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Multivalent live-attenuated or inactivated vaccines are often used to control the bovine viral diarrhea disease (BVD). Still, they retain inherent disadvantages and do not provide the expected protection. This study developed a new vaccine prototype, including the external segment of the E2 viral protein from five different subgenotypes selected after a massive screening. The E2 proteins of every subgenotype (1aE2, 1bE2, 1cE2, 1dE2, and 1eE2) were produced in mammalian cells and purified by IMAC. An equimolar mixture of E2 proteins formulated in an oil-in-water adjuvant made up the vaccine candidate, inducing a high humoral response at 50, 100, and 150 µg doses in sheep. A similar immune response was observed in bovines at 50 µg. The cellular response showed a significant increase in the transcript levels of relevant Th1 cytokines, while those corresponding to the Th2 cytokine IL-4 and the negative control were similar. High levels of neutralizing antibodies against the subgenotype BVDV1a demonstrated the effectiveness of our vaccine candidate, similar to that observed in the sera of animals vaccinated with the commercial vaccine. These results suggest that our vaccine prototype could become an effective recombinant vaccine against the BVD.
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Anticuerpos Antivirales , Diarrea Mucosa Bovina Viral , Vacunas de Subunidad , Vacunas Sintéticas , Vacunas Virales , Animales , Bovinos , Vacunas Virales/inmunología , Vacunas de Subunidad/inmunología , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Vacunas Sintéticas/inmunología , Diarrea Mucosa Bovina Viral/prevención & control , Diarrea Mucosa Bovina Viral/inmunología , Diarrea Mucosa Bovina Viral/virología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/sangre , Ovinos , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/genética , Citocinas/metabolismo , Virus de la Diarrea Viral Bovina/inmunología , Virus de la Diarrea Viral Bovina/genética , Virus de la Diarrea Viral Bovina Tipo 1/inmunología , Virus de la Diarrea Viral Bovina Tipo 1/genéticaRESUMEN
Bovine viral diarrhea virus (BVDV) affects cattle worldwide causing severe productive and economic loss. In this study, we investigated the subgenotypes of BVDV circulating in cattle samples from the Aysén region, an active cattle breeding area located in southern Chile. Partial amplification of the 5' untranslated region (UTR) was performed by polymerase chain reaction (PCR), and twelve samples were analyzed by Sanger sequencing and phylogenetic analysis. Eight samples were identified as belonging to Pestivirus bovis subgenotype 1e, three to 1-b, and one to 1-d. The phylogenetic analyses performed revealed a marked distance between these now-identified strains and those previously reported in the country. These findings support the need to continually expand the analysis of the variability of the viral phylogeny for the currently circulating BVDV strains and to update the vaccines recommended for this livestock area and surrounding areas.
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Virus de la Diarrea Viral Bovina , Animales , Bovinos , Chile/epidemiología , Filogenia , Virus de la Diarrea Viral Bovina/genética , Regiones no Traducidas 5' , DiarreaRESUMEN
BACKGROUND & AIMS: Metabolic syndrome (MetS) is characterized by obesity, glucose intolerance, and hepatic steatosis. Alterations in the gut microbiome play important roles in the development of MetS. However, the mechanisms by which this occurs are poorly understood. Dual oxidase 2 (DUOX2) is an antimicrobial reduced nicotinamide adenine dinucleotide phosphate oxidase expressed in the gut epithelium. Here, we posit that epithelial DUOX2 activity provides a mechanistic link between the gut microbiome and the development of MetS. METHODS: Mice carrying an intestinal epithelial-specific deletion of dual oxidase maturation factor 1/2 (DA IEC-KO), and wild-type littermates were fed a standard diet and killed at 24 weeks. Metabolic alterations were determined by glucose tolerance, lipid tests, and body and organ weight measurements. DUOX2 activity was determined by Amplex Red. Intestinal permeability was determined by fluorescein isothiocyanate-dextran, microbial translocation assessments, and portal vein lipopolysaccharide measurements. Metagenomic analysis of the stool microbiome was performed. The role of the microbiome was assessed in antibiotic-treated mice. RESULTS: DA IEC-KO males showed increased body and organ weights accompanied by glucose intolerance and increased plasma lipid and liver enzyme levels, and increased adiposity in the liver and adipose tissue. Expression of F4/80, CD68, uncoupling protein 1, carbohydrate response element binding protein, leptin, and adiponectin was altered in the liver and adipose tissue of DA IEC-KO males. DA IEC-KO males produced less epithelial H2O2, had altered relative abundance of Akkermansiaceae and Lachnospiraceae in stool, and showed increased portal vein lipopolysaccharides and intestinal permeability. Females were protected from barrier defects and MetS, despite producing less H2O2. Antibiotic depletion abrogated all MetS phenotypes observed. CONCLUSIONS: Intestinal epithelial inactivity of DUOX2 promotes MetS in a microbiome-dependent manner.
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Microbioma Gastrointestinal , Intolerancia a la Glucosa , Síndrome Metabólico , Animales , Femenino , Masculino , Ratones , Antibacterianos , Oxidasas Duales , Peróxido de Hidrógeno , Lipopolisacáridos , Obesidad/metabolismoRESUMEN
There are emerging concerns about the potential cerebral cortex injury from aspartame due to the accumulation of the various neurotoxic metabolic components in the central nervous system after long-term dietary exposure. The aim of this study was to evaluate the effect of oral aspartame consumption on cerebral cortex injury in the rat brain, and further evaluate the various underlying molecular mechanisms, with a special focus on oxidative stress, inflammation, mitochondrial dysfunction, and apoptosis pathways. Sprague Dawley rats (nineteen, female) were randomly sub-divided into three groups: (i) normal diet with vehicle: control group (five rats), (ii) low dose of aspartame group (LA): seven rats received 30 mg/kg body weight (bw) daily doses of aspartame, (iii) high dose of aspartame group (HA): seven rats received 60 mg/kg bw daily doses of aspartame. After 8 weeks, the LA and HA groups showed lower expression levels of brain-derived neurotrophic factor (BDNF), antioxidant enzyme activity (SOD2, CAT), antioxidant marker (Nrf2), inflammatory response (IκB), mitochondrial biogenesis (Sirt1, PGC1α, Nrf1, TFAM), mitochondrial DNA (mtDNA) copy number, and apoptosis-related proteins (Bax, Caspase-3) expressions. Aspartame administration also elevated oxidative stress levels (Malondialdehyde, MDA), 8-hydroxy-2-deoxy guanosine (8-OHdG), PGE2 and COX-2 expressions, pro-inflammatory cytokines (TNFα, IL6, IL1ß), antioxidant marker expression (Keap1), inflammatory responses (iNOS, NFκB), and glial fibrillary acidic protein (GFAP) levels in the cerebral cortex of the rats, thereby contributing to the reduced survival of pyramidal cells and astrocyte glial cells of the cerebral cortex. Therefore, these findings imply that aspartame-induced neurotoxicity in rats' cerebral cortex could be regulated through four mechanisms: inflammation, enhanced oxidant stress, decreased mitochondrial biogenesis, and apoptosis pathways.
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Bacterial DNA gyrase, a type IIA DNA topoisomerase that plays an essential role in bacterial DNA replication and transcription, is a clinically validated target for discovering and developing new antibiotics. In this article, based on a supercoiling-dependent fluorescence quenching (SDFQ) method, we developed a high-throughput screening (HTS) assay to identify inhibitors targeting bacterial DNA gyrase and screened the National Institutes of Health's Molecular Libraries Small Molecule Repository library containing 370,620 compounds in which 2891 potential gyrase inhibitors have been identified. According to these screening results, we acquired 235 compounds to analyze their inhibition activities against bacterial DNA gyrase using gel- and SDFQ-based DNA gyrase inhibition assays and discovered 155 new bacterial DNA gyrase inhibitors with a wide structural diversity. Several of them have potent antibacterial activities. These newly discovered gyrase inhibitors include several DNA gyrase poisons that stabilize the gyrase-DNA cleavage complexes and provide new chemical scaffolds for the design and synthesis of bacterial DNA gyrase inhibitors that may be used to combat multidrug-resistant bacterial pathogens. Additionally, this HTS assay can be applied to screen inhibitors against other DNA topoisomerases.
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Bacterial DNA gyrase, an essential enzyme, is a validated target for discovering and developing new antibiotics. Here we screened a pool of polyphenols and discovered that digallic acid is a potent DNA gyrase inhibitor. We also found that several food additives based on gallate, such as dodecyl gallate, potently inhibit bacterial DNA gyrase. Interestingly, the IC50 of these gallate derivatives against DNA gyrase is correlated with the length of hydrocarbon chain connecting to the gallate. These new bacterial DNA gyrase inhibitors are ATP competitive inhibitors of DNA gyrase. Our results also show that digallic acid and certain gallate derivatives potently inhibit E. coli DNA topoisomerase IV. Several gallate derivatives have strong antimicrobial activities against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA). This study provides a solid foundation for the design and synthesis of gallate-based DNA gyrase inhibitors that may be used to combat antibacterial resistance.
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Girasa de ADN , Staphylococcus aureus Resistente a Meticilina , ADN Bacteriano , Inhibidores de Topoisomerasa II/farmacología , Escherichia coliRESUMEN
(1) Background: The psychoactive and non-psychoactive constituents of cannabis, Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), synergistically reduce allodynia in various animal models of neuropathic pain. Unfortunately, THC-containing drugs also produce substantial side-effects when administered systemically. We examined the effectiveness of targeted spinal delivery of these cannabis constituents, alone and in combination. (2) Methods: The effect of acute intrathecal drug delivery on allodynia and common cannabinoid-like side-effects was examined in a mouse chronic constriction injury (CCI) model of neuropathic pain. (3) Results: intrathecal THC and CBD produced dose-dependent reductions in mechanical and cold allodynia. In a 1:1 combination, they synergistically reduced mechanical and cold allodynia, with a two-fold increase in potency compared to their predicted additive effect. Neither THC, CBD nor combination THC:CBD produced any cannabis-like side-effects at equivalent doses. The anti-allodynic effects of THC were abolished and partly reduced by cannabinoid CB1 and CB2 receptor antagonists AM281 and AM630, respectively. The anti-allodynic effects of CBD were partly reduced by AM630. (4) Conclusions: these findings indicate that intrathecal THC and CBD, individually and in combination, could provide a safe and effective treatment for nerve injury induced neuropathic pain.
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Cannabidiol , Cannabinoides , Cannabis , Alucinógenos , Neuralgia , Analgésicos/efectos adversos , Animales , Cannabidiol/efectos adversos , Agonistas de Receptores de Cannabinoides/farmacología , Cannabinoides/efectos adversos , Modelos Animales de Enfermedad , Dronabinol/efectos adversos , Alucinógenos/efectos adversos , Hiperalgesia/inducido químicamente , Hiperalgesia/tratamiento farmacológico , Ratones , Neuralgia/tratamiento farmacológicoRESUMEN
Cereals play an important role in global food security. Data from the UN Food and Agriculture Organization projects increased consumption of cereals from 2.6 billion tonnes in 2017 to approximately 2.9 billion tonnes by 2027. However, cereals are prone to contamination by toxigenic fungi, which lead to mycotoxicosis. The current methods for mycotoxin control involve the use of chemical preservatives. However, there are concerns about the use of chemicals in food preservation due to their effects on the health, nutritional quality, and organoleptic properties of food. Therefore, alternative methods are needed that are affordable and simple to use. The fermentation technique is based on the use of microorganisms mainly to impart desirable sensory properties and shelf-life extension. The lactic acid bacteria (LAB) are generally regarded as safe (GRAS) due to their long history of application in food fermentation systems and ability to produce antimicrobial compounds (hydroxyl fatty acids, organic acids, phenyllactic acid, hydrogen peroxide, bacteriocins, and carbon dioxide) with a broad range of antifungal activity. Hence, LAB can inhibit the growth of mycotoxin-producing fungi, thereby preventing the production of mycotoxins. Fermentation is also an efficient technique for improving nutrient bioavailability and other functional properties of cereal-based products. This review seeks to provide evidence of the potential of LAB from African fermented cereal-based products as potential biological agents against mycotoxin-producing fungi.
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BACKGROUND & AIMS: Chronic colonic inflammation leads to dysplasia and cancer in patients with inflammatory bowel disease. We have described the critical role of innate immune signaling via Toll-like receptor 4 (TLR4) in the pathogenesis of dysplasia and cancer. In the current study, we interrogate the intersection of TLR4 signaling, epithelial redox activity, and the microbiota in colitis-associated neoplasia. METHODS: Inflammatory bowel disease and colorectal cancer data sets were analyzed for expression of TLR4, dual oxidase 2 (DUOX2), and NADPH oxidase 1 (NOX1). Epithelial production of hydrogen peroxide (H2O2) was analyzed in murine colonic epithelial cells and colonoid cultures. Colorectal cancer models were carried out in villin-TLR4 mice, carrying a constitutively active form of TLR4, their littermates, and villin-TLR4 mice backcrossed to DUOXA-knockout mice. The role of the TLR4-shaped microbiota in tumor development was tested in wild-type germ-free mice. RESULTS: Activation of epithelial TLR4 was associated with up-regulation of DUOX2 and NOX1 in inflammatory bowel disease and colorectal cancer. DUOX2 was exquisitely dependent on TLR4 signaling and mediated the production of epithelial H2O2. Epithelial H2O2 was significantly increased in villin-TLR4 mice; TLR4-dependent tumorigenesis required the presence of DUOX2 and a microbiota. Mucosa-associated microbiota transferred from villin-TLR4 mice to wild-type germ-free mice caused increased H2O2 production and tumorigenesis. CONCLUSIONS: Increased TLR4 signaling in colitis drives expression of DUOX2 and epithelial production of H2O2. The local milieu imprints the mucosal microbiota and imbues it with pathogenic properties demonstrated by enhanced epithelial reactive oxygen species and increased development of colitis-associated tumors. The inter-relationship between epithelial reactive oxygen species and tumor-promoting microbiota requires a 2-pronged strategy to reduce the risk of dysplasia in colitis patients.
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Colitis Ulcerosa/complicaciones , Neoplasias Asociadas a Colitis/patología , Oxidasas Duales/metabolismo , Microbioma Gastrointestinal/inmunología , Receptor Toll-Like 4/metabolismo , Animales , Azoximetano/administración & dosificación , Azoximetano/toxicidad , Carcinogénesis/inducido químicamente , Carcinogénesis/inmunología , Carcinogénesis/patología , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/inmunología , Colitis Ulcerosa/microbiología , Neoplasias Asociadas a Colitis/inmunología , Neoplasias Asociadas a Colitis/microbiología , Colon/efectos de los fármacos , Colon/inmunología , Colon/microbiología , Colon/patología , Conjuntos de Datos como Asunto , Sulfato de Dextran/administración & dosificación , Sulfato de Dextran/toxicidad , Modelos Animales de Enfermedad , Vida Libre de Gérmenes , Humanos , Peróxido de Hidrógeno/metabolismo , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , NADPH Oxidasa 1/metabolismo , Receptor Toll-Like 4/genéticaRESUMEN
Noncoding circular RNAs are widespread in the tree of life. Particularly, intron-containing circular RNAs which apparently upregulate their parental gene expression. Entamoeba histolytica, the causative agent of dysentery and liver abscesses in humans, codes for several noncoding RNAs, including circular ribosomal RNAs, but no intron containing circular RNAs have been described to date. Divergent RT-PCR and diverse molecular approaches, allowed us to detect bona fide full-length intronic circular RNA (flicRNA) molecules. Self-splicing reactions, RNA polymerase II inhibition with Actinomycin D, and second step of splicing-inhibition with boric acid showed that the production of flicRX13 (one of the flicRNAs found in this work, and our test model) depends on mRNA synthesis and pre-mRNA processing instead of self-splicing. To explore the cues and factors involved in flicRX13 biogenesis in vivo, splicing assays were carried out in amoeba transformants where splicing factors and Dbr1 (intron lariat debranching enzyme 1) were silenced or overexpressed, or where Rabx13 wild-type and mutant 5'ss (splice site) and branch site minigene constructs were overexpressed. Whereas SF1 (splicing factor 1) is not involved, the U2 auxiliary splicing factor, Dbr1, and the GU-rich 5'ss are involved in postsplicing flicRX13 biogenesis, probably by Dbr1 stalling, in a similar fashion to the formation of ciRNAs (circular intronic RNAs), but with distinctive 5'-3'ss ligation points. Different from the reported functions of ciRNAs, the 5'ss GU-rich element of flicRX13 possibly interacts with transcription machinery to silence its own gene in cis. Furthermore, introns of E. histolytica virulence-related genes are also processed as flicRNAs.
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Entamoeba histolytica/genética , Entamoeba histolytica/metabolismo , Intrones , Empalme del ARN , ARN/genética , ARN/metabolismo , Silenciador del Gen , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , ARN CircularRESUMEN
AIM: To investigate the effects of plecanatide and dolcanatide on maintenance of paracellular permeability, integrity of tight junctions and on suppression of visceral hypersensitivity. METHODS: Transport of fluorescein isothiocyanate (FITC)-dextran was measured to assess permeability across cell monolayers and rat colon tissues. Effects of plecanatide and dolcanatide on the integrity of tight junctions in Caco-2 and T84 monolayers and on the expression and localization of occludin and zonula occludens-1 (ZO-1) were examined by immunofluorescence microscopy. Anti-nociceptive activity of these agonists was evaluated in trinitrobenzene sulfonic acid (TNBS)-induced inflammatory as well as in non-inflammatory partial restraint stress (PRS) rat models. Statistical significance between the treatment groups in the permeability studies were evaluated using unpaired t-tests. RESULTS: Treatment of T84 and Caco-2 monolayers with lipopolysaccharide (LPS) rapidly increased permeability, which was effectively suppressed when monolayers were also treated with plecanatide or dolcanatide. Similarly, when T84 and Caco-2 monolayers were treated with LPS, cell surface localization of tight junction proteins occludin and ZO-1 was severely disrupted. When cell monolayers were treated with LPS in the presence of plecanatide or dolcanatide, occludin and ZO-1 were localized at the cell surface of adjoining cells, similar to that observed for vehicle treated cells. Treatment of cell monolayers with plecanatide or dolcanatide without LPS did not alter permeability, integrity of tight junctions and cell surface localization of either of the tight junction proteins. In rat visceral hypersensitivity models, both agonists suppressed the TNBS-induced increase in abdominal contractions in response to colorectal distension without affecting the colonic wall elasticity, and both agonists also reduced colonic hypersensitivity in the PRS model. CONCLUSION: Our results suggest that activation of GC-C signaling might be involved in maintenance of barrier function, possibly through regulating normal localization of tight junction proteins. Consistent with these findings, plecanatide and dolcanatide showed potent anti-nociceptive activity in rat visceral hypersensitivity models. These results imply that activation of GC-C signaling may be an attractive therapeutic approach to treat functional constipation disorders and inflammatory gastrointestinal conditions.
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Estreñimiento/tratamiento farmacológico , Agonistas de la Guanilato Ciclasa C/farmacología , Síndrome del Colon Irritable/tratamiento farmacológico , Receptores de Enterotoxina/metabolismo , Dolor Visceral/tratamiento farmacológico , Administración Oral , Animales , Células CACO-2 , Colon/citología , Colon/efectos de los fármacos , Colon/patología , Estreñimiento/patología , Dextranos/farmacocinética , Femenino , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/farmacocinética , Agonistas de la Guanilato Ciclasa C/uso terapéutico , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Síndrome del Colon Irritable/etiología , Síndrome del Colon Irritable/patología , Lipopolisacáridos/farmacología , Masculino , Péptidos Natriuréticos/farmacología , Péptidos Natriuréticos/uso terapéutico , Nocicepción/efectos de los fármacos , Péptidos/farmacología , Péptidos/uso terapéutico , Permeabilidad/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Ácido Trinitrobencenosulfónico/toxicidad , Dolor Visceral/inducido químicamente , Dolor Visceral/patologíaRESUMEN
Postmortem computed tomography (CT) has been extensively used in the last decade for identification purposes and in various anthropologic studies. Postmortem CT measurements of scapulae, analyzed using logistic discriminant function developed in this study, showed 94.5% accuracy in estimating sex. Data analyzed using the Dabbs and Moore-Jansen (2010) discriminant function and the discriminant function generated in this study provided nearly identical results with disagreement in only one case. Height and weight were not statically significant in sex prediction. The results of this study show that data obtained from volume rendered postmortem CT images can be considered reliable and treated as a practical option to standard anthropological methods, especially in mass fatalities as a rapid triage tool for sex determination.
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Tomografía Computarizada Multidetector , Escápula/diagnóstico por imagen , Determinación del Sexo por el Esqueleto/métodos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Análisis Discriminante , Femenino , Antropología Forense , Humanos , Imagenología Tridimensional , Modelos Logísticos , Masculino , Maryland , Persona de Mediana Edad , Reproducibilidad de los Resultados , Escápula/anatomía & histología , Adulto JovenRESUMEN
Andes virus is the main causative agent of Hantavirus cardiopulmonary syndrome in South America. There are currently no vaccines or treatments against Andes virus. However, there are several evidences suggesting that antibodies against Andes virus envelope glycoproteins may be enough to confer full protection against Hantavirus cardiopulmonary syndrome. The goal of the present work was to express, purify and characterize the extracellular domains of Andes virus glycoproteins Gn and Gc. We generated two adenoviral vectors encoding the extracellular domains of Andes virus glycoproteins Gn and Gc. Both molecules were expressed by adenoviral transduction in SiHa cells. We found that sGc ectodomain was mainly secreted into the culture medium, whereas sGn was predominantly retained inside the cells. Both molecules were expressed at very low concentrations (below 1 µg/mL). Treatment with the proteasome inhibitor ALLN raised sGc concentration in the cell culture medium, but did not affect expression levels of sGn. Both ectodomains were purified by immobilized metal ion affinity chromatography, and were recognized by sera from persons previously exposed to Andes virus. To our knowledge, this is the first work that addresses the expression and purification of Andes virus glycoproteins Gn and Gc. Our results demonstrate that sGn and sGc maintain epitopes that are exposed on the surface of the viral envelope. However, our work also highlights the need to explore new strategies to achieve high-level expression of these proteins for development of a vaccine candidate against Andes virus.
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Orthohantavirus/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas del Envoltorio Viral/aislamiento & purificación , Proteínas del Envoltorio Viral/metabolismo , Línea Celular Tumoral , Electroforesis en Gel de Poliacrilamida , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genéticaRESUMEN
BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is caused by multiple factors including hepatic oxidative stress, lipotoxicity, and mitochondrial dysfunction. Obesity is among the risk factors for NAFLD alongside type 2 diabetes mellitus and hyperlipidemia. α- mangostin (α-MG) extracts from the pericarps of mangosteen (Garcinia mangostana Linn.) may regulate high fat diet-induced hepatic steatosis; however the underlying mechanisms remain unknown. The aim of this study was to investigate the regulatory effect of α-MG on high fat diet-induced hepatic steatosis and the underlying mechanisms related to mitochondrial functionality and apoptosis in vivo and in vitro. METHODS: Sprague Dawley (SD) rats were fed on either AIM 93-M control diet, a high-fat diet (HFD), or high-fat diet supplemented with 25 mg/day mangosteen pericarp extract (MGE) for 11 weeks. Thereafter, the following were determined: body weight change, plasma free fatty acids, liver triglyceride content, antioxidant enzymes (superoxide dismutase, SOD; glutathione, GSH; glutathione peroxidase, GPx; glutathione reductase GRd; catalase, CAT) and mitochondrial complex enzyme activities. In the in vitro study, primary liver cells were treated with 1 mM free fatty acid (FFA) (palmitate: oleate acid = 2:0.25) to induce steatosis. Thereafter, the effects of α-MG (10 µM, 20 µM, 30 µM) on total and mitochondria ROS (tROS, mitoROS), mitochondria bioenergetic functions, and mitochondrial pathway of apoptosis were examined in the FFA-treated primary liver cells. RESULTS: The MGE group showed significantly decreased plasma free fatty acids and hepatic triglycerides (TG) and thiorbarbituric acid reactive substances (TBARS) levels; increased activities of antioxidant enzymes (SOD, GSH, GPx, GRd, CAT); and enhanced NADH-cytochrome c reductase (NCCR) and succinate-cytochrome c reductase (SCCR) activities in the liver tissue compared with HFD group. In the in vitro study, α-MG significantly increased mitochondrial membrane potential, enhanced cellular oxygen consumption rate (OCR), decreased tROS (total ROS) and mitoROS (mitochondrial ROS) levels ; reduced Ca2+ and cytochrome c (cyt c) release from mitochondria, and reduced caspases 9 and 3 activities compared with control group. CONCLUSION: These findings demonstrate α-MG attenuated hepatic steatosis in high fat-diet fed rats potentially through enhanced cellular antioxidant capacity and improved mitochondrial functions as well as suppressed apoptosis of hepatocytes. The findings of study represent a novel nutritional approach on the use of α-MG in the prevention and management of NAFLD.
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ERΔ3 transgenic mice expressing a dominant negative estrogen receptor α (ERα) variant lacking the second zinc finger in the DNA binding domain were developed to examine its potential to inhibit estrogen action in vivo. To investigate if ERΔ3 expression influences uterine carcinogenesis, ERΔ3 transgenic mice were exposed to diethylstilbestrol (DES) on post-natal days 1-5. Neonatal DES treatment induced uterine adenocarcinomas in 81% of 8-month-old ERΔ3 mice compared to 49% of wild-type females (p<0.016). ERΔ3 did not inhibit the expression of the estrogen-responsive progesterone receptor and lactoferrin genes in the presence of ERα or modify their expression in ERα knockout (αERKO) mice. Higher circulating 17ß-estradiol levels and non-classical signaling by ERΔ3 may be related to the earlier incidence of uterine cancer. These findings indicate that expression of this ERα variant can influence determining events in uterine cancer development and its natural occurrence in the human uterus would unlikely be protective.
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Carcinógenos/toxicidad , Dietilestilbestrol/toxicidad , Receptor alfa de Estrógeno/genética , Estrógenos/toxicidad , Neoplasias Uterinas/genética , Animales , Animales Recién Nacidos , Estradiol/sangre , Receptor alfa de Estrógeno/metabolismo , Femenino , Masculino , Ratones , Ratones Transgénicos , Progesterona/sangre , Neoplasias Uterinas/inducido químicamente , Neoplasias Uterinas/metabolismoRESUMEN
The pathogenesis of inflammation in the central nervous system (CNS), which contributes to numerous neurodegenerative diseases and results in encephalopathy and neuroinflammation, is poorly understood. Sphingolipid metabolism plays a crucial role in maintaining cellular processes in the CNS, and thus mediates the various pathological consequences of inflammation. For a better understanding of the role of sphingosine kinase activation during neuroinflammation, we developed a bacterial lipopolysaccharide (LPS)-induced brain injury model. The onset of the inflammatory response was observed beginning 4 hours after intracerebral injection of LPS into the lateral ventricles of the brain. A comparison of established neuroinflammatory parameters such as white matter rarefactions, development of cytotoxic edema, astrogliosis, loss of oligodendrocytes, and major cytokines levels in wild type and knockout mice suggested that the neuroinflammatory response in SphK1-/- mice was significantly upregulated. At 6 hours after intracerebroventricular injection of LPS in SphK1-/- mice, the immunoreactivity of the microglia markers and astrocyte marker glial fibrillary acidic protein (GFAP) were significantly increased, while the oligodendrocyte marker O4 was decreased compared to WT mice. Furthermore, western blotting data showed increased levels of GFAP. These results suggest that SphK1 activation is involved in the regulation of LPS induced brain injury. RESEARCH HIGHLIGHTS: ⢠Lipopolysaccharide (LPS) intracerebral injection induces severe neuroinflammation. ⢠Sphingosine kinase 1 deletion worsens the effect of the LPS. ⢠Overexpression of SphK1 might be a potential new treatment approach to neuroinflammation.
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Enfermedades del Sistema Nervioso Central/inducido químicamente , Inflamación/inducido químicamente , Lipopolisacáridos/farmacología , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Animales , Secuencia de Bases , Cartilla de ADN , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
BACKGROUND: Oocytes in humans, mice and other mammals lack identifiable centrioles. The proximal centriole brought in by the fertilizing sperm in humans and most other mammals appears to gives rise to the centrioles at the spindle poles in the zygote, and is believed to indicate that centrioles are inherited through the paternal lineage. However, both the proximal and distal sperm centrioles degenerate in mice and other rodents. A bipolar mitotic spindle nucleates from multiple centrosome-like structures in the mouse zygote and centrioles are not seen until the blastocyst stage, suggesting that centrioles are inherited through the maternal lineage in mice. We previously identified speriolin as a spermatogenic cell-specific binding partner of Cdc20 that co-localizes with pericentrin in mouse spermatocytes and is present in the centrosome in round spermatids. METHOD: The nature and localization of speriolin in mouse and human sperm and the fate of speriolin following fertilization in the mouse were determined using immunofluorescence microscopy, immunoelectron microscopy and western blotting. RESULTS: Speriolin surrounds the intact proximal centriole in human sperm, but is localized at the periphery of the disordered distal centriole in mouse sperm. Human speriolin contains an internal 163-amino acid region not present in mouse that may contribute to localization differences. Speriolin is carried into the mouse oocyte during fertilization and remains associated with the decondensing sperm head in zygotes. The speriolin spot appears to undergo duplication or splitting during the first interphase and is detectable in 2-cell embryos. CONCLUSIONS: Speriolin is a novel centrosomal protein present in the connecting piece region of mouse and human sperm that is transmitted to the mouse zygote and can be detected throughout the first mitotic division.
Asunto(s)
Proteínas Portadoras/fisiología , Proteínas de Ciclo Celular/fisiología , Centrosoma/metabolismo , Espermatozoides/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/análisis , Proteínas Portadoras/química , Proteínas de Ciclo Celular/análisis , Proteínas de Ciclo Celular/química , Secuencia Conservada , Humanos , Masculino , Ratones , Análisis de Secuencia de Proteína , Solubilidad , Espermatozoides/ultraestructura , Cigoto/metabolismoRESUMEN
Phosphoglycerate kinase 2 (PGK2), an isozyme that catalyzes the first ATP-generating step in the glycolytic pathway, is encoded by an autosomal retrogene that is expressed only during spermatogenesis. It replaces the ubiquitously expressed phosphoglycerate kinase 1 (PGK1) isozyme following repression of Pgk1 transcription by meiotic sex chromosome inactivation during meiotic prophase and by postmeiotic sex chromatin during spermiogenesis. The targeted disruption of Pgk2 by homologous recombination eliminates PGK activity in sperm and severely impairs male fertility, but does not block spermatogenesis. Mating behavior, reproductive organ weights (testis, excurrent ducts, and seminal vesicles), testis histology, sperm counts, and sperm ultrastructure were indistinguishable between Pgk2(-/-) and wild-type mice. However, sperm motility and ATP levels were markedly reduced in males lacking PGK2. These defects in sperm function were slightly less severe than observed in males lacking glyceraldehyde-3-phosphate dehydrogenase, spermatogenic (GAPDHS), the isozyme that catalyzes the step preceding PGK2 in the sperm glycolytic pathway. Unlike Gapdhs(-/-) males, the Pgk2(-/-) males also sired occasional pups. Alternative pathways that bypass the PGK step of glycolysis exist. We determined that one of these bypass enzymes, acylphosphatase, is active in mouse sperm, perhaps contributing to phenotypic differences between mice lacking GAPDHS or PGK2. This study determined that PGK2 is not required for the completion of spermatogenesis, but is essential for sperm motility and male fertility. In addition to confirming the importance of the glycolytic pathway for sperm function, distinctive phenotypic characteristics of Pgk2(-/-) mice may provide further insights into the regulation of sperm metabolism.