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Electron. j. biotechnol ; 52: 1-12, July. 2021. tab, ilus, graf
Article in English | LILACS | ID: biblio-1283167


BACKGROUND: Chronic lymphocytic leukaemia (CLL) is a neoplasm of B-cells characterized by variable prognosis. Exploring the proteome of CLL cells may provide insights into the disease. Therefore, eleven proteomics experiments were conducted on eleven primary CLL samples. RESULTS: We reported a CLL proteome consisting of 919 proteins (false discovery rate (FDR) 1%) whose identification was based on the sequencing of two or more distinct peptides (FDR of peptide sequencing 1%). Mass spectrometry-based protein identification was validated for four different proteins using Western blotting and specific antibodies in different CLL samples. Small sizes of nucleolin (~57 kDa and ~68 kDa) showed a potential association with good prognosis CLL cells (n = 8, p < 0.01). Compared with normal B-cells, CLL cells over-expressed thyroid hormone receptor-associated protein 3 (THRAP3; n = 9; p = 0.00007), which is implicated in cell proliferation; and heterochromatin protein 1-binding protein 3 (HP1BP3; n = 10; p = 0.0002), which promotes cell survival and tumourogenesis. A smaller form of HP1BP3, which may correspond to HP1BP3 isoform-2, was specifically identified in normal B-cells (n = 10; p = 0.0001). HP1BP3 and THRAP3 predicted poor prognosis of CLL (p 0.05). Consistently, THRAP3 and HP1BP3 were found to be associated with cancer-related pathways (p 0.05). CONCLUSIONS: Our findings add to the known proteome of CLL and confirm the prognostic importance of two novel cancer-associated proteins in this disease.

Leukemia, Lymphocytic, Chronic, B-Cell , Biomarkers, Tumor/analysis , Mass Spectrometry , Transcription Factors/analysis , Nuclear Proteins/analysis , Blotting, Western , Chromatography, Liquid , Proteomics , DNA-Binding Proteins/analysis
Electron. j. biotechnol ; 52: 59-66, July. 2021. ilus, tab
Article in English | LILACS | ID: biblio-1283592


BACKGROUND: Many human genetic diseases arise from point mutations. These genetic diseases can theoretically be corrected through gene therapy. However, gene therapy in clinical application is still far from mature. Nearly half of the pathogenic single-nucleotide polymorphisms (SNPs) are caused by G:C>A:T or T:A>C:G base changes and the ideal approaches to correct these mutations are base editing. These CRISPR-Cas9-mediated base editing does not leave any footprint in genome and does not require donor DNA sequences for homologous recombination. These base editing methods have been successfully applied to cultured mammalian cells with high precision and efficiency, but BE4 has not been confirmed in mice. Animal models are important for dissecting pathogenic mechanism of human genetic diseases and testing of base correction efficacy in vivo. Cytidine base editor BE4 is a newly developed version of cytidine base editing system that converts cytidine (C) to uridine (U). RESULTS: In this study, BE4 system was tested in cells to inactivate GFP gene and in mice to introduce single-base substitution that would lead to a stop codon in tyrosinase gene. High percentage albino coat-colored mice were obtained from black coat-colored donor zygotes after pronuclei microinjection. Sequencing results showed that expected base changes were obtained with high precision and efficiency (56.25%). There are no off-targeting events identified in predicted potential off-target sites. CONCLUSIONS: Results confirm BE4 system can work in vivo with high precision and efficacy, and has great potentials in clinic to repair human genetic mutations.

Animals , Mice , Adenosine Deaminase , Cytosine , CRISPR-Cas Systems , Gene Editing/methods , Base Sequence , Blotting, Western , Models, Animal , Real-Time Polymerase Chain Reaction , Mutation
Bol. latinoam. Caribe plantas med. aromát ; 20(3): 315-323, may. 2021. ilus, tab
Article in English | LILACS | ID: biblio-1343489


To investigate effectsof Yangyinyiqi Mixture on pulmonary fibrosis caused by bleomycin. SD ratswere divided randomly into: model group(distilled water,1 mL·0.1 kg-1), dexamethasone acetate group (dexamethasone acetate, the dosage was reduced gradually), low-dose group (Yangyinyiqi Mixture, 11 g·kg-1), moderate-dose group (Yangyinyiqi Mixture, 22 g·kg-1), high-dose group (Yangyinyiqi Mixture, 44 g·kg-1) and control group (distilled water, 1 mL·0.1 kg-1). Yangyinyiqi Mixture and dexamethasone acetate were intragastrically administrated. Lung tissue was collected for histopathological examination. Compared with control group, collagen markedly increased and HYP content significantly increased on 7th day in model group (p<0.01). On 28th day, collagen was diffusely deposited, alveolar was destroyed, and HYP content significantly increased (p<0.01). Compared with model group, bleomycin-induced suffering injury caused MMP-9 expression levels to rapidly increase (7and 14 days, p<0.01). TIMP-1 markedly increased (7and 14 days, p<0.01) and stayed at a high level to28th day. Yangyinyiqi Mixture exerted an effect against pulmonary fibrosis, which could involved prevention of collagen deposition through inhibitingMMP-9 and TIMP-1 expression.

El trabajo investiga los efectos de la mezcla Yangyinyiqi sobre la fibrosis pulmonary causada por bleomicina. Ratas SD se dividieron aleatoriamente en: grupo modelo (agua destilada, 1 mL·0.1 kg-1), grupo acetate de dexametasona (acetate de dexametasona, la dosis se redujo gradualmente), grupo de dosis baja (mezcla Yangyinyiqi, 11 g·kg-1), grupo de dosis moderada (mezcla Yangyinyiqi, 22 g·kg-1), grupo de dosis alta (mezcla Yangyinyiqi, 44 g·kg-1) y grupo control (agua destilada, 1 Ml·0.1 kg-1). La mezcla de Yangyinyiqi y el acetate de dexametasona se administraron por vía intragástrica. Se recolectó tejido pulmonary para examen histopatológico. En comparación con el grupo control, el colágeno aumentó notablemente y el contenido de HYP aumentó significativamente el séptimo día en el grupo modelo (p<0.01). El día 28, el colágeno se depositó difusamente, se produjo destrucción alveolar y el contenido de HYP aumento significativamente (p<0.01). En comparación con el grupo modelo, la lesión inducida por bleomicina causó que los niveles de expression de MMP-9 aumentaron rápidamente (7 y 14 días, p<0.01). TIMP-1 aumentó notablemente (7 y 14 días, p<0.01) y se mantuvo en un nivel alto hasta el día 28. La mezcla Yangyinyiqi ejerció un efecto contra la fibrosis pulmonary, lo que podría implicar la prevención del deposito de colágenio mediante la inhibición de la expression de MMP-9 y TIMP-1.

Animals , Male , Rats , Pulmonary Fibrosis/drug therapy , Drugs, Chinese Herbal/administration & dosage , Tissue Inhibitor of Metalloproteinases/metabolism , Matrix Metalloproteinase 9/metabolism , Bleomycin , Dexamethasone/administration & dosage , Blotting, Western , Rats, Sprague-Dawley , Matrix Metalloproteinase 1 , Disease Models, Animal , Hydroxyproline/analysis
Electron. j. biotechnol ; 51: 8-16, May. 2021. tab, graf, ilus
Article in English | LILACS | ID: biblio-1343314


BACKGROUND: Myogenic regulatory factors (MRFs) such as MyoD, Myf6 and Myf5 play a vital role in the growth and development of muscles. Jeju Native Pig (JNP) is the top ranker in Korea amongst the indigenous livestock reared for meat purpose. Few studies covering transcript abundance of the MRFs and related to their co-expression with Pax7 in JNP have been conducted. Despite having better quality pork, JNP does not have a comparative growth rate with respect to western breeds. Therefore, the present study was designed with the objective to study the relative transcript levels of MRFs in the postnatal myogenesis of longissimus dorsi muscles in JNP and Berkshire breeds. RESULTS: Relative transcript levels were analyzed by qRT-PCR and blot expression analysis through Western blotting. Immunocytochemistry was performed to analyze their expressions at cellular levels. ToppCluster aided in the analysis of gene ontology of biological processes. The quantitative transcript levels of MyoD and Pax7 were significantly (P < 0.05) higher in Berkshire than in JNP. Myotube formation was observed under the co-expression of MyoD and Pax7. ToppCluster helped in the understanding of the linking of biological processes of the MRFs with the different signaling pathways. MyBPH had significantly (P < 0.05) high transcript levels during the chosen age groups in JNP than Berkshire. CONCLUSIONS: The current study can be helpful in understanding the genetic basis for myogenesis in postnatal stage. Moreover, it can act as stepping stone for the identification of marker genes related to body growth and meat quality in JNP.

Animals , Swine , Myogenic Regulatory Factors/metabolism , Muscle Development/genetics , Immunohistochemistry , Genetic Markers , Blotting, Western , Myogenic Regulatory Factors/genetics , PAX7 Transcription Factor/metabolism , Real-Time Polymerase Chain Reaction , Gene Ontology , Pork Meat
Electron. j. biotechnol ; 51: 40-49, May. 2021. tab, ilus, graf
Article in English | LILACS | ID: biblio-1343322


BACKGROUND: Scavenger receptor class B (SRB) is a multifunctional protein in animals that participates in physiological processes, including recognition of a wide range of ligands. Astaxanthin is a major carotenoid found in shrimp. However, the molecular mechanism of astaxanthin and SRB protein binding has not been reported. RESULTS: In the present study, a member of the SRB subfamily, named PmSRB, was identified from the transcriptome of black tiger shrimp (Penaeus monodon). The open reading frame of PmSRB was 1557 bp in length and encoded 518 amino acids. The structure of PmSRB included a putative transmembrane structure at the N-terminal region and a CD36 domain. Multiple sequence alignment indicated that the CD36 domain were conserved. Phylogenetic analysis showed four separate branches (SRA, SRB, SRC, and croquemort) in the phylogenetic tree and that PmSRB was clustered with SRB of Eriocheir sinensis. Quantitative real-time polymerase chain reaction showed that the PmSRB gene was widely expressed in all tissues tested, with the highest expression level observed in the lymphoid organ and brain. Subcellular localization analysis revealed that PmSRB-GFP (green fluorescent protein) fusion proteins were predominantly localized in the cell membrane. The recombinant proteins of PmSRB showed binding activities against astaxanthin in vitro. CONCLUSIONS: PmSRB was identified and characterized in this study. It is firstly reported that PmSRB may take as an important mediator of astaxanthin uptake in shrimp.

Animals , Penaeidae , Receptors, Scavenger/metabolism , In Vitro Techniques , Blotting, Western , Chromatography, High Pressure Liquid , Sequence Alignment , Xanthophylls , Receptors, Scavenger/isolation & purification , Receptors, Scavenger/genetics , Real-Time Polymerase Chain Reaction/methods , Transcriptome
Vaccimonitor (La Habana, Print) ; 30(1)ene.-abr. 2021. graf
Article in Spanish | LILACS, CUMED | ID: biblio-1150250


La fiebre tifoidea causada por Salmonella Paratyphi A (fiebre paratifoidea) es indistinguible de la producida por Salmonella Typhi y el grado de incidencia ha aumentado en los últimos años, especialmente en el sudeste asiático. Por otro lado, la diarrea y otras complicaciones entéricas causadas por Salmonella Enteritidis y Salmonella Typhimurium continúan siendo un problema de salud grave, especialmente en países subdesarrollados. Las vacunas continúan siendo la forma más efectiva de prevenir estas enfermedades. Existen vacunas basadas en el polisacárido capsular de Salmonella Typhi que protegen contra la fiebre tifoidea; sin embargo, no hay vacunas efectivas licenciadas para uso en humanos que prevengan las enfermedades producidas por los serotipos de Salmonella no tifoideas. El desarrollo de una formulación con capacidad para proteger contra estas enfermedades sigue siendo un desafío para la comunidad científica. En este trabajo se evaluó, mediante Western blot, la reactividad de los sueros de ratones inmunizados por vía subcutánea con formulaciones basadas en vesículas de membrana externa derivadas de Salmonella Paratyphi A, Salmonella Enteritidis y Salmonella Typhimurium, contra los respectivos lisados celulares, para identificar la formulación que induce la mejor respuesta inmunológica cruzada. Los resultados obtenidos indicaron una alta reactividad de todos los sueros a los lisados, sin una diferencia aparente entre ellos. Sin lugar a dudas, se deberán realizar pruebas de inmunogenicidad seguidas de pruebas de retos cruzados para identificar un candidato vacunal. Estos resultados sugieren que las vesículas de membrana externa empleadas en este estudio están compuestas por antígenos posiblemente conservados en los tres serotipos de Salmonella y que pueden inducir una respuesta inmune de amplio espectro y protección cruzada(AU)

Typhoid fever caused by Salmonella Paratyphi A (paratyphoid fever) is indistinguishable from that caused by Salmonella Typhi and the degree of incidence has increased in recent years, especially in Southeast Asia. On the other hand, diarrhea and other enteric complications caused by Salmonella Enteritidis and Salmonella Typhimurium continue to be a serious health problem, especially in underdeveloped countries. Vaccines continue to be the most effective way to prevent these diseases. There are vaccines based on Salmonella Typhi capsular polysaccharide, which protects against typhoid fever; however, there are no effective vaccines licensed for use in humans to prevent disease caused by nontyphoidal Salmonella serotypes. Developing a formulation capable of protecting against these diseases remains a challenge for the scientific community. In this work, the reactivity of the sera of mice immunized subcutaneously with formulations based on Outer Membrane Vesicles (OMV) derived from Salmonella Paratyphi A, Salmonella Enteritidis and Salmonella Typhimurium, was evaluated by Western blot, against the respective cell lysates to identify the formulation that induces the best cross immune response. The results obtained indicated a high reactivity of all the sera to the lysates; without an apparent difference between them. Undoubtedly, immunogenicity tests followed by cross-challenge tests should be performed to identify a vaccine candidate. These results suggest that the OMV used in this study are composed of possibly conserved antigens in the three Salmonella serotypes and that they can induce a broad-spectrum immune response and cross protection(AU)

Mice , Salmonella paratyphi A , Typhoid Fever/transmission , Blotting, Western/methods , Vaccines
Electron. j. biotechnol ; 50: 16-22, Mar. 2021. ilus, tab
Article in English | LILACS | ID: biblio-1292419


BACKGROUND: Cecropin P1, acting as an antimicrobial, has a broad-spectrum antibacterial activity with some antiviral and antifungal properties. It is a promising natural alternative to antibiotics which is originally isolated from the pig intestinal parasitic nematode Ascaris suum. Many studies have shown that Cecropin P1 is helpful for the prevention or treatment of clinical diseases. Therefore, it is very necessary to establish a safe, nontoxic, and efficient expression method of Cecropin P1. RESULTS: The results indicated that the recombinant protein was about 5.5 kDa showed by Tricine­SDS­ PAGE and Western blot. And Cecropin P1 was efficiently secreted and expressed after 12 h of induction, with an increasing yield over the course of the induction. Its maximum concentration was 7.83 mg/L after concentration and purification. In addition, in vitro experiments demonstrated that Cecropin P1 not only exerted a strong inhibitory effect on Escherichia coli, Salmonella sp., Shigella sp., and Pasteurella sp., but also displayed an antiviral activity against PRRSV NADC30-Like strain. CONCLUSIONS: Collectively, the strategy of expressing Cecropin P1 in Saccharomyces cerevisiae is harmless, efficient, and safe for cells. In addition, the expressed Cecropin P1 has antiviral and antibacterial properties concurrently.

Peptides/pharmacology , Saccharomyces cerevisiae/drug effects , Anti-Bacterial Agents/pharmacology , Antiviral Agents/pharmacology , Peptides/chemistry , In Vitro Techniques , Recombinant Proteins , Microbial Sensitivity Tests , Blotting, Western
Univ. salud ; 23(1): 76-82, ene.-abr. 2021. tab, graf
Article in Spanish | LILACS, COLNAL | ID: biblio-1157012


Resumen Introducción: El virus de la Hepatitis E (HVE) es de ácido ribonucleico desnudo, los genotipos 3 y 4 pueden presentarse como una zoonosis transmitida por agua o alimentos contaminados. En la zona del eje cafetero-Colombia, no se ha descrito la presencia de anticuerpos para este virus en la comunidad. Objetivo: Determinar la prevalencia de anticuerpos anti-HVE de tipo Inmuniglobulinas G (IgG) en muestras de suero de un laboratorio clínico del Eje Cafetero. Materiales y métodos: En un periodo de dos meses se analizaron 90 sueros de pacientes atendidos en un laboratorio clínico de la ciudad de Armenia, se utilizaron tres técnicas diferentes para la caracterización de los anticuerpos y se compararon sus resultados. Resultados: De los 90 sueros evaluados, la técnica de ELISA de anticuerpos totales ELISA IgG anti HVE Recom Well marca Mikrogen identificó 2 sueros positivos (2,2%), la Prueba ELISA IgG HVE versión ULTRA® marca Diapro evidenció una muestra equivoca (1,1%). La prueba western blot Recom line HVE marca Mikrogen detectó 4 muestras positivas (4,4%). Conclusiones: Se encontró una prevalencia de anticuerpos HVE IgG que oscila entre 0 y 4,4% dependiendo de la prueba comercial utilizada, evidenciando circulación del virus y un posible ciclo infecciosos en la región.

Abstract Introduction: Hepatitis E virus (HEV) is a nonenveloped, RNA virus. HEV genotypes 3 and 4 are considered zoonosis transmitted by contaminated water and/or food. The presence of antibodies against this virus have not been described in communities inhabiting the "Coffee Axis" region of Colombia. Objective: To determine the prevalence of anti-Hepatitis E IgG in serum samples analyzed in a clinical laboratory from the Colombian Coffee Axis. Materials and methods: 90 serum samples from patients treated at a clinical laboratory in the city of Armenia (Quindio) were analyzed and compared through three different methods that characterize antibodies. Results: The Mikrogen recomWell ELISA kit (IgG anti-HEV) identified two positive sera (2.2%). The Diapro HEV IgG ELISA (version ULTRA®) test registered a false positive sample (1.1%). The Mikrogen recom Line HVE western blot assay detected 4 positive samples (4.4%). Conclusions: Depending on the commercial kit used, the prevalence of anti-HEV IgG antibodies fluctuated between 0% to 4.4%, which demonstrates that the virus is circulating and that a possible infectious cycle in this region exists.

Hepatitis E virus , Immunoglobulin G , Enzyme-Linked Immunosorbent Assay , Blotting, Western
Article in Chinese | WPRIM | ID: wpr-879849


OBJECTIVE@#To study the role and mechanism of histone deacetylase 1 (HDAC1) and histone deacetylase 2 (HDAC2) in mouse neuronal development.@*METHODS@#The mice with Synapsin1-Cre recombinase were bred with @*RESULTS@#The mice with @*CONCLUSIONS@#Deletion of

Animals , Blotting, Western , Histone Deacetylase 1/genetics , Histone Deacetylase 2 , Histone Deacetylases/genetics , Immunohistochemistry , Mice , Neurons/metabolism , Signal Transduction
Electron. j. biotechnol ; 47: 89-99, sept. 2020. ilus, tab, graf
Article in English | LILACS | ID: biblio-1253101


BACKGROUND: Koelreuteria henryi Dummer is an indigenous plant in Taiwan. The species has been used in traditional folk medicine for the promotion of liver functions and for treating malaria and urethritis. The present study investigated the antioxidant activity of the flower extract of Koelreuteria henryi Dummer. The extraction conditions were optimized by the contents of total phenolic acids and total flavonoids, and antioxidant activity assays. Moreover, an in vitro study for investigating antioxidant activity of K. henryi flower extract was demonstrated by hydrogen peroxide (H2O2)-induced apoptosis. RESULTS: K. henryi flower extracted for 150 min showed high contents of total phenolic acids and total flavonoids. In an in vitro model, L929 cells were pretreated with K. henryi flower extract, and then treated with H2O2 to induce oxidative damage. Results demonstrated that H2O2-induced apoptosis was inhibited by the treatment of 200 µg/ml K. henryi flower extract through the mitochondria-mediated pathway and mitogen-activated protein kinase (MAPK) pathway. The caspase 8/9 activity and expression of p-p38 and pERK were repressed by K. henryi flower extract. In addition, the prevention of H2O2-induced apoptosis by K. henryi flower extract activated the nuclear factor-erythroid 2-related factor (Nrf2) stress response pathway to transcript heme oxygenase 1 (HO-1). Also, K. henryi flower extract prevented H2O2-induced apoptosis through HO-1 production, as evident by the use of HO-1 inhibitor. CONCLUSIONS: The present study demonstrated that K. henryi flower extract could inhibit the H2O2-induced apoptosis in L929 cells through the activation of the Nrf2/HO-1 pathway.

Plant Extracts/pharmacology , Oxidative Stress/drug effects , Sapindaceae/chemistry , Antioxidants/pharmacology , Flavonoids/analysis , Blotting, Western , Apoptosis , Flowers/chemistry , Heme Oxygenase-1 , NF-E2-Related Factor 2 , Caspase 8 , Hydrogen Peroxide
Electron. j. biotechnol ; 47: 17-28, sept. 2020. ilus, graf
Article in English | LILACS | ID: biblio-1253006


BACKGROUND: Cichoric acid (CA) is extracted from Echinacea purpurea. It is well known and widely used for its immunological function. However, the effect of CA on peripheral blood mononuclear cells (PBMCs) from yaks is still unclear. This study investigated the potential influences of CA on the proliferation, cytokine induction, and apoptosis of PBMCs from Datong yak in vivo, and aimed to provide a basis for exploring the pharmacological activities of CA on yaks. RESULTS: In this study, CA promoted PBMCs proliferation by combining concanavalin A (Con A) and exhibited a dose-dependent effect as demonstrated by a Cell Counting Kit-8. The concentration of 60 µg/ml CA was the best and promoted the transformation from the G0/G1 phase to the S and G2/M phases with Con A. Furthermore, 60 µg/ml CA significantly increased IL-2, IL-6, and IFN-γ levels and PCNA, CDK4 and Bcl-2 expression levels, but it significantly inhibited the TP53, Bax, and Caspase-3 expression levels. Transcriptome analysis revealed a total of 6807 differentially expressed genes (DEGs) between the CA treatment and control groups. Of these genes, 3788 were significantly upregulated and 3019 were downregulated. Gene Ontology and pathway analysis revealed that DEGs were enriched in cell proliferation and immune function signaling pathways. The expression level of some transcription factors (BTB, Ras, RRM_1, and zf-C2H2) and genes (CCNF, CCND1, and CDK4) related to PBMCs proliferation in yaks were significantly promoted after CA treatment. By contrast, anti-proliferation-associated genes (TP53 and CDKN1A) were inhibited. CONCLUSIONS: In summary, CA could regulate the immune function of yaks by promoting proliferation and inhibiting inflammation and apoptosis of PBMCs.

Animals , Cattle , Succinates/pharmacology , Caffeic Acids/pharmacology , Leukocytes, Mononuclear/drug effects , Echinacea/chemistry , Cell Proliferation/drug effects , Transcription Factors , Enzyme-Linked Immunosorbent Assay , Leukocytes, Mononuclear/cytology , Blotting, Western , Cytokines , Apoptosis/drug effects , Concanavalin A/pharmacology , Real-Time Polymerase Chain Reaction , RNA-Seq
Int. j. morphol ; 38(4): 919-923, Aug. 2020. tab, graf
Article in English | LILACS | ID: biblio-1124877


Letrozole (Letro) is a drug commonly used for breast cancer treatment since it can decrease estrogen level. In experimental animal, the Letro has been used to induce the polycystic ovarian syndrome (PCOS) model. Tyrosine phosphorylation (TyrPho) is an essential process in various biological functions both normal and abnormal conditions especially reproduction. Although some side effects of Letro are reported, the alterations of TyrPho responsible for liver and kidney functions have never been demonstrated. In this study, the blood serum, liver, and kidney of control and PCOS rats induced with Letro (orally, 1 mg/ KgBW) for consecutive 21 days were used to determine the serum biochemical components and to investigate the TyrPho expression using western blot analysis. Histopathology of such tissues was observed by Masson's trichrome staining. The results showed that Letro did not affect histological structures but significantly increased the serum levels of urea nitrogen, cholesterol, triglyceride, HDL, LDL, ALT, AST, and alkaline phosphatase. Additionally, the TyrPho protein expressions of 32 and 27 kDas in liver and of 55 and 43 kDas in kidney were increased while of a kidney 26 kDa was decreased as compared to those of control. In conclusion, this recent study indicated that the changes of TyrPho proteins in liver and kidney induced with Letro associated with their functions by alteration of serum biochemical levels.

El letrozol (Letro) es un medicamento utilizado comúnmente para el tratamiento del cáncer de mama, debido a que puede disminuir el nivel de estrógeno. En animales de experimentación, el Letro se ha utilizado para inducir el modelo de síndrome de ovario poliquístico (PCOS). La fosforilación de tirosina (TyrPho) es un proceso esencial en diversas funciones biológicas, tanto en condiciones normales como anormales, especialmente en la reproducción. A pesar de informes que indican algunos efectos secundarios de Letro, no se han demostrado las alteraciones de TyrPho responsables de las funciones hepáticas y renales. En este estudio, el suero sanguíneo, el hígado y el riñón control y las ratas PCOS inducidas con Letro (por vía oral, 1 mg / KgBW) durante 21 días consecutivos se usaron para determinar los componentes bioquímicos del suero y para investigar la expresión de TyrPho usando análisis de transferencia Western. La histopatología de los tejidos se observó mediante la tinción tricrómica de Masson. Los resultados mostraron que Letro no afectó las estructuras histológicas, pero aumentó significativamente los niveles séricos de urea, colesterol, triglicéridos, HDL, LDL, ALT, AST y fosfatasa alcalina. Además, las expresiones de la proteína TyrPho de 32 y 27 kDas en el hígado y de 55 y 43 kDas en el riñón aumentaron mientras que en un riñón disminuyeron 26 kDa en comparación con el control. En conclusión, este estudio indicó que los cambios de las proteínas TyrPho en el hígado y los riñones inducidos con Letro se asociaron con sus funciones mediante la alteración de los niveles bioquímicos en suero.

Animals , Female , Rats , Polycystic Ovary Syndrome/chemically induced , Letrozole/adverse effects , Kidney/drug effects , Liver/drug effects , Phosphorylation/physiology , Tyrosine/metabolism , Blotting, Western , Rats, Wistar , Disease Models, Animal , Electrophoresis, Polyacrylamide Gel
Int. j. morphol ; 38(2): 400-405, abr. 2020. graf
Article in English | LILACS | ID: biblio-1056454


Accumulating evidence from preclinical and clinical studies indicates prenatal exposure to stress or excess glucocorticoids can affect offspring brain. Glucocorticoid receptor (GR) is an important target of glucocorticoid. Therefore the aim of the present study was to investigate the expression of GR in prenatally stressed adult offspring and the relationship between GR expression and behavior in offspring. Pregnant rats received restraint stress during the last week of pregnancy. Hippocampal glucocorticoid receptor expression levels in the offspring were detected on postnatal 60 (P60).Cognition function was also detected. It shows significantly lower hippocampal GR expression was observed in female prenatally stressed offspring compared with their controls at P60. Corresponding to the expression of GR, female prenatally stressed offspring exhibited poorer spatial learning and memory abilities in the Barnes maze than control, This suggests that cognitive impairment in prenatally stressed rat offspring attribute lower hippocampal GR expression.

La evidencia acumulada de estudios preclínicos y clínicos indica que la exposición prenatal al estrés, o el exceso de glucocorticoides puede afectar el desarrollo cerebral de las crías. El receptor de glucocorticoides (RG) es un objetivo importante de los glucocorticoides. Por lo tanto, el objetivo del presente estudio fue investigar la expresión de RG en crías adultas estresadas durante el período prenatal y la relación entre la expresión de RG y el comportamiento de las crías. Las ratas preñadas recibieron niveles de estrés restringido, durante la última semana de embarazo. Se determinaron niveles de expresión del receptor de glucocorticoides del hipocampo y niveles de función cognitiva en las crías. En comparación con el grupo control se observó una expresión de RG en el hipocampo, significativamente menor en las crías estresadas prenatalmente, en comparación con los controles en P60. En referencia a la expresión de RG, las crías estresadas prenatalmente exhibieron habilidades de memoria y aprendizaje espacial menores, en el laberinto de Barnes que el grupo control. Esto sugiere que el deterioro cognitivo en crías de ratas estresadas prenatalmente muestran una menor expresión de RG en el hipocampo.

Animals , Female , Pregnancy , Rats , Prenatal Exposure Delayed Effects , Receptors, Glucocorticoid/metabolism , Cognitive Dysfunction , Hippocampus/metabolism , Stress, Physiological , Immunohistochemistry , Blotting, Western , Rats, Sprague-Dawley
Arq. neuropsiquiatr ; 78(1): 21-27, Jan. 2020. graf
Article in English | LILACS | ID: biblio-1088987


Abstract Objective: The phytohormone abscisic acid (ABA) as a signaling molecule exists in various types of organisms from early multicellular to animal cells and tissues. It has been demonstrated that ABA has an antinociceptive effect in rodents. The present study was designed to assess the possible role of PKA and phosphorylated ERK (p-ERK) on the antinociceptive effects of intrathecal (i.t.) ABA in male Wistar rats. Methods: The animals were cannulated intrathecally and divided into different experimental groups (n=6‒7): Control (no surgery), vehicle (received ABA vehicle), ABA-treated groups (received ABA in doses of 10 or 20 µg/rat), ABA plus H.89 (PKA inhibitor)-treated group which received the inhibitor 15 min prior to the ABA injection. Tail-flick and hot-plate tests were used as acute nociceptive stimulators to assess ABA analgesic effects. p-ERK was evaluated in the dorsal portion of the spinal cord using immunoblotting. Results: Data showed that a microinjection of ABA (10 and 20 µg/rat, i.t.) significantly increased the nociceptive threshold in tail flick and hot plate tests. The application of PKA inhibitor (H.89, 100 nM/rat) significantly inhibited ABA-induced analgesic effects. Expression of p-ERK was significantly decreased in ABA-injected animals, which were not observed in the ABA+H.89-treated group. Conclusions: Overall, i.t. administration of ABA (10 µg/rat) induced analgesia and p-ERK down-expression likely by involving the PKA-dependent mechanism.

Resumo Objetivo: O ácido fito-hormônio abscísico (ABA) existe como molécula sinalizadora em vários tipos de organismos, de multicelulares a células e tecidos animais. Foi demonstrado que o ABA tem efeito antinociceptivo em roedores. O presente estudo foi desenhado para avaliar o possível papel da PKA e da ERK fosforilada (p-ERK) nos efeitos antinociceptivos do ABA intratecal (i.t.) em ratos Wistar machos. Métodos: Os animais foram canulados por via i.t. e divididos em diferentes grupos experimentais (n=6‒7): controle (sem cirurgia), veículo (veículo ABA recebido), grupos tratados com ABA (recebeu ABA em doses de 10 ou 20 µg/rato), grupo tratado com ABA mais H.89 (inibidor de PKA) que recebeu o inibidor 15 minutos antes da injeção de ABA. Os testes de movimento da cauda e placa quente foram utilizados como estimuladores nociceptivos agudos para avaliar os efeitos analgésicos da ABA. A p-ERK foi avaliada na porção dorsal da medula espinhal por imunotransferência. Resultados: A microinjeção de ABA (10 e 20 µg/rato, i.t.) aumentou significativamente o limiar nociceptivo nos testes de movimento da cauda e placa quente. A aplicação de inibidor de PKA (H.89, 100 nM/rato) inibiu significativamente os efeitos analgésicos induzidos por ABA. A expressão de p-ERK diminuiu significativamente em animais injetados com ABA que não foram observados no grupo tratado com ABA+H.89. Conclusões: No geral, a administração i.t. de ABA (10 µg/rato) induziu a analgesia e expressão negativa de p-ERK provavelmente envolvendo mecanismo dependente de PKA.

Animals , Male , Plant Growth Regulators/pharmacology , Spinal Cord/metabolism , Abscisic Acid/pharmacology , Cyclic AMP-Dependent Protein Kinases/drug effects , Extracellular Signal-Regulated MAP Kinases/drug effects , Analgesics/pharmacology , Reference Values , Spinal Cord/drug effects , Time Factors , Blotting, Western , Reproducibility of Results , Rats, Wistar , Cyclic AMP-Dependent Protein Kinases/analysis , Extracellular Signal-Regulated MAP Kinases/analysis , Intracellular Signaling Peptides and Proteins/pharmacology
Rev. Assoc. Med. Bras. (1992) ; 66(1): 42-47, Jan. 2020. graf
Article in English | LILACS | ID: biblio-1091906


SUMMARY OBJECTIVE ADAMTS4 is a member of the ADAMTS4 family, which secretes proteinases. The mechanism of tumor metastasis may be correlated to its promotion of angiogenesis. It was determined whether ADAMTS4 participates in colorectal cancer progression. Methods The expression in clinical samples and CRC cell lines was investigated. Using immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and RT-PCR, the expression of ADAMTS4 was determined in colorectal tumors of different cancer stages and anatomic sites, and in three cell lines of different aggressiveness. Results The overexpression of ADAMTS4 was observed in tissue samples by IHC, and this was mainly located in the cytoplasm, as detected by FISH. The qRT-PCR and western blot analyses further supported the clinical sample findings. Conclusion The present data support the notion that the overexpression of ADAMTS4 in CRC might be useful as a non-invasive biomarker for detecting CRC in patients.

RESUMO OBJETIVO ADAMTS4 é um membro da família ADAMTS4, que secreta proteinases. O mecanismo da metástase do tumor pode ser correlacionado a sua promoção da angiogênese. Determinou-se se ADAMTS4 participa na progressão do câncer colorretal. Métodos A expressão em amostras clínicas e linhas de células CRC foi investigada. Usando a imuno-histoquímica (IHC), a hibridação fluorescente in situ (HFIS) e o RT-PCR, a expressão de ADAMTS4 foi determinada em tumores colorretais de diferentes estágios do câncer e locais anatômicos, e em três linhas de células de níveis de agressividade distintos. Resultados A superexpressão de ADAMTS4 foi observada em amostras de tecido por IHC, e esta foi localizada principalmente no citoplasma, como detectado pelo HFIS. O qRT-PCR e a análise de wester blot corroboraram os resultados clínicos da amostra. Conclusão Os dados atuais corroboram a noção de que a superexpressão de ADAMTS4 no CRC pode ser útil como um biomarcador não invasivo para a detecção de CRC em pacientes.

Humans , Male , Female , Aged , Colorectal Neoplasms/pathology , ADAMTS4 Protein/analysis , Prognosis , Reference Values , RNA, Messenger/analysis , Immunohistochemistry , Colorectal Neoplasms/genetics , Biomarkers, Tumor , Gene Expression Regulation, Neoplastic , Up-Regulation , Blotting, Western , Analysis of Variance , In Situ Hybridization, Fluorescence , Disease Progression , Cell Line, Tumor , Middle Aged
Braz. j. med. biol. res ; 53(1): e8883, Jan. 2020. tab, graf
Article in English | LILACS | ID: biblio-1055486


Opa-interacting protein 5 antisense transcript 1 (OIP5-AS1) is one kind of cytoplasmic long non-coding RNA (lncRNA), which has been demonstrated to play a critical function in multiple cancers. However, the detailed mechanism of OIP5-AS1 in the regulation of cervical cancer progression is still obscure. Here, we demonstrated that lncRNA OIP5-AS1 was upregulated in cervical cancer and was correlated with poor prognosis by bioinformatics studies. OIP5-AS1 depletion inhibited cell proliferation and promoted cell apoptosis in cervical cancer cells. Furthermore, we clarified that ROCK1 was the downstream effector of OIP5-AS1 and OIP5-AS1 acted as a molecular sponge of miR-143-3p. Finally, we verified that OIP5-AS1 exerted its function in the regulation of cervical cancer progression via interacting with miR-143-3p to regulate ROCK1 expression. Our study revealed novel mechanisms about how lncRNA OIP5-AS1 executed its function in cervical cancer and thus provided potential therapeutic targets for the disease.

Humans , Female , Uterine Cervical Neoplasms/pathology , Apoptosis/physiology , MicroRNAs/metabolism , Cell Proliferation/physiology , rho-Associated Kinases/metabolism , RNA, Long Noncoding/metabolism , Gene Expression Regulation, Neoplastic , Up-Regulation , Uterine Cervical Neoplasms/metabolism , Blotting, Western , Apoptosis/genetics , Reverse Transcriptase Polymerase Chain Reaction , MicroRNAs/genetics , Cell Line, Tumor , Cell Proliferation/genetics , rho-Associated Kinases/genetics , RNA, Long Noncoding/genetics
Article in English | LILACS, SES-SP, SESSP-IALPROD, SES-SP | ID: biblio-1121320


Difficulties in confirming and discriminating human T-cell lymphotropic virus type 1 (HTLV-1) and HTLV-2 infections by serological Western blot (WB) assays (HTLV Blot 2.4; MP Biomedicals) have been reported in Brazil, mainly in HIV/AIDS patients, with a large number of WB-indeterminate and WB-positive but HTLV-untypeable results. Nonetheless, a line immunoassay (LIA) (INNO-LIA HTLV-I/II; Fujirebio) provided enhanced specificity and sensitivity for confirming HTLV-1/2 infections. To add information concerning the improved ability of the LIA in relation to WB when applied to samples of individuals from different risk groups from Brazil, we performed the present study. Three groups were analyzed group 1 (G1), with 62 samples from HIV/AIDS patients from São Paulo, SP (48 WB indeterminate and 14 HTLV untypeable); group 2 (G2), with 24 samples from patients with hepatitis B or hepatitis C from São Paulo (21 WB indeterminate and 3 HTLV untypeable; 17 HIV seropositive); and group 3 (G3), with 25 samples from an HTLV outpatient clinic in Salvador, Bahia (16 WB indeterminate and 9 HTLV untypeable; all HIV seronegative). Overall, the LIA confirmed HTLV-1/2 infection (HTLV-1, HTLV-2, or HTLV) in 66.1% (G1), 83.3% (G2), and 76.0% (G3) of samples. Interestingly, the majority of WB-indeterminate results were confirmed by the LIA as being HTLV-2 positive in G1 and G2 but not in G3, in which the samples were defined as being HTLV-1 or HTLV positive. These results agree with the virus types that circulate in such patients of different regions in Brazil and emphasize that the LIA is the bes

Humans , Male , Female , Adolescent , Adult , Middle Aged , Aged , Aged, 80 and over , Young Adult , HTLV-I Infections/diagnosis , HTLV-II Infections/diagnosis , Hepatitis C , AIDS-Related Opportunistic Infections/diagnosis , Hepatitis B , Immunoassay , Blotting, Western , Sensitivity and Specificity , Coinfection
Article in English | WPRIM | ID: wpr-828988


Objective@#The definite diagnosis of human and animal prion diseases depends on the examination of special pathological changes and/or detection of PrP in the brain tissues of suspected cases. Thus, developing methods to obtain PrP antibody with good specificity and sensitivity is fundamental for prion identification.@*Methods@#We prepared a PrP-specific polyclonal antibody (pAb P54) in a -knockout mouse model immunization with recombinant full-length human PrP protein residues 23-231. Thereafter, we verified that pAb in Western blot, immunohistochemistry (IHC), and immunofluorescent (IFA) assays.@*Results@#Western blot illustrated that the newly prepared pAb P54 could react with recombinant PrP protein, normal brain PrP from healthy rodents and humans, and pathological PrP in the brains of experimental rodents infected with scrapie and humans infected with different types of prion diseases. The electrophoretic patterns of brain PrP and PrP observed after their reaction with pAb P54 were nearly identical to those produced by commercial PrP monoclonal antibodies. Three glycosylated PrP molecules in the brain homogenates were clearly demonstrated in the reactions of these molecules with pAb P54. IHC assay revealed apparent PrP deposits in the GdnCl-treated brain slices of 139A-infected mice and 263K-infected hamsters. IFA tests with pAb P54 also showed clear green signals surrounding blue-stained cell nuclei.@*Conclusion@#The newly prepared pAb P54 demonstrated reliable specificity and sensitivity and, thus, may have potential applications not only in studies of prion biology but also in the diagnosis of human and experimental rodent prion diseases.

Animals , Antibodies , Allergy and Immunology , Blotting, Western , Fluorescent Antibody Technique , Immunization , Immunohistochemistry , Mice , Mice, Knockout , PrPC Proteins , Allergy and Immunology , PrPSc Proteins , Allergy and Immunology , Prion Proteins , Allergy and Immunology , Recombinant Proteins , Allergy and Immunology
Article in Chinese | WPRIM | ID: wpr-880820


OBJECTIVE@#To explore the protective effect of SBi4211 (heptamidine), an inhibitor of S100B, against central nervous system injury induced by HIV-1 envelope protein gp120.@*METHODS@#In an @*RESULTS@#In the cell co-culture system, SBi4211 treatment significantly inhibited gp120-induced expression of S100B, RAGE and GFAP in U251 cells (@*CONCLUSIONS@#SBi4211 can protect neurons from gp120-induced neurotoxicity possibly by inhibiting the S100B/ RAGE-mediated signaling pathway.

Animals , Astrocytes , Blotting, Western , Central Nervous System , HIV Envelope Protein gp120 , Mice , Neurons , S100 Calcium Binding Protein beta Subunit , Signal Transduction
Chinese Journal of Biotechnology ; (12): 2083-2091, 2020.
Article in Chinese | WPRIM | ID: wpr-878468


Rabbit haemorrhagic disease virus (RHDV) and myxoma virus (MYXV), are two pathogens that have harmful effect on rabbit breeding and population decline of European rabbits in their native range, causing rabbit haemorrhagic disease (rabbit fever) and myxomatosis, respectively. The capsid protein VP60 of the RHDV represents the major antigenic protein. To develop a recombinant bivalent vaccine candidate that can simultaneously prevent these two diseases, we used the nonessential gene TK (thymidine kinase) of MYXV as the insertion site to construct a recombinant shuttle vector p7.5-VP60-GFP expressing the RHDV major capsid protein (VP60) and the selectable marker GFP. Then the shuttle vector p7.5-VP60-GFP was transfected into rabbit kidney cell line RK13 which was previously infected with MYXV. After homologous recombination, the recombinant virus expressing GFP was screened under a fluorescence microscope and named as rMV-VP60-GFP. Finally, the specific gene-knock in and expression verification of the vp60 and gfp genes of the recombinant virus was confirmed by PCR and Western blotting. The results showed that these two genes were readily knocked into the MYXV genome and also successfully expressed, indicating that the recombinant MYXV expressing the vp60 of RHDV was generated. Protection against MYXV challenge showed that the recombinant virus induced detectable antibodies against MYXV which would shed light on development of the effective vaccine.

Animals , Blotting, Western , Caliciviridae Infections/veterinary , Hemorrhagic Disease Virus, Rabbit/immunology , Rabbits , Vaccines, Synthetic/immunology , Viral Structural Proteins/genetics