Nevertheless, the discovery of bat herpesviruses is geographically uneven. Of the various bat species tested to date, only a few were from the New World. Seeking to investigate the distribution and diversity of herpesviruses circulating in neotropical bats, we carried out molecular screening of 195 blood DNA samples from 11 species of three bat families (Phyllostomidae, Mormoopidae, and Molossidae). Using polymerase chain reaction amplification, with degenerate consensus primers targeting highly conserved amino acid motifs of the herpesvirus DNA polymerase and Glycoprotein B genes, we characterized novel viral sequences from all tested species. BLAST searches, pairwise nucleotide and amino acid sequence comparisons, as well as phylogenetic analyses confirmed that they all belonged to the Herpesviridae family, of the Beta- and Gammaherpesvirinae subfamilies. Fourteen partial DNA polymerase gene sequences, of which three beta- and 11 gamma-herpesviruses, were detected. A total of 12 partial Glycoprotein B gene sequences, all gamma-herpesviruses, were characterized. Every sequence was specific to a bat species and in some species (Desmodus rotundus, Carollia perspicillata, and Pteronotus rubiginosus) multiple viruses were found. Phylogenetic analyses of beta- and gammaherpesvirus sequences led to the identification of bat-specific clades. Those composed of sequences obtained from different bat species belonging to distinct subfamilies follow the taxonomy of bats. This study confirms the astonishing diversity of bat herpesviruses and broadens our knowledge of their host range. Nevertheless, it also emphasizes the fact that, to better appreciate the evolutionary history of these viruses, much remains to be done at various taxonomic levels.

Asunto(s)

Quirópteros/virología , Herpesviridae/genética , Filogenia , Proteínas Virales/genética , Animales , ADN Polimerasa Dirigida por ADN/genética , Exodesoxirribonucleasas/genética , Guyana Francesa , Gammaherpesvirinae/genética , Glicoproteínas/genética , Martinica

RESUMEN

Articular cartilage is an avascular tissue with a structure that allows it to support and cushion the overload of the surfaces in contact. It maintains its metabolic functions due to the contribution of different signaling pathways. However, several factors play a role in its deterioration, allowing to the development of osteoarthritis (OA), and one of the major factors is genetic. Our goal was to identify gene-gene interactions (epistasis) between five signaling pathways involved in the articular cartilage metabolism as possible indicators of OA risk. We applied the Multifactor-Dimensionality Reduction (MDR) method to identify and characterize the epistasis between 115 SNPs located in 73 genes related to HIF-1α, Wnt/ß-catenin, cartilage extracellular matrix metabolism, oxidative stress, and uric acid transporters. Ninety three patients diagnosed with primary knee OA and 150 healthy controls were included in the study. Genotyping was performed with the OpenArray system, the statistical analysis was carried out with the STATA software v14, and epistasis was analyzed with the MDR software v3.0.2. The MDR analysis revealed that the best interaction model was between polymorphisms rs17786744 of the STC1 gene and rs2615977 of the COL11A1 gene, with an entropy value of 4.44%, CVC 8/10, OR 5.60, 95% CI 3.27-9.59, p < 0.0001. Under this interaction model, we identified high and low risk genotypes involved in OA development. Our results suggest complex interactions between STC1 and COL11A1 genes that might have an impact on genetic susceptibility to develop OA. Further studies are required to confirm it.

Asunto(s)

Colágeno Tipo XI/genética , Glicoproteínas/genética , Osteoartritis de la Rodilla/genética , Adulto , Alelos , Estudios de Casos y Controles , Epistasis Genética/genética , Femenino , Frecuencia de los Genes , Predisposición Genética a la Enfermedad/genética , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Reducción de Dimensionalidad Multifactorial/métodos , Osteoartritis/genética , Polimorfismo de Nucleótido Simple/genética , Factores de Riesgo , Programas Informáticos

RESUMEN

KEY MESSAGE: SBTX has defensive role against C. kikuchii, and therefore, its constituent genes SBTX17 and SBTX27 are promising candidates to engineer pathogen resistant plants. Soybean (Glycine max [L.] Merr.) is economically the most important legume crop in the world. Its productivity is strongly affected by fungal diseases, which reduce soybean production and seed quality and cause losses of billions of dollars worldwide. SBTX is a protein that apparently takes part in the defensive chemical arsenal of soybean against pathogens. This current study provides data that reinforce this hypothesis. Indeed, SBTX inhibited in vitro the mycelial growth of Cercospora kikuchii, it is constitutively located in the epidermal region of the soybean seed cotyledons, and it is exuded from mature imbibed seeds. Moreover, RT-qPCR analysis of the SBTX associated genes, SBTX17 and SBTX27, which encode for the 17 and 27 kDa polypeptide chains, showed that both genes are expressed in all studied plant tissues during the soybean development, with the highest levels found in the mature seeds and unifoliate leaves. In addition, to assess a local response of the soybean secondary leaves from 35-day-old plants, they were inoculated with C. kikuchii and treated with salicylic acid. It was verified using RT-qPCR that SBTX17 and SBTX27 genes overexpressed in leaves compared to controls. These findings strongly suggest that SBTX has defensive roles against C. kikuchii. Therefore, SBTX17 and SBTX27 genes are promising candidates to engineer pathogen resistant plants.

Asunto(s)

Ascomicetos , Resistencia a la Enfermedad/genética , Glycine max/metabolismo , Glicoproteínas/fisiología , Enfermedades de las Plantas/microbiología , Ácido Salicílico/farmacología , Proteínas de Soja/fisiología , Ascomicetos/efectos de los fármacos , Ascomicetos/crecimiento & desarrollo , Cotiledón/genética , Cotiledón/metabolismo , Expresión Génica , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/genética , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicoproteínas/farmacología , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Hojas de la Planta/microbiología , Regiones Promotoras Genéticas , Semillas/genética , Semillas/metabolismo , Proteínas de Soja/genética , Proteínas de Soja/metabolismo , Proteínas de Soja/farmacología , Glycine max/genética , Glycine max/crecimiento & desarrollo , Glycine max/microbiología , Regulación hacia Arriba

RESUMEN

Prostate cancer (PCa) is one of the most prevalent male tumours. Stanniocalcin-1 (STC1) is a glycoprotein and, although the role of STC1 in human cancer is poorly understood, it is suggested to be involved in the development and progression of different neoplasms. This study investigated the protein expression profile of STC1 in PCa and benign prostatic hyperplasia (BPH) samples and STC1 signalling during cell proliferation and cell death in vitro using cell lines. We found higher levels of STC1 in PCa when compared to BPH tissue and that STC1 inhibited forskolin stimulation of cAMP in PC-3 cells. A monoclonal antibody against STC1 was effective in reducing cell proliferation, in promoting cell cycle arrest, and in increasing apoptosis in the same cells. Since STC1 acts as a regulator of prostatic tissue signalling, we suggest that this protein is a novel candidate biomarker for prostate tumour clinical progression and a potential therapeutic target.

Asunto(s)

Biomarcadores de Tumor/metabolismo , Colforsina/farmacología , Glicoproteínas/genética , Glicoproteínas/metabolismo , Hiperplasia Prostática/metabolismo , Neoplasias de la Próstata/metabolismo , Apoptosis , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Progresión de la Enfermedad , Humanos , Masculino , Células PC-3 , Hiperplasia Prostática/genética , Neoplasias de la Próstata/genética , Regulación hacia Arriba

RESUMEN

Although the discovery of glycogen in the liver, attributed to Claude Bernard, happened more than 160 years ago, the mechanism involved in the initiation of glucose polymerization remained unknown. The discovery of glycogenin at the core of glycogen's structure and the initiation of its glucopolymerization is among one of the most exciting and relatively recent findings in Biochemistry. This review focuses on the initial steps leading to the seminal discoveries of proteoglycogen and glycogenin at the beginning of the 1980s, which paved the way for subsequent foundational breakthroughs that propelled forward this new research field. We also explore the current, as well as potential, impact this research field is having on human health and disease from the perspective of glycogen storage diseases. Important new questions arising from recent studies, their links to basic mechanisms involved in the de novo glycogen biogenesis, and the pervading presence of glycogenin across the evolutionary scale, fueled by high throughput -omics technologies, are also addressed.

Asunto(s)

Glucosiltransferasas/metabolismo , Glucógeno/metabolismo , Glicoproteínas/metabolismo , Animales , Glucosa/metabolismo , Glucosiltransferasas/química , Glucosiltransferasas/genética , Glucógeno/química , Enfermedad del Almacenamiento de Glucógeno/enzimología , Enfermedad del Almacenamiento de Glucógeno/genética , Enfermedad del Almacenamiento de Glucógeno/metabolismo , Glicoproteínas/química , Glicoproteínas/genética , Glicosilación , Humanos , Hígado/enzimología , Hígado/metabolismo , Polimerizacion

RESUMEN

trans-Sialidase and cruzipain are important virulence factors from Trypanosoma cruzi, the etiological agent of Chagas disease, that have highly antigenic domains in their structure and were reported as potential tools for diagnosis of the illness. The aim of the present study is to assess the possibility of using cruzipain and the catalytic domain of trans-sialidase in a Surface Plasmon Resonance-based immunosensor for the diagnosis of chronic Chagas disease. Immunoassays carried out with canine sera verified that cruzipain allows the detection of anti-Trypanosoma cruzi antibodies whereas recombinant trans-sialidase did not yield specific detections, due to the high dilutions of serum used in the immunoassays that hinder the possibility to sense the specific low titer antibodies. The developed cruzipain-based biosensor, whose price per assay is comparable to a commercial enzyme-linked immunosorbent assay (ELISA), was successfully applied for the rapid quantification of specific antibodies against Trypanosoma cruzi in fresh human sera showing an excellent agreement with ELISA.

Asunto(s)

Anticuerpos Antiprotozoarios/sangre , Enfermedad de Chagas/diagnóstico , Enfermedad de Chagas/veterinaria , Ensayo de Inmunoadsorción Enzimática/métodos , Trypanosoma cruzi/aislamiento & purificación , Animales , Enfermedad de Chagas/sangre , Enfermedad de Chagas/parasitología , Cisteína Endopeptidasas/análisis , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/inmunología , Enfermedades de los Perros/sangre , Enfermedades de los Perros/diagnóstico , Enfermedades de los Perros/parasitología , Perros , Glicoproteínas/análisis , Glicoproteínas/genética , Glicoproteínas/inmunología , Humanos , Neuraminidasa/análisis , Neuraminidasa/genética , Neuraminidasa/inmunología , Proteínas Protozoarias/análisis , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Trypanosoma cruzi/genética , Trypanosoma cruzi/inmunología , Factores de Virulencia/sangre , Factores de Virulencia/genética , Factores de Virulencia/inmunología

RESUMEN

CONTEXT: ALS deficiency (ACLSD), caused by mutations in IGFALS, is characterized by a mild short stature, low concentrations of IGF-I and IGFBP-3, and a normal growth hormone (GH) stimulation test response. To our knowledge, no larger deletions have been reported. CASE DESCRIPTION: A 17-year-old adolescent male was evaluated due to delayed puberty and short stature. He had a height of 154.4 cm (SDS -2.84), a weight of 53.3 kg (SDS -1.41), a BMI of 22.4 kg/m2 (SDS +0.31), a Tanner 2 pubertal stage with a testicular volume of 10 mL, and a bone age of 16 years (SDS -1.33). After biochemical evaluation, low IGF-I levels, undetectable IGFBP-3 levels, and a normal response to the GH stimulation test were observed, suggesting GH insensitivity. ACLSD was confirmed by ALS measurement (116 ng/mL, SDS -3.19) and genetic analysis of IGFALS. An apparently homozygous missense variant, p. Pro624Leu, was found in exon 2 of the proband; this mutation was observed on one allele of the proband's father but was absent in the mother and siblings. Deletion/duplication analysis by multiplex ligation-dependent probe amplification (MLPA) was consistent with a deletion encompassing a significant part of exon 2 on one allele in the proband and in his mother and siblings. CONCLUSION: This is the first report of a large deletion in a patient with ACLSD. Deletion/duplication analysis should be considered in the genetic study of ACLSD, especially when homozygosity for a pathogenic variant cannot be confirmed by the study of the parents or when no variants are found but ALS concentrations are very low.

Asunto(s)

Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Proteínas Portadoras/genética , Exones , Glicoproteínas/genética , Mutación Missense , Eliminación de Secuencia , Adolescente , Femenino , Humanos , Masculino , Linaje

RESUMEN

The Semliki Forest virus (SFV) viral vector has been widely used for transient protein expression. This study aimed to analyze comprehensively the capacity of SFV vector to express rabies lyssavirus glycoprotein (RVGP) in mammalian cells. The assessed parameters were transfection strategy, multiplicity of infection (MOI), harvest time and mammalian cell host. Two transfection approaches, electroporation and lipofection were evaluated to obtain the recombinant SFV, and the electroporation was found to be the most effective. Viral quantification by RT-qPCR was performed to elucidate the relation between the amount of recombinant virus utilized in the infection process and the production levels of the heterologous protein. Four different multiplicities of infection (MOIs = 1; 10; 15; 50) were evaluated using five mammalian cell lines: BHK-21, HuH-7, Vero, L929, and HEK-293T. Protein expression was assessed at two harvest times after infection (24 and 48 h). The recombinant protein generated was characterized by western blot, dot blot, and indirect immunofluorescence (IIF), while its concentration was determined by enzyme-linked immunosorbent assay (ELISA). Similar expression patterns were observed in cell lines BHK-21, HEK-293T, L929, and Vero, with higher RVGP production in the first 24 h. The BHK-21 cells showed yields of up to 4.3 µg per 106 cells when lower MOIs (1 and 10) were used. The HEK-293 T cells also showed similar production (4.3 µg per 106 cells) with MOI of 1, while the L929 and Vero cell lines showed lower expression rates of 2.82 and 1.26 µg per 106 cells, respectively. These cell lines showed lower expression levels at 48 h after infection compared to 24 h. Controversially, in the case of the HuH-7 cell line, RVGP production was higher at 48 h after infection (4.0 µg per 106 cells) and using MOIs of 15 and 50. This work may contribute to optimize the RVGP production using SFV system in mammalian cells. This study can also substantiate for example, the development of approaches that use of SFV for applications for other protein expressions and suggests values for relevant parameters and cell lines of this biotechnique.

Asunto(s)

Glicoproteínas/genética , Glicoproteínas/metabolismo , Virus de la Rabia/metabolismo , Virus de los Bosques Semliki/genética , Animales , Línea Celular , Chlorocebus aethiops , Electroporación , Regulación Viral de la Expresión Génica , Células HEK293 , Humanos , Ingeniería de Proteínas , Virus de la Rabia/genética , Transfección , Células Vero , Proteínas Virales/genética , Proteínas Virales/metabolismo

RESUMEN

Cryptosporidium parvum is a protozoan parasite of the phylum Apicomplexa responsible for cryptosporidiosis in calves, a disease that causes significant diarrhea and impairs gain of body weight, generating important production losses. As to now, no effective drugs or vaccines are available for the treatment or prevention of bovine cryptosporidiosis. Several reports suggest that development of a vaccine to prevent cryptosporidiosis is feasible, but relatively few vaccine candidates have been characterized and tested. The most prominent C. parvum antigen is gp60, an O-glycosylated mucin-like protein tethered to the parasite membrane by a glycosylphosphatidylinositol (GPI) anchor. Gp60 has been shown to be involved in essential mechanisms for the survival of C. parvum, such as recognition, adhesion to, and invasion of host cells. This work was aimed at expressing gp60 in Tetrahymena thermophila, a ciliated protozoon with numerous advantages for the heterologous expression of eukaryotic proteins, as a first approach for the development of a recombinant vaccine for bovine cryptosporidiosis. T. thermophila-expressed gp60 localized to the protozoon cell surface and oral apparatus, and partitioned into the Triton X-114 detergent phase. This indicates that the protein entered the reticuloendothelial system of the ciliate, and suggests it contains a GPI-anchor. Homogenates of gp60-expressing T. thermophila cells were recognized by sera from calves naturally infected with C. parvum demonstrating their immunoreactivity. In summary, the heterologous expression of gp60, a C. parvum-encoded GPI-anchored protein, has been successfully demonstrated in the ciliate T. thermophila.

Asunto(s)

Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Glicoproteínas/genética , Glicoproteínas/inmunología , Tetrahymena thermophila/genética , Animales , Bovinos , Enfermedades de los Bovinos/inmunología , Enfermedades de los Bovinos/prevención & control , Criptosporidiosis/inmunología , Criptosporidiosis/prevención & control , Cryptosporidium parvum/genética , Vacunas Sintéticas/sangre , Vacunas Sintéticas/genética