RESUMO
The human hematopoietic stem/progenitor cell 117 (HSPC117) protein is an essential component of protein complexes and has been identified to be involved in many important functions. However, how this gene expression is regulated and whether the HSPC117 gene affects cell migration is still unknown. The aim of this study was to identify whether HSPC117 mRNA expression is regulated by epigenetic modification and whether HSPC117 expression level affects the expression of matrix metalloproteinase 2 (MMP 2), matrix metalloproteinase 14 (MMP 14), and tissue inhibitor of metalloproteinases 2 (TIMP 2), and further affects human placenta choriocarcinoma cell (JEG-3) migration speed. In our epigenetic modification experiment, JEG-3 cells were cultured in medium with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC), the histone deacetylase (HDAC) inhibitor trichostatin A (TSA), or both inhibitors. Then, the HSPC117 mRNA and protein expressions were assessed using real-time quantitative PCR (qPCR) and Western blot assay. The results showed that, compared to the control, HSPC117 mRNA expression was increased by TSA or 5-aza-dC. The highest HSPC117 expression level was found after treatment with both 5-aza-dC and TSA. Further, in order to investigate the effect of HSPC117 on MMP 2, MMP 14, and TIMP 2 mRNA expressions, pEGFP-C1-HSPC117 plasmids were transfected into JEG-3 cells to improve the expression of HSPC117 in the JEG-3 cells. Then, the mRNA expression levels of MMP 2, MMP 14, TIMP 2, and the speed of cell migration were assessed using the scratch wound assay. The results showed that over-expression of HSPC117 mRNA reduced MMP 2 and MMP 14 mRNA expression, while TIMP 2 mRNA expression was up-regulated. The scratch wound assay showed that the migration speed of JEG-3 cells was slower than the non-transfected group and the C1-transfected group. All of these results indicate that HSPC117 mRNA expression is regulated by epigenetic modification; over-expression of HSPC117 decreases MMP 2 and MMP 14 transcription, reduces cell migration speed, and increases TIMP 2 transcription.
Assuntos
Epigênese Genética , Proteínas/metabolismo , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Decitabina , Epigênese Genética/efeitos dos fármacos , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Metilação/efeitos dos fármacos , Proteínas/genética , RNA Mensageiro/metabolismo , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismoRESUMO
Gram-negative bacterial infections pose a significant threat to public health. Toll-like receptor 4 (TLR4) recognizes bacterial lipopolysaccharide (LPS) and induces innate immune responses, autophagy, and cell death, which have major impacts on the body's physiological homeostasis. However, the role of TLR4 in bacterial LPS-induced autophagy and apoptosis in large mammals, which are closer to humans than rodents in many physiological characteristics, remains unknown. So far, few reports focus on the relationship between TLR, autophagy, and apoptosis in large mammal levels, and we urgently need more tools to further explore their crosstalk. Here, we generated a TLR4-enriched mammal model (sheep) and found that a high-dose LPS treatment blocked autophagic degradation and caused strong innate immune responses and severe apoptosis in monocytes/macrophages of transgenic offspring. Excessive accumulation of autophagosomes/autolysosomes might contribute to LPS-induced apoptosis in monocytes/macrophages of transgenic animals. Further study demonstrated that inhibiting TLR4 downstream NF-κB or p38 MAPK signaling pathways reversed the LPS-induced autophagy activity and apoptosis. These results indicate that the elevated TLR4 aggravates LPS-induced monocytes/macrophages apoptosis by leading to lysosomal dysfunction and impaired autophagic flux, which is associated with TLR4 downstream NF-κB and MAPK signaling pathways. This study provides a novel TLR4-enriched mammal model to study its potential effects on autophagy activity, inflammation, oxidative stress, and cell death. These findings also enrich the biological functions of TLR4 and provide powerful evidence for bacterial infection.
Assuntos
Lipopolissacarídeos , NF-kappa B , Humanos , Animais , Ovinos , NF-kappa B/metabolismo , Lipopolissacarídeos/farmacologia , Receptor 4 Toll-Like/metabolismo , Apoptose , Mamíferos/metabolismo , AutofagiaRESUMO
The intramuscular fat (IMF) content and fatty acid composition are important meat quality traits that are mostly affected by the cattle breed. Muscle, adipose tissue and liver are important organs involved in the development of intramuscular adipose tissue. Thus, we hypothesized that there were marked differences in the adipogenesis and lipid metabolism of these tissues between Wagyu-cross and Holstein steers during the finishing phases. To test this hypothesis, we analyzed the expression levels of adipogenesis- and lipid metabolism-related genes in longissimus muscle (LM), subcutaneous fat (SCF) and liver from Wagyu-cross and Holstein steers at 26 months of age. The IMF content and fatty acid profile of LM were determined. Wagyu-cross steers had a higher IMF content and MUFA percentages in the LM than Holstein steers (P<0.05). The relative expression of FGF2, COL1A1, SREBP1c, SCD1, GRP78 and LEP was greater in the LM of Wagyu-cross steers than in Holstein steers (P<0.05). In contrast, Holstein steer SCF had higher (P<0.05) mRNA expression levels of FABP4 and ADIPOQ than Wagyu-cross steers. In the liver, the expression of SREBP1c and GRP78 in Wagyu-cross steers was significantly higher than that in Holstein steers (P<0.05). The results demonstrate that both intramuscular adipogenesis and fibrogenesis are enhanced in Wagyu-cross steers compared with Holstein steers during the finishing phase and that IMF deposition is positively correlated with the maturity of SCF and hepatic lipid accumulation in Wagyu-cross steers.
Assuntos
Adipogenia/genética , Tecido Adiposo/metabolismo , Expressão Gênica , Metabolismo dos Lipídeos/genética , Fígado/metabolismo , Músculo Esquelético/metabolismo , Animais , Bovinos , Ácidos Graxos/metabolismo , Masculino , Especificidade da Espécie , Gordura Subcutânea/metabolismoRESUMO
Insulin-like growth factor 1 (IGF1), also known as somatomedin C, is essential for the regulation of animal growth and development. In many species, the IGF1 gene can be alternatively spliced into multiple transcripts, encoding different pre-pro-IGF1 proteins. However, the exact alternative splicing patterns of IGF1 and the sequence information of different splice variants in sheep are still unclear. In this study, four splice variants (class 1-Ea, class 1-Eb, class 2-Ea, and class 2-Eb) were obtained, but no IGF1 Ec, similar to that found in other species, was discovered. Bioinformatics analysis showed that the four splice variants shared the same mature peptide (70 amino acids) and possessed distinct signal peptides and E peptides. Tissue expression analysis indicated that the four splice variants were broadly expressed in all tested tissues and were most abundantly expressed in the liver. In most tissues and stages, the expression of class 1-Ea was highest, and the expression of other splice variants was low. Overall, levels of the four IGF1 splice variants at the fetal and lamb stages were higher than those at the adult stage. Overexpression of the four splice variants significantly increased fibroblast proliferation and inhibited apoptosis (P < 0.05). In contrast, silencing IGF1 Ea or IGF1 Eb with siRNA significantly inhibited proliferation and promoted apoptosis (P < 0.05). Among the four splice variants, class 1-Ea had a more evident effect on cell proliferation and apoptosis. In summary, the four ovine IGF1 splice variants have different structures and expression patterns and might have different biological functions.
RESUMO
Reproductive traits are important factors in sheep production. The Booroola fecundity (FecB) gene-the first major gene for prolificacy identified in sheep-has a positive effect on ovulation rates and litter size under natural reproductive conditions. However, the effect of the FecB gene on reproductive performance under assisted reproduction, which uses many artificial hormones, remains unclear. In the present study, we evaluated the effect of FecB (BMPR-1B mutation) on reproductive performance under assisted reproduction, and examined offspring body weight at birth and weaning and survival rate at weaning. There were no differences among three genotype groups (homozygous carrier, BB; heterozygous carrier, B+; non-carrier, ++) in terms of estrus detection rate, time to estrus onset, or estrus duration following estrus synchronization (Pâ¯>â¯0.05). The pregnancy rates at 60â¯d were similar among three genotype groups after artificial insemination (Pâ¯>â¯0.05). However, the B allele had an additive effect on litter size (one copy resulted in an increase of 0.88 lambs and two copies produced an additional 0.41 lambs; Pâ¯<â¯0.01), and increased lambing and fecundity rates (Pâ¯<â¯0.01). After multiple ovulation, the average numbers of recovered embryos per ewe were 9.16⯱â¯0.79, 8.20⯱â¯0.77, and 8.44⯱â¯0.61 in the BB, B+, and ++ ewes, respectively (Pâ¯>â¯0.05). There were no differences in the fertilization rate or numbers of grade 1-2 embryos among different groups (Pâ¯>â¯0.05). The birth and weaning weights of lambs from BB and B+ ewes were lower than those of lambs born from ++ ewes (Pâ¯<â¯0.01) owing to the high fecundity. The survival rate of lambs at weaning did not differ among groups (Pâ¯>â¯0.05). Our results indicated that the presence of the B allele had an additive effect on litter size after artificial insemination, but it did not influence the parameters of estrus synchronization and multiple ovulation. Furthermore, the higher prolificacy in ewes carrying the B allele was associated with a reduction in offspring body weight at birth and weaning.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Fertilidade/genética , Reprodução/genética , Técnicas de Reprodução Assistida/veterinária , Ovinos/genética , Animais , Peso Corporal/genética , Cruzamento , Cruzamentos Genéticos , Feminino , Genótipo , Vigor Híbrido/genética , Tamanho da Ninhada de Vivíparos/genética , Mutação , Gravidez , Resultado da Gravidez/genética , Resultado da Gravidez/veterinária , Resultado do TratamentoRESUMO
Toll-like receptor 4 (TLR4) is a critical pattern recognition receptor that plays a critical role in the host innate immune system's recognition of Gram-negative bacteria. Since it is the lipopolysaccharide (LPS) receptor, it links the activated inflammatory response with autophagy and oxidative stress. Autophagy, or type II programmed cell death, was reported to have defensive functions in response to the production of inflammatory cytokines and oxidative stress. To explore the relationship between autophagy, inflammation, and oxidative stress, a TLR4-enriched transgenic (Tg) animal model (sheep) was generated. Autophagy activity in the Tg blood monocytes was significantly higher than in the wild-type animal under LPS stress, and it returned to normal after transfection of TLR4 siRNA. Pretreatment with 3-methyladenine (3-MA) inhibited autophagy and enhanced oxidative stress and the production of TNF-α. The LPS-induced reactive oxygen species (ROS) level was markedly increased in the Tg group at an early stage before quickly returning to normal values. In addition, suppressing ROS production by N-acetyl-L-cysteine down-regulated the number of intracellular autophagosomes and the expression of Beclin-1, ATG5, and cytokines IL-1ß, IL-6, and TNF-α. Further mechanistic investigation suggested that the TLR4-associated p38 mitogen-activated protein kinase (MAPK) signaling pathway was involved in autophagy and oxidative stress. P38 MAPK promotes intracellular autophagy, ROS production, and inflammatory response. Moreover, TLR4 over-expression suppressed oxidative stress and the production of inflammatory cytokines and increased autophagy activity in vivo. Taken together, our results showed that LPS induced autophagy, which was related to TLR4-mediated ROS production through the p38 MAPK signaling pathway. In addition, our study also provided a novel transgenic animal model to analyze the effects of TLR4 on autophagy, oxidative stress, and inflammatory responses.
RESUMO
It is known that the male hypogonadism plays an important role in regulating adipose metabolism. In the present study, fifteen pairs of full male sibs were divided into a castrated group and an intact group with a paired experiment design. The pigs were slaughtered at an age of 175days. The carcass characteristics and fat deposit of the studied animal were measured, and the hormone and serum lipid levels of the peripheral blood samples were determined, and the differentially expressed genes of the back fat between the two groups were screened with porcine genome array. Our results showed that the absence of male gonadal steroids attributed to castration significantly raised the serum lipid levels and increased fat accumulation in the pigs. A total of 225 differentially expressed genes were identified between the boars and barrows and 135 of them were upregulated. The analysis of Gene Ontology categories and KEGG pathway indicated that these differentially expressed genes were mainly involved in metabolism of lipid, carbohydrate, amino acid, xenobiotics biodegradation, and immune diseases pathways. Our results indicated that there were higher capacity of fatty acid of synthesis, enhanced uptaking capacity of fatty acids and cholesterol, inhibited lipolysis, and enhanced carbohydrate metabolism in the adipose tissue of barrows compared to boars. The findings of the present study provide new insight into the mechanisms of adipose metabolism induced by hormone deficiency.
Assuntos
Tecido Adiposo/metabolismo , Perfilação da Expressão Gênica/métodos , Hormônios Esteroides Gonadais/deficiência , Lipídeos/sangue , Animais , Composição Corporal , Hormônios Esteroides Gonadais/sangue , Masculino , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Transdução de Sinais , Suínos , Transcriptoma , Regulação para CimaRESUMO
Genetic modification provides a means to enhancing disease resistance in animals. Toll-like receptor 4 (TLR4), a member of the TLR family, is critical for the recognition of lipopolysaccharide (LPS)/endotoxin from Gram-negative bacteria by host immune cells, which initiates cell activation and subsequently triggers a proinflammatory response to the invading pathogens. In this study, the first generation of genetically modified (GM) sheep overexpressing TLR4 was produced by microinjection for better disease resistance. Compared with wild-type (WT) rams, the GM rams have similar growth performance, basic semen quality and spermatozoon ultrastructure. The offspring birth rates after cervical artificial insemination were also similar between GM (90.32%) and WT (92.38%) rams. Overall, the presence and expression of the TLR4 transgene in the genome did not appear to interfere with normal semen production, reproductive traits and the ability of transgene transmission to offspring. The expression levels of TLR4, tumor necrosis factor and interferon gamma genes in monocyte/macrophages from GM sheep were significantly higher than that from WT sheep at early stages after LPS stimulation. The GM offspring born from the founder transgenic ram inseminated ewes had similar survival rate with WT offspring (88.89% vs 84.86%) at weaning. The TLR4 transgene showed no deleterious effects on growth performance, reproductive traits and offspring survivability of GM rams. Therefore, the GM sheep overexpressing TLR4 provide a powerful experimental model for analyzing function of TLR4 in vivo during infection and inflammation.
Assuntos
Animais Geneticamente Modificados , Ovinos/genética , Ovinos/fisiologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Animais , Longevidade , Preservação do SêmenRESUMO
Metal ion homeostasis and heavy metal detoxification systems are regulated by certain genes associated with metal ion transport. Metallothionein (MT) and metal response element binding transcription factor 1 (MTF1) are important regulatory proteins involved in the mediation of intracellular metal ion balance. Differences in the zincbinding affinities of the zinc fingers of MTF1 and the α and ßdomains of MT facilitate their regulation of Zn2+ concentration. Alterations in the intracellular concentration of Zn2+ influence the MTF1 zinc finger number, and MTF1 containing certain zinc finger numbers regulates the expression of corresponding target genes. The present review evaluates the association between zinc finger number in MTF1 protein, MTF1 target genes and the mechanism underlying MT regulation of the zinc finger number in MTF1.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Íons/metabolismo , Metalotioneína/metabolismo , Fatores de Transcrição/metabolismo , Zinco/metabolismo , Animais , Regulação da Expressão Gênica , Humanos , Estresse Oxidativo , Ligação Proteica , Elementos de Resposta , Fator MTF-1 de TranscriçãoRESUMO
Cryopreservation has been widely utilized in livestock and human embryos, which allows for storage of worthy embryos for a long period of time, although it is still uncertain as how long embryos can be cryopreserved in liquid nitrogen. The aims of this study were to evaluate the effects of long-term cryopreservation on birth rate of transferred sheep embryos at morula or blastocyst stage, and to investigate growth performance and viability of their offspring. A total of 373 sheep embryos from the same batch, which had been cryopreserved by conventional procedure for 0.5 yr (n=259) or 7.5 yr (n=114), respectively, were transferred to 373 recipient ewes. In parallel, artificial inseminations, acting as controls, were conducted in the same month in both years (n=81 and n=110) that embryo transfers were performed. Results showed that there were no significant differences in birth rate between short-term cryopreservation group (cryopreserved for 0.5 yr in 2003) and long-term cryopreservation group (cryopreserved for 7.5 yr in 2010) either at the morula or blastocyst stage (p>0.05). No specific differences in birth weight were observed among short-term cryopreservation, artificial insemination 1 (performed in 2003), long-term cryopreservation and artificial insemination 2 (performed in 2010) group (p>0.05). The weaning weights were similar between the short-term cryopreservation and long-term cryopreservation group (p>0.05). The mortality rates of the offspring were similar in both groups as well (p>0.05). We concluded that the long-term cryopreservation did not appear to adversely affect birth rate of the embryos, growth performance and viability of their offspring. Our results indicated that the cryopreserved sheep embryos should be stable in liquid nitrogen for at least 7.5 years.